thromboplastin and Hemophilia-A

Tissue factor pathway inhibitor (TFPI) is an alternatively spliced anticoagulant protein that primarily dampens the initiation phase of coagulation before thrombin is generated. As such, TFPI's actions are localized to cells expressing TF and to sites of injury, where it is an important regulator of bleeding in hemophilia. The major splice isoforms TFPIα and TFPIβ localize to different sites within and surrounding the vasculature. Both forms directly inhibit factor Xa (FXa) via their Kunitz 2 domain and inhibit TF-FVIIa via their Kunitz 1 domain in a tight complex primarily localized to cells. By forming complexes localized to distinct cellular microenvironments and engaging additional cell surface receptors, TFPI alters cellular trafficking and signaling pathways driven by coagulation proteases of the TF pathway. TFPIα, which circulates in complex with FV and protein S, also serves an inhibitor of FXa independent of the TF initiation complex and prevents the formation of an active prothrombinase. This regulation of thrombin generation in the context of vessel injury is effectively blocked by antibodies to Kunitz 2 domain of TFPI and exploited as a therapy to restore efficient hemostasis in hemophilia.

Topics: Blood Coagulation; Factor Xa; Hemophilia A; Humans; Lipoproteins; Thrombin; Thromboplastin

Regulatory mechanisms responsible for limiting blood clot formation are critical for maintaining normal haemostasis. Dysregulation can lead to bleeding (e.g. haemophilia) or thrombosis. New findings showing that tissue factor pathway inhibitor-alpha (TFPIα) binds coagulation factor V(a) and inhibits prothrombinase assembly highlights that our understanding of the initiation of coagulation is evolving. Work over the past decade on the biochemistry of FV activation has laid the groundwork for deciphering the mechanistic bases that may underpin how TFPIα mediates these anticoagulant effects. Collectively, these new findings are re-shaping our thinking about how coagulation is initiated at the site of injury. These ideas could have important clinical implications and help identify new ways to bias the coagulation response for the treatment of haemophilia and other disorders of the haemostatic process.

Topics: Blood Coagulation; Factor V; Hemophilia A; Hemostatics; Humans; Lipoproteins; Thrombin; Thromboplastin

Vascular injury-induced access of blood to tissue factor (TF) leads to the formation of a TF-FVII/FVIIa complex and the triggering of blood coagulation. The activated TF-dependent pathway is regulated by Tissue Factor Pathway Inhibitor (TFPI), which binds and inhibits FXa, but more importantly forms an inactive quaternary complex with TF-FVIIa-FXa, effectively shutting off the TF activity. The old view of TF residing in extravascular sites exclusively has recently been challenged by several reports on TF expression in various blood cells. The latter arena has unfortunately been marred by many contradictions, apparently related to inferior tools and/or study design, notably the widespread use of antibodies with inferior and misleading specificity and TF activity assays of low sensitivity/specificity. Our own studies along with many other reports, compels the conclusion that in blood of healthy individuals TF is exclusively associated with and expressed in circulating monocytes. In this short review the distribution of TF and TFPI in blood is discussed.

Topics: Animals; Blood Cells; Blood Coagulation; Blood Platelets; Cell-Derived Microparticles; Coagulants; Eosinophils; Hemophilia A; Humans; Lipoproteins; Monocytes; Neutrophils; Signal Transduction; Thromboplastin

Binding of factor VIIa (FVIIa) to tissue factor (TF) and the subsequent initiation of the clotting cascade is essential for hemostasis. However, the aberrant expression of FVIIa-TF contributes to thrombosis. Despite the tremendous progress made in the past 25years in understanding the molecular mechanisms involved in the interaction between FVIIa and TF, there is less known about the cell biology of these proteins. Availability of hemophilic mice (by specific knock-out of FVIII or FIX genes) and novel TF transgenic mice has allowed us in recent years to investigate the importance of TF-FVIIa-induced coagulation from wound healing to sepsis. This supplement explores new aspects of TF-FVIIa biology, with a particular focus on structural biology, cell biology and animal models.

Topics: Animals; Cell-Derived Microparticles; Disease Models, Animal; Factor IX; Factor VIIa; Hemophilia A; Hemostasis; Humans; Mice; Mice, Knockout; Sepsis; Thromboplastin; Wound Healing

Plasma coagulation factor VIIa (FVIIa) initiates the coagulation cascade by binding to its cofactor, tissue factor (TF) on cell surfaces, which eventually leads to fibrin deposition and platelet activation. Recent studies showed that FVIIa also binds to endothelial cell protein C receptor (EPCR), a known cellular receptor for anticoagulant protein C\\activated protein C, on the endothelium. The present article reviews our current knowledge of FVIIa interaction with EPCR and discusses the potential significance of this interaction in hemostasis, treatment of bleeding disorders with pharmacological doses of FVIIa and FVIIa clearance.

Topics: Binding Sites; Blood Coagulation Factors; Endothelial Cells; Endothelium, Vascular; Factor VIIa; Fibrin; Gene Expression Regulation; Hemophilia A; Hemostasis; Humans; Models, Biological; Receptors, Cell Surface; Thromboplastin

Wound healing involves a number of physiologic mechanisms including coagulation, inflammation, formation of granulation tissue, and tissue remodeling. Coagulation with robust thrombin generation leading to fibrin formation is necessary for wound healing. It is less clear if there is a requirement for ongoing coagulation to support tissue remodeling. We have studied wound healing in mice with defects in both the initiation (low tissue factor) and propagation (hemophilia B) phases. In hemophilia B mice, dermal wound healing is delayed; this delay is associated with bleeding into the granulation tissue. Mice can be treated with replacement therapy (factor IX) or bypassing agents (factor VIIa) to restore thrombin generation. If treated just prior to wound placement, mice will have normal hemostasis in the first day of wound healing. As the therapeutic agents clear, the mice will revert to hemophilic state. If the primary role of coagulation in wound healing is to provide a stable platelet/fibrin plug that is loaded with thrombin, then treating hemophilic animals just prior to wound placement should restore normal wound healing. The results from this study did not support that hypothesis. Instead the results show that restoring thrombin generation only at the time of wound placement did not improve the delayed wound healing. In preliminary studies on low tissue factor mice, there also appears to be a delay in wound healing with evidence of bleeding into the granulation tissue. The current data suggests that ongoing coagulation function needs to be maintained to support a normal wound healing process.

Topics: Animals; Blood Coagulation; Disease Models, Animal; Factor VIIa; Hemophilia A; Hemophilia B; Mice; Mice, Transgenic; Models, Biological; Signal Transduction; Thrombin; Thromboplastin; Wound Healing

Treatment choices for haemophilia patients with inhibitors are suboptimal. Tissue factor-bearing microparticles home to thrombi in a cell adhesion molecule-dependent fashion. Their potential utility as a procoagulant is discussed along with the challenges of evaluating this approach in mouse models of haemophilia.

Topics: Animals; Bone Marrow; Cell-Derived Microparticles; Endothelium, Vascular; Hemophilia A; Hemostasis; Humans; Leukocytes; Membrane Glycoproteins; Mice; Mice, Transgenic; Models, Biological; Thrombin; Thromboplastin

Recombinant activated factor VIIa (rFVIIa) has many clinical applications for patients with congenital bleeding disorders and in a variety of clinical settings. Additional studies in the future are ongoing and should provide the clinical anesthesiologist an additional option during certain bleeding states. Specific recommendations as to timing of administration and frequent monitoring of ionized calcium status are suggested at this time. Optimization of fibrinogen levels, platelet levels, pH, and body temperature will enhance efficacy of rFVIIa.

Topics: Cerebral Hemorrhage; Factor VIIa; Hemophilia A; Hemophilia B; Humans; Postpartum Hemorrhage; Recombinant Proteins; Thromboplastin; Warfare; Wounds and Injuries

Tissue factor (TF) is a transmembrane receptor for Factor VII/VIIa (FVII/VIIa). It is constitutively expressed by cells surrounding blood vessels. The endothelium physically separates this potent "activator" from its circulating ligand FVII/FVIIa and prevents inappropriate activation of the clotting cascade. Breakage of the endothelial barrier leads to exposure of extravascular TF and rapid activation of the clotting cascade. TF is also expressed in certain tissues, such as the heart and brain, and provides additional hemostatic protection to these tissues. Small amounts of TF are also present in blood in the form of microparticles, which are small membrane vesicles derived from activated and apoptotic cells. Levels of microparticle TF increase in a variety of diseases, such as sepsis and cancer, and this so-called "blood-borne" TF may contribute to thrombosis associated with these diseases. Recombinant FVIIa has been developed as an effective hemostatic drug for the treatment of hemophilia patients with inhibitory antibodies. In addition, it is used for patients with bleeding that do not respond to conventional therapy. However, the mechanism by which recombinant FVIIa restores hemostasis has not been clearly defined. In conclusion, the TF:FVIIa complex is essential for hemostasis and recombinant FVIIa is an effective hemostatic drug.

Topics: Animals; Cell-Derived Microparticles; Endothelium, Vascular; Factor VIIa; Hemophilia A; Hemostasis; Hemostatics; Humans; Recombinant Proteins; Thromboplastin; Thrombosis

The correct diagnosis of factor VIII deficiency and the assessment of severity of the disease are essential for a patient-tailored treatment strategy. An optimal diagnostic procedure comprises sensitive and specific screening methods and factor VIII activity assays. Different screening reagents show variable characteristics and receiver operator characteristic curves are presented showing the relation between sensitivity and specificity of eleven activated partial thromboplastin time reagents. The details of the three methods for factor VIII activity assay, one-stage and two-stage assay and chromogenic assays, are discussed. The chromogenic assay seems to be more sensitive than the one-stage assay with regard to the detection of severe haemophilia. Discrepant results obtained with one-stage and two-stage assays are reviewed and discussed.

Topics: Blood Coagulation; Chromogenic Compounds; Clinical Laboratory Techniques; Hemophilia A; Humans; Phenotype; Reagent Kits, Diagnostic; Reproducibility of Results; Sensitivity and Specificity; Thromboplastin

The molecular mechanisms responsible for the hemostatic efficacy of recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark) in platelet-related bleeding disorders remain unclear. The general concept is that rFVIIa locally enhances thrombin generation at the site of injury, where tissue factor (TF) has become exposed. However, a growing amount of evidence shows that rFVIIa is also able to exert its activity in a manner independent of TF. Using an in vitro flow model, we recently showed that TF-independent thrombin generation is responsible for increased platelet deposition onto injured vessels following rFVIIa administration. Furthermore, it has been shown that rFVIIa can restore platelet aggregation in Glanzmann's thrombasthenia (GT) patients via TF-independent thrombin generation. However, the mechanism behind TF-independent thrombin generation remains to be elucidated. It is postulated that, in vivo, both the TF-dependent and TF-independent thrombin generation induced by rFVIIa contribute to the control of hemorrhage in patients with platelet-related bleeding disorders and, perhaps, other causes of hemorrhagic diatheses.

Topics: Blood Coagulation; Blood Platelet Disorders; Factor VIIa; Hemophilia A; Hemostasis; Humans; Platelet Activation; Recombinant Proteins; Thromboplastin

For hemophilia patients with inhibitors against FVIII or FIX, the development of recombinant factor VIIa (rFVIIa) raises the possibility of a therapeutic alternative whose availability and convenience of treatment are comparable to those of FVIII or FIX. In support of this new concept for the treatment of bleeding episodes, pharmacological doses of FVIIa have been shown to induce hemostasis. Pharmacological doses of rFVIIa enhance thrombin generation on thrombin-activated platelets, thereby facilitating the formation of strong, well-structured fibrin plugs resistant to premature proteolysis. Modified rFVIIa molecules with a stronger hemostatic potential have been produced. Inhibition of the FVII-TF-dependent pathway (TFPI and rFVIIai) has been tried in attempts to prevent thrombosis, with promising results in animal models so far not confirmed in clinical trials.

Topics: Factor VIIa; Hemophilia A; Hemostatic Disorders; Hemostatics; Humans; Recombinant Proteins; Thromboplastin

Recombinant activated coagulation factor VII (rFVIIa) was developed for the treatment of patients with hemophilia who have developed inhibitors against the factor they are missing. Hemophilia is a serious bleeding disorder and patients with hemophilia develop repeated spontaneous CNS, joint and muscle bleeding. Any trauma, even mild events, may cause life-threatening bleeding, and without treatment, these patients have a life expectancy of about 16 years. Thus, hemophilia can be regarded as a model of severe bleeding, and an agent capable of inducing hemostasis in severe hemophilia independent of the hemophilia proteins (FVIII or FIX) may also be effective in patients without hemophilia who experience serious bleeds. The availability of rFVIIa stimulated research on the role of FVII and tissue factor (TF) in the hemostatic process. As a result, a picture partly different from the one suggested by previous models has emerged. These previous models basically neglected the role of cells and cell membranes. The importance of platelets and platelet membrane phospholipids in hemostasis has been demonstrated, and the new concept of the hemostatic process, focusing on cell surfaces, has been outlined.

Topics: Cerebral Hemorrhage; Clinical Trials as Topic; Dose-Response Relationship, Drug; Factor VII Deficiency; Factor VIIa; Hemophilia A; Hemophilia B; Hemostatics; Infusions, Intravenous; Recombinant Proteins; Thromboplastin

Topics: Antigen-Antibody Complex; Biomarkers; Hemophilia A; Humans; Immunoassay; Partial Thromboplastin Time; Reference Values; Specimen Handling; Thromboplastin; von Willebrand Diseases

We have developed a cell-based model of hemostasis. This model suggests that the defect in hemophilia is specifically a failure of platelet-surface factor Xa (FXa) generation, leading to a failure of platelet surface thrombin generation. Activation of FX by FVIIa/tissue factor (TF) does not compensate for a lack of FXa activation on the platelet surface by the FVIIIa/FIXa complex. This is because plasma protease inhibitors prevent FXa from moving through the fluid phase from the TF-bearing cell to the platelet surface. We have previously proposed a platelet-dependent mechanism of action for high-dose factor VIIa (FVIIa; Novoseven, Novo Nordisk, Copenhagen, Denmark). Our data suggest that, when present at high levels, FVIIa binds to activated platelets and activates small amounts of FX independent of TF. This platelet-surface FXa can partially restore platelet-surface thrombin generation in hemophilia. Recently, van't Veer and colleagues reported results from an in vitro model in which coagulation reactions were initiated by relipidated TF. The authors concluded that high-dose FVIIa may exert a hemostatic effect in hemophilia by overcoming inhibition of FVIIa/TF activity by zymogen FVII. By contrast, we found that plasma levels of FVII did not slow thrombin generation in a model system initiated with cell-associated TF. This discrepancy highlights the potential differences between the studies of the coagulation reactions assembled on living cells compared to phospholipid vesicles. Our data suggest that in a cellular system high-dose FVIIa acts primarily by enhancing the rate of thrombin generation on platelet surfaces and not by overcoming inhibition by zymogen FVII of TF-dependent activation of FX.

Topics: Animals; Blood Coagulation; Factor VII; Factor VIIa; Factor X Deficiency; Factor Xa; Hemophilia A; Hemostasis; Hemostatics; Humans; Recombinant Proteins; Thromboplastin

Tissue factor (TF), a transmembrane protein, is the main initiator of blood clotting in vivo which functions by complexing the enzyme, factor VIIa, which then activates its natural substrates, factors IX and X. TF functions by increasing the affinity of factor VIIa for the surface and increasing the catalytic rate of factor VIIa. TF is not proteolytically activated, but is regulated by its exposure to blood, for example by cellular expression after stimulation with endotoxin, tumour necrosis factor, or interleukin I. The function of TF is modulated by the surrounding phospholipid surface. Anionic phospholipids stimulate TF:VIIa activity by lowering the apparent KM. Since the enzyme is localized on a two-dimensional surface, the apparent enzymatic parameters are dependent on the transport of substrate to the surface. This, in turn, is a function of enzyme density and flow conditions.

Topics: Biological Transport; Blood Coagulation; Catalysis; Chemical Phenomena; Chemistry, Physical; Endothelium, Vascular; Enzyme Activation; Factor VIIa; Hemophilia A; Humans; Kinetics; Membrane Lipids; Models, Biological; Phospholipids; Regional Blood Flow; Surface Properties; Thromboplastin

Management of patients with factor VIII (and IX) inhibitors includes management of acute bleeds and methods to induce immune suppression and tolerance and to detect patients at risk of developing inhibitors. The methods used over the years to treat acute bleeding have been more or less successful. The best method is to raise factor VIII levels by human or porcine factor VIII concentrate, but this is not usually possible. Prothrombin complex concentrates, activated or non-activated, have enjoyed some success as factor VIII by passing agents, but the development of recombinant activated factor VII represents a new and promising method of inducing haemoslasis at the site of bleeding whilst minimizing the risk of disseminated intravascular coagulation. Alternatively, the use of tissue factor is under consideration to exploit the extrinsic system. Methods to induce immunological tolerance by use of the 'Bonn' regime or by the introduction of immunomodulation with the 'Malmö' regime of extracorporeal immunodepletion, cyclophosphamide, and intravenous immunoglobulin continue to be attempted with significant but variable success. Gradually the inhibitor problem is being contained, but it is still an important complication of haemophilia therapy.

Topics: Animals; Antifibrinolytic Agents; Blood Coagulation Factors; Combined Modality Therapy; Factor IX; Factor VIIa; Factor VIII; Hemophilia A; Hemophilia B; Hemorrhage; Humans; Immune Tolerance; Immunosuppressive Agents; Isoantibodies; Swine; Thromboplastin

A revised hypothesis of coagulation integrates all the factors known to be involved into a single pathway that is initiated by factor VIIa/tissue factor and in which "contact" factors are not required. A key point is that the initial hemostatic response must be "consolidated" by the progressive local generation of factor Xa and thrombin.

Topics: Blood Coagulation; Hemophilia A; Humans; Models, Biological; Thromboplastin

Topics: Blood Coagulation; Blood Coagulation Factors; Hemophilia A; Humans; Prothrombin; Thrombin; Thromboplastin

Topics: Amino Acid Sequence; Blood Coagulation; Blood Coagulation Factors; Disseminated Intravascular Coagulation; Factor VII; Hemophilia A; Humans; Lipoproteins; Molecular Sequence Data; Thromboplastin

The role of factor VII in the haemostatic mechanism as well as thrombosis has recently gained new interest. Today's concept that factor VII may be a key regulator in the initiation of blood coagulation is based on studies that provide new evidence for a mandatory activation of factor VII to factor VIIa in blood. Exposure of thromboplastin to the circulation may not trigger activation of blood coagulation before the one chain factor VII is converted to the active two chain form of factor VIIa. A hypothetical model is proposed for the initiation and subsequent activation steps of the blood coagulation process. In this model, it is suggested that circulating activators of factor VII activate inactive complexes of thromboplastin-factor VII. Subsequently, newly generated factor Xa will accelerate this reaction and thereafter be the most potent activator of factor VII. This model would also fit with the clinical observation that moderate factor VII deficiency may be associated with thrombotic episodes discussed in this communication. This article also discusses the role of recombinant factor VIIa in the treatment of factor VIII deficiency patients with acquired factor VIII inhibitors, factor VII and ischemic heart disease and the factor VII-phospholipid complex, and the regulation of the thromboplastin-factor VIIa complex by factor Xa and extrinsic pathway inhibitor (EPI).

Topics: Blood Coagulation; Coronary Disease; Factor VII; Factor VIIa; Hemophilia A; Hemostasis; Humans; Phospholipids; Recombinant Proteins; Thromboplastin

Factor VIII inhibitors are antibodies of the IgG class that neutralize the procoagulant activity of FVIII. They arise as alloantibodies in multitransfused haemophilia A patients and spontaneously in some individuals. Therapeutic strategy must be separated into two concepts: treatment of bleeding episodes and treatment of the inhibitor itself. Treatment of an haemorrhagic complication is dependent on the inhibitor titre and the status of the patient as a 'high' or 'low' responder. The choice has to be made between massive amounts of human FVIII concentrate, porcine FVIII and plasma preparations with 'bypassing' activity. Adjuvant therapy such as plasma exchange or extracorporeal passage of plasma over a Protein A sepharose column might be helpful. More recent products are still under evaluation such as FVIIa or recombinant tissue factor. Intravenous immunoglobulins have proven to be helpful mainly in patients with spontaneous inhibitors. Treatment of the inhibitor itself must be started as soon as possible. Several approaches are being used, immunosuppressive therapy or induction of immune tolerance using high intermediate or low doses of FVIII concentrate.

Topics: Antibodies; Blood Coagulation Factors; Factor VIIa; Factor VIII; Hemophilia A; Humans; Immunoglobulin G; Immunosuppressive Agents; Thromboplastin

Topics: Blood Coagulation; Blood Platelets; Blood Vessels; Collagen; Enzyme Activation; Factor IX; Factor VII; Factor X; Hemophilia A; Hemophilia B; Hemostasis; Humans; Kininogens; Monocytes; Muscle, Smooth, Vascular; Thrombin; Thromboplastin

Factor VII appears to be the key regulatory protein in the initiation of both the intrinsic and extrinsic systems of coagulation. The single chain, or zymogen form, of factor VII possesses enzymatic activity which makes it an ideal candidate for the initiation of coagulation following vascular injury. A number of interactions between the intrinsic and extrinsic systems of coagulation have been identified. It appears that factor VII is capable of directly activating factor IX and vice versa. The study of factor VII variants with associated thromboembolic complications may provide a number of answers regarding the initiation and regulation of the blood coagulation process.

Topics: Blood Coagulation; Blood Coagulation Tests; Cerebral Hemorrhage; Enzyme Activation; Factor IX; Factor VII; Factor VII Deficiency; Hemarthrosis; Hemophilia A; Humans; Lipoproteins; Liver Diseases; Neutralization Tests; Partial Thromboplastin Time; Radioimmunoassay; Thromboplastin; Thrombosis; Vitamin K Deficiency

Factor VII, a single-chain zymogen, has sufficient proteolytic activity to initiate blood coagulation. The reason that coagulation does not occur continuously is that the zymogen like its 2-chain derivative enzyme, factor VIIa, absolutely requires tissue factor. The latter, a lipid-dependent glycoprotein, is not normally present in the blood. Upon tissue injury, however, coagulation is initiated by the activation of factors IX and X. The relationship of these events to the possibility that hemophilia A and B are tissue factor-dependent diseases is discussed.

Topics: Blood Coagulation; Factor IX; Factor IXa; Factor VII; Factor VII Deficiency; Factor VIIa; Factor X; Factor Xa; Hemophilia A; Humans; Kinetics; Thromboplastin; Thrombosis

Topics: Blood Coagulation Disorders; Blood Transfusion; Calcium; Factor V Deficiency; Factor VIII; Factor X Deficiency; Factor XI Deficiency; Factor XII Deficiency; Factor XIII Deficiency; Female; Hemophilia A; Hemophilia B; Humans; Hypoprothrombinemias; Male; Pedigree; Thromboplastin

Topics: Blood Coagulation Factors; Cells, Cultured; Disseminated Intravascular Coagulation; Hemophilia A; Humans; Saliva; Thromboplastin; Trypsin; Trypsin Inhibitors; Vitamin K

Topics: Angioedema; Animals; Antithrombins; Blood Coagulation; Blood Coagulation Factors; Blood Platelets; Complement System Proteins; Enzyme Precursors; Factor VIII; Factor XII; Female; Fibrin; Fibrinogen; Fibrinolysis; Hemophilia A; Hemostasis; Humans; Kallikreins; Male; Platelet Adhesiveness; Thrombin; Thromboplastin; Thrombosis; von Willebrand Diseases

Topics: Aminocaproates; Blood Coagulation Disorders; Blood Coagulation Tests; Blood Proteins; Hemophilia A; Hemorrhagic Disorders; Humans; Plasminogen; Thromboplastin

Topics: Adult; Antibodies; Anticoagulants; Antifibrinolytic Agents; Blood Coagulation Disorders; Blood Coagulation Tests; Blood Platelets; Blood Transfusion; Factor VIII; Female; Hemophilia A; Humans; Lupus Erythematosus, Systemic; Male; Middle Aged; Prothrombin Time; Thrombin; Thromboplastin

Topics: Chromosome Aberrations; Chromosome Disorders; Factor VIII; Female; Genes, Regulator; Hemophilia A; Heterozygote; Homozygote; Humans; Prothrombin; Thromboplastin; Trisomy

Topics: Adrenal Cortex Hormones; Antibodies; Calcium; Chromosomes; Epinephrine; Factor IX; Factor VIII; Hemophilia A; Humans; Phospholipids; Progesterone; Spleen; Thromboplastin; Thyroxine; von Willebrand Diseases

Topics: Aminocaproates; Blood Coagulation Disorders; Blood Coagulation Factors; Blood Coagulation Tests; Blood Transfusion; Factor V Deficiency; Factor VII Deficiency; Factor VIII; Fibrinogen; Freezing; Hemophilia A; Hemophilia B; Hemorrhage; Hemostasis; Humans; Hypoprothrombinemias; Plasma; Plasma Volume; Prothrombin; Prothrombin Time; Thromboplastin; Vitamin K

Topics: Adult; Blood Coagulation Tests; Blood Transfusion; Factor VIII; Hemophilia A; Humans; Male; Surgical Procedures, Operative; Thromboplastin

Topics: Blood Coagulation Factors; Blood Coagulation Tests; Blood Transfusion; Cerebral Hemorrhage; Epistaxis; Factor VIII; Gastrointestinal Hemorrhage; Hemarthrosis; Hematuria; Hemophilia A; Hemorrhage; Humans; Oral Hemorrhage; Thromboplastin; Wounds and Injuries

Most patients with severe hemophilia A suffer from a profoundly compromised hemostatic response. In addition to both the delayed and slow development of a clot, previous studies have documented that severe hemophilia A is also associated with reduced clot stability.. We examined whether the clot stability in hemophiliacs could be improved by treatment with tranexamic acid (TXA) in combination with recombinant factor VIII (rFVIII).. Baseline blood samples were obtained from eight males with severe hemophilia A. Thereafter, a bolus injection of rFVIII was administered to increase the functional level of FVIII to approximately 50%. After 10 min, blood was collected followed by an intravenous injection of TXA. A third blood sample was obtained after a further 10 min. Whole blood clotting profiles were determined by thrombelastography using minimal tissue factor activation. Clot formation was assessed by both clot initiation and clot propagation. At clot termination, the maximum clot firmness and area under the elasticity curve were used to illustrate clot stability. Tissue-plasminogen activator was included in those experiments designed to assess clot stability.. As expected, rFVIII increased clot formation, whereas TXA had no effect upon this parameter. Assays including tissue-plasminogen activator revealed that rFVIII increased the maximum clot firmness 3-fold; whereas the presence of TXA induced an additional 4-fold increase. The area under the elasticity curve increased 5-fold after rFVIII and 24-fold after addition of TXA.. The study demonstrates that simultaneous treatment with TXA and rFVIII significantly improves the clot stability in patients with hemophilia A.

Topics: Adult; Antifibrinolytic Agents; Blood Coagulation; Drug Therapy, Combination; Factor VIII; Fibrinolysis; Hemophilia A; Humans; Male; Recombinant Proteins; Severity of Illness Index; Thrombelastography; Thromboplastin; Tissue Plasminogen Activator; Tranexamic Acid; Treatment Outcome; Whole Blood Coagulation Time

Topics: Acute Disease; Child; Clinical Trials as Topic; Factor VIII; Hemarthrosis; Hemophilia A; Hemophilia B; Humans; Male; Placebos; Prednisone; Thromboplastin

Compared with conventional coagulation tests and factor-specific assays, viscoelastic hemostatic assays (VHAs) can provide a more thorough evaluation of clot formation and lysis but have several limitations including clot deformation. In this proof-of-concept study, we test a noncontact technique, termed resonant acoustic rheometry (RAR), for measuring the kinetics of human plasma coagulation. Specifically, RAR utilizes a dual-mode ultrasound technique to induce and detect surface oscillation of blood samples without direct physical contact and measures the resonant frequency of the surface oscillation over time, which is reflective of the viscoelasticity of the sample. Analysis of RAR results of normal plasma allowed defining a set of parameters for quantifying coagulation. RAR detected a flat-line tracing of resonant frequency in hemophilia A plasma that was corrected with the addition of tissue factor. Our RAR results captured the kinetics of plasma coagulation and the newly defined RAR parameters correlated with increasing tissue factor concentration in both healthy and hemophilia A plasma. These findings demonstrate the feasibility of RAR as a novel approach for VHA, providing the foundation for future studies to compare RAR parameters to conventional coagulation tests, factor-specific assays, and VHA parameters.

Topics: Acoustics; Blood Coagulation; Blood Coagulation Tests; Hemophilia A; Humans; Kinetics; Thromboplastin

Mim8 is a next generation bispecific antibody developed for the prophylactic treatment of hemophilia A. The sensitivity of activated plasma thromboplastin time (APTT), assays measuring factor VIII activity (FVIII:C) and thrombin generation to plasma containing Mim8 was assessed.. Congenital severe hemophilia A plasma was spiked with Mim8 at 0 μg/mL to 20 μg/mL. APTT was measured with 4 reagents, one-stage FVIII with 9 reagents, and chromogenic FVIII with 6 assays. Thrombin generation was assessed in a low tissue factor system.. At 1-μg/mL Mim8, the APTT was shortened to within normal limits and one-stage FVIII:C was >1.00 IU/mL with all APTT reagents. Modified one-stage assays calibrated with Mim8 reference material calibrated recovered within 20% of the target Mim8 concentration with most APTT reagents. Factor VIII:C of bovine only chromogenic assays was <0.04 IU/mL at all Mim8 concentrations. Factor VIII:C of human only chromogenic assay was >4.00 IU/mL at 20-μg/mL Mim8. Factor VIII:C of hybrid bovine FX, human FIXa chromogenic assays ranged from 0.076 to >3.00 IU/mL at 20-μg/mL Mim8. Normal thrombin generation was restored at 5-μg/mL Mim8.. APTT-based assays are sensitive to Mim8 and should not be performed in the presence of the drug. Chromogenic assays containing human proteins or hybrid human/bovine proteins demonstrated variable sensitivity to Mim8. Bovine only chromogenic assays were largely insensitive to the presence of Mim8. Thrombin generation normalized at increased Mim8 concentrations. Modified one-stage and chromogenic assays could be used to quantify the Mim8 concentration in plasma.

Topics: Animals; Antibodies, Bispecific; Cattle; Factor VIII; Hemophilia A; Hemostatics; Humans; Indicators and Reagents; Partial Thromboplastin Time; Thrombin; Thromboplastin

Mim8 is a novel antifactor IXa/antifactor X bispecific antibody in clinical development for prophylactic treatment of hemophilia A with and without inhibitors. Patients treated with Mim8 may need supplementary bleed treatment under certain conditions such as surgery or major trauma.. This study aimed to better understand the response of Mim8 in thrombin generation assays (TGAs) alone or in combination with other hemostatic proteins.. We used TGAs with different activators (tissue factor (TF) and activated factor XI) to better understand the similarities and differences between the mode of action of Mim8 and factor VIII (FVIII). Following this, we investigated the effects of mixing Mim8 with the main bleed treatment options for persons with hemophilia A with or without inhibitors: FVIII, activated factor VII (FVIIa), and activated prothrombin complex concentrates (aPCC).. The results indicated that for patients without inhibitors, Mim8 does not interfere with FVIII's mode of action. For patients with inhibitors, Mim8 mixed with aPCC results in a strong synergistic effect causing thrombin generation far exceeding the normal levels. Contrary to this, mixing Mim8 with FVIIa results in a more controlled additive effect, visible only when using TF as a trigger, which does not exceed the normal level of thrombin generation.. These findings support the use of approved clinical doses of FVIIa for bleed treatment of patients with FVIII inhibitors treated with Mim8. Additionally, the findings suggest that concomitant use of FVIII and Mim8 is safe for managing breakthrough bleeds.

Topics: Factor IX; Factor VIIa; Factor VIII; Hemophilia A; Hemorrhage; Hemostatics; Humans; Recombinant Proteins; Thrombin; Thromboplastin

 Hemophilia A (HA) is characterized by decreased or absent factor VIII (FVIII) activity. Current FVIII assays are based on clotting time and thus only provide information about the initiation of coagulation. In contrast, thrombin generation assays (TGAs) can be used to measure the full coagulation spectrum of initiation, propagation, and termination that provide information on the whole course of thrombin generation and inhibition. However, the commercially available TG kits lack sensitivity for measurements of hemophilia plasma within lower FVIII ranges, which is essential for explaining differences in bleeding phenotypes in hemophiliacs at clinically low levels of FVIII..  Optimization of the TGA for measurements of low FVIII levels in severe HA patients..  TGA measurements were performed in severe HA pooled plasma (.  TGA initiated by tissue factor (TF) alone at varying concentrations was unable to significantly differentiate between FVIII levels below 20%. In contrast, TGA activation with low concentrations of TF in presence of FXIa appeared to be highly sensitive for FVIII changes both in high and low ranges. In addition, a representative TGA curve at trough levels could only be produced using the dual TF/FXIa TGA..  We propose a critical optimization for the setup of the TGA for measurements in severe HA plasma. The dual TF/FXIa TGA shows increased sensitivity, especially in lower FVIII ranges, which allows for better individual characterization at baseline, prediction of interventions, and follow-up.

Topics: Blood Coagulation Tests; Factor VIII; Factor XIa; Hemophilia A; Hemostatics; Humans; Thrombin; Thromboplastin

Plasma-derived FVIII/VWF complex was reported to be less sensitive to inhibitors than FVIII preparations devoid of VWF.. To compare the efficacy of FVIII/VWF complex (Fanhdi) and five different VWF-free FVIII preparations in restoring thrombin generation and activation of thrombin-activatable fibrinolysis inhibitor (TAFI) in haemophilic plasma, with and without inhibitor, and in cell-based models.. Experiments were performed in haemophilic plasma supplemented with inhibitory IgG or in plasma samples obtained from haemophilia A patients without (n = 11) and with inhibitor (n = 12). Thrombin generation was evaluated by calibrated automated thrombography (CAT) under standard conditions, in the presence of activated protein C (APC) or thrombomodulin (TM), and in cell-based models including endothelial cells, either alone or in combination with platelets or tissue factor-expressing blood mononuclear cells. The kinetics of TAFI activation was determined by a two-stage functional assay in the absence and in the presence of APC.. In haemophilic plasma without inhibitor, Fanhdi enhanced thrombin generation and TAFI activation as well as recombinant (2nd-4th generation) and plasma-derived FVIII preparations devoid of VWF. On the contrary, in plasma with inhibitor, Fanhdi displayed a greater ability to restore thrombin generation and TAFI activation under all tested conditions. Notably, in cell-based models including endothelial cells, Fanhdi proved more efficient than all other preparations in improving thrombin generation even in the absence of inhibitor.. The greater pro-haemostatic activity of FVIII/VWF complex, either in haemophilic plasma with inhibitor or in the presence of endothelial cells, may offer therapeutic advantages.

Topics: Carboxypeptidase B2; Coagulants; Combined Modality Therapy; Endothelial Cells; Factor VIII; Fibrinolysis; Hemophilia A; Hemostasis; Humans; Immunoglobulin G; Kinetics; Plasma; Protein C; Thrombin; Thrombomodulin; Thromboplastin; Treatment Outcome; von Willebrand Factor

Circulating microparticles (MPs) are procoagulant due to the surface containing phosphatidylserine (PS), which facilitates coagulation. We investigated if MPs improve hemostasis in HA plasma models. MPs isolated from pooled normal human plasma were added to severe, moderate and mild HA plasma models (0%, 2.5%, 20% FVIII). The MPs' effect on hemostasis was evaluated by calibrated automated thrombogram (CAT) and overall hemostasis potential (OHP) assays, while fibrin structure was imaged by standard confocal, stimulated emission depletion (STED) microscopy and scanning electron microscopy (SEM). MPs partially restored thrombin generation and fibrin formation in all HA plasma models. The procoagulant effect of MPs requires PS exposure, to a less extent of contact pathway activation, but not tissue factor exposure or in vitro stimulation of MPs. MPs partially normalized the fibrin structure, and using super-resolution STED, MPs attached to fibrin were clearly resolved. In summary, our results demonstrate that PS positive MPs could improve hemostasis in HA plasma models.

Topics: Blood Coagulation; Cell-Derived Microparticles; Factor VIII; Fibrin; Hemophilia A; Hemostasis; Humans; Microscopy, Confocal; Microscopy, Electron, Scanning; Phosphatidylserines; Thrombin; Thromboplastin

In haemophilia, thrombin generation and fibrin deposition upon vascular injury critically depend on the tissue factor (TF)-driven coagulation pathway. TF expression by monocytes/macrophages and circulating microvesicles contributes to haemostasis, thrombosis and inflammation. Inflammation is a hallmark of blood-induced joint disease. The aim of this study is to correlate TF production by whole-blood monocytes with inflammatory markers and clinical parameters in patients with moderate-to-severe haemophilia A or B (n = 43) in comparison to healthy males (n = 23). Monocyte TF antigen and microvesicle-associated TF procoagulant activity (MV TF PCA) were measured immediately after blood draw (baseline) and following incubation of whole blood with buffer or lipopolysaccharide (LPS) using two-colour flow cytometry and chromogenic FXa generation assay, respectively. Patients with HIV or uncontrolled HBV/HCV infections were excluded. TF was hardly detectable and not different in baseline and buffer-treaded samples from both groups. Stimulation with LPS, however, induced monocyte TF production, with increased TF-specific mean fluorescence intensity (P = 0.08) and MV TF PCA (P < 0.05) in patients compared to controls. Patients also had elevated hs-CRP and IL-6 serum levels (P < 0.001), which correlated with LPS-induced TF parameters. Further exploratory analyses revealed that the presence of systemic (low-grade) inflammation and boosted LPS-induced monocyte TF production were mainly restricted to patients with clinically controlled HBV and/or HCV infection (n = 16), who were older and also had a significantly worse orthopaedic joint score than patients with no history of viral hepatitis (P < 0.01). Our study delineates a previously unrecognised link between systemic inflammation and inducible monocyte TF production in patients with haemophilia A or B.

Topics: Adult; Case-Control Studies; Female; Hemophilia A; Humans; Inflammation; Lipopolysaccharides; Male; Middle Aged; Monocytes; Secretory Pathway; Severity of Illness Index; Thromboplastin; Young Adult

Topics: Blood Coagulation; Factor VIII; Hemophilia A; Hemophilia B; Humans; Thromboplastin; Tissue Distribution

Activated factor VII (FVIIa) is pertinent to the initiation of blood coagulation. Proteolytic and amidolytic activity of FVIIa are greatly enhanced by its cofactor, tissue factor (TF).. We aimed to generate a single-domain antibody (sdAb) that recognizes free FVIIa rather than TF-bound FVIIa.. A llama-derived phage library was used to screen for anti-FVIIa sdAbs.. This observation is compatible with the view that FVIIa functions independently of TF under these conditions. In conclusion, we have generated a sdAb that specifically blocks TF-independent activity of FVIIa. This antibody can be used to gain insight into the roles of TF-bound and TF-free FVIIa.

Topics: Animals; Anticoagulants; Blood Coagulation; Coagulants; Disease Models, Animal; Factor VIIa; Factor VIII; Female; Hemophilia A; Humans; Male; Mice, Inbred C57BL; Protein Binding; Single-Domain Antibodies; Thromboplastin

Topics: Animals; Antibodies, Monoclonal, Humanized; Disease Models, Animal; Factor VIIa; Female; Hemophilia A; Hemorrhage; Hemostasis; Rabbits; Thromboplastin

Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic pathway that negatively regulates thrombin production during coagulation. Under haemophilic conditions, where the intrinsic coagulation pathway is impaired, inhibition of TFPI may improve clotting.. We investigated the ex vivo effects of a human TFPI neutralizing antibody, marstacimab (previously PF-06741086), in coagulation assays including rotational thromboelastometry (ROTEM), thrombin generation assay (TGA) and the dilute prothrombin time (dPT) assay, performed in haemophilic whole blood and plasmas. We compared the effects of marstacimab to the effects of recombinant coagulation factors and investigated the reproducibility of marstacimab in restoring haemostasis by comparing its effect in whole blood collected from the same study participants on differing days.. Citrated whole blood and plasmas obtained from haemophilia participants were supplemented ex vivo with vehicle, marstacimab, recombinant FVIII (rFVIII) or recombinant factor IX (rFIX) and analysed in ROTEM, TGA and the dPT assay using low tissue factor concentrations to trigger coagulation.. Marstacimab induced pro-coagulant responses in ROTEM parameters including reduction in clotting times and increases in angle. Similarly, participant plasmas supplemented with marstacimab exhibited improvements in TGA parameters, including reduced lag times, increased peak thrombin concentrations and reductions in dPT clotting time. Concentrations of marstacimab tested showed activity comparable to addition of rFVIII or rFIX and were reproducible.. These studies show the ex vivo potency of marstacimab in restoring haemostasis in whole blood and plasmas from haemophilia participants and comparability to ex vivo reconstitution with recombination coagulation factors.

Topics: Antibodies, Monoclonal, Humanized; Blood Coagulation; Female; Hemophilia A; Humans; Male; Plasma; Thromboplastin

Tissue factor pathway inhibitor-alpha (TFPI-α) is a Kunitz-type serine protease inhibitor, which suppresses coagulation by inhibiting the tissue factor (TF)/factor VIIa complex as well as factor Xa. In static plasma-phospholipid systems, TFPI-α thus suppresses both factor Xa and thrombin generation. In this article, we used a microfluidics approach to investigate how TFPI-α regulates fibrin clot formation in platelet thrombi at low wall shear rate. We therefore hypothesized that the anticoagulant effect of TFPI-α in plasma is a function of the local procoagulant strength-defined as the magnitude of thrombin generation under flow, due to local activities of TF/factor VIIa and factor Xa. To test this hypothesis, we modulated local coagulation by microspot coating of flow channels with 0 to 100 pM TF/collagen, or by using blood from patients with haemophilia A or B. For blood or plasma from healthy subjects, blocking of TFPI-α enhanced fibrin formation, extending from a platelet thrombus, under flow only at <2 pM coated TF. This enhancement was paralleled by an increased thrombin generation. For mouse plasma, genetic deficiency in TFPI enhanced fibrin formation under flow also at 0 pM TF microspots. On the other hand, using blood from haemophilia A or B patients, TFPI-α antagonism markedly enhanced fibrin formation at microspots with up to 100 pM coated TF. We conclude that, under flow, TFPI-α is capable to antagonize fibrin formation in a manner dependent on and restricted by local TF/factor VIIa and factor Xa activities.

Topics: Animals; Anticoagulants; Blood Coagulation; Blood Platelets; Coagulants; Collagen; Crosses, Genetic; Factor VIIa; Factor Xa; Female; Fibrin; Healthy Volunteers; Hemophilia A; Hemophilia B; Heterozygote; Humans; Lipoproteins; Male; Mice; Mice, Inbred C57BL; Perfusion; Thromboplastin; Thrombosis

Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell-derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed.

Topics: Adult; Aged; Aged, 80 and over; Animals; Blood Coagulation; Blood Coagulation Factors; Blood Platelets; Cardiopulmonary Bypass; Carrier Proteins; Cysteine Endopeptidases; Factor IX; Factor VIIa; Factor VIII; Factor X; Hemophilia A; Hemorrhage; Hemostasis; Humans; Hydroxyeicosatetraenoic Acids; Lipoproteins; Male; Mice; Mice, Inbred C57BL; Middle Aged; Neoplasm Proteins; Phospholipids; Surface Plasmon Resonance; Thrombin; Thromboplastin

Topics: Adolescent; Adult; Blood Coagulation; Child; Child, Preschool; Factor VIII; Hemophilia A; Hemorrhage; Humans; Indicators and Reagents; Middle Aged; Partial Thromboplastin Time; Recombinant Proteins; Thrombelastography; Thromboplastin; Young Adult

Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa.

Topics: Allosteric Regulation; Animals; Biological Therapy; Blood Coagulation; Disease Models, Animal; Factor VIIa; Female; Hemophilia A; Humans; Kinetics; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Dynamics Simulation; Protein Engineering; Recombinant Proteins; Thromboplastin

Thrombin generation test (TGT) is a global haemostasis assay with a potential to predict bleeding tendencies and treatment effects in patients with haemophilia. Despite 15 years of clinical research, the diagnostic value of TGT remains controversial, possibly due to suboptimal sensitivity to coagulation deficiencies, robustness and reproducibility.. The goal of this study was to explore the effect of calcium chloride (CaCl. Normal and factor-deficient plasmas supplemented with lacking coagulation factor and different CaCl. Thrombin peak height (TPH) was strongly CaCl. Variations in CaCl

Topics: Blood Chemical Analysis; Calcium Chloride; Dose-Response Relationship, Drug; Hemophilia A; Hemorrhage; Hemostasis; Thrombin; Thromboplastin

ESSENTIALS: The role of tissue factor (TF) in recombinant factor VIIa (rFVIIa) therapy in hemophilia is unclear. An acquired mouse hemophilia model with very low or normal levels of human TF was used in the study. rFVIIa is equally effective in correcting the bleeding in mice expressing low or normal levels of TF. Pharmacological doses of rFVIIa restore hemostasis in hemophilia independent of TF.. Recombinant factor VIIa (rFVIIa) has been used widely for treating hemophilia patients with inhibitory autoantibodies against factor VIII or IX. Its mechanism of action is not entirely known. A majority of in vitro studies suggested that pharmacological concentrations of rFVIIa restore hemostasis in hemophilia in a phospholipid-dependent manner, independent of tissue factor (TF). However, a few studies suggested that a TF-dependent mechanism has a primary role in correction of bleeding by rFVIIa in hemophilia patients. Here, we investigated the potential contribution of TF in rFVIIa-induced hemostasis in hemophilia employing a model system of FVIII antibody-induced hemophilia in TF transgenic mice.. Mice expressing low levels of human TF (LTF mice), mice expressing relatively high levels of human TF (HTF mice) and wild-type mice (WT mice) had neutralizing anti-FVIII antibodies administered in order to induce hemophilia in these mice. The mice were then treated with varying concentrations of rFVIIa. rFVIIa-induced hemostasis was evaluated with the saphenous vein bleeding model.. Administration of FVIII inhibitory antibodies induced the hemophilic bleeding phenotype in all three genotypes. rFVIIa administration rescued the bleeding phenotype in all three genotypes. No significant differences were observed in rFVIIa-induced correction of bleeding between LTF and HTF mice that had FVIII antibodies administered.. Our results provide strong evidence supporting the suggestion that the hemostatic effect of pharmacological doses of rFVIIa stems from a TF-independent mechanism.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Coagulants; Disease Models, Animal; Dose-Response Relationship, Drug; Factor VIIa; Factor VIII; Genotype; Hemophilia A; Hemostasis; Humans; Mice, Inbred C57BL; Mice, Transgenic; Phenotype; Recombinant Proteins; Thromboplastin

There is a clinical need to develop safe therapeutic strategies to mitigate bleeding. Previously, we found that a novel zymogen-like factor Xa variant (FXa-I16L) was effective in correcting the coagulation defect in hemophilic mice. Here we expand the mutational framework to tune the FX(a) zymogen-like state. Alteration of FXa zymogenicity yields variants (V17M, I16L, I16M, V17T, V17S, and I16T) with a wide range (≤1000-fold) of reduced function toward physiologic substrates and inhibitors. The extent of zymogen-like character, including resistance to antithrombin III, correlates well with plasma half-life (<2 minutes to >4 hours). Importantly, biologic function, including that of the most zymogen-like variant (FXa-I16T), was greatly enhanced when bound to FVa membranes. This resulted in improvement of clotting times and thrombin generation in hemophilic plasma. The FXa variants were remarkably effective in mouse injury models. In these systems, the data show that the more active the protease, the more difficult it is to overcome the protective mechanism of circulating inhibitors to achieve a therapeutic benefit. Depending on the treatment situation, the more zymogen-like variants (V17S and I16T) were most useful when given before injury whereas variants exhibiting greater activity but shorter half-lives (I16L and I16M) were most effective when administered after injury. This new class of FXa variants provides a useful and flexible platform for selectively bioengineering biologic function and half-life to target different clinical bleeding scenarios.

Topics: Animals; Blood Coagulation; Catalytic Domain; DNA Mutational Analysis; Enzyme Precursors; Factor Xa; Half-Life; Hemophilia A; Hemostatics; Mice; Mice, Inbred BALB C; Mutant Proteins; Prothrombin; Thromboplastin

Conventional coagulation factor assays are associated with certain limitations, as they do not always reflect the clinical heterogeneity of bleeding in hemophilic patients or correctly reflect the individual patient response to treatment with bypassing agents or novel factor concentrates. The thrombin generation assay (TGA) is currently being assessed as a possible method for characterizing bleeding phenotypes in individuals with hemophilia.. This study assessed the robustness and sensitivity of the TGA for measuring the activity of recombinant factor VIII (rFVIII), recombinant factor IX (rFIX) and their glycoPEGylated derivatives, N8-GP and N9-GP, in vitro.. Factor-deficient plasma was spiked with 0.13-130 IU dL(-1) rFVIII or N8-GP (hemophilia A [HA] plasma), or rFIX or N9-GP (hemophilia B [HB] plasma). A calibrated automated thrombogram triggered with tissue factor (TF) or activated FXI (FXIa) was used to measure thrombin generation over time. Endogenous thrombin potential, peak thrombin, velocity index, lag time and time to peak thrombin were analyzed.. FXIa-triggered assays were not affected by glycoPEGylation and were sufficiently sensitive to differentiate between spiked samples mimicking severe and moderate HB and HA; TF-triggered assays were not sufficiently sensitive for this distinction in HA. Both FXIa-triggered and TF-triggered assays had an acceptable level of variability (≤ 20%), although TF-triggered assays were associated with greater variability.. FXIa-triggered TGA reactions produced more robust and sensitive results than TF-triggered TGA reactions, and have the potential for use in monitoring patients treated with glycoPEGylated or non-PEGylated coagulation factor concentrates. These promising results merit confirmation with clinical samples to correlate in vitro and in vivo data.

Topics: Blood Coagulation Tests; Drug Monitoring; Factor IX; Factor VIII; Factor XIa; Hemophilia A; Humans; Polyethylene Glycols; Recombinant Proteins; Sensitivity and Specificity; Thrombin; Thromboplastin

Assays to determine the optimal hemostatic effects of bypass therapy in hemophilia A (HA) patients with inhibitors are difficult to compare. Clot waveform analysis (CWA), based on the continuous monitoring of routine coagulation parameters (prothrombin time/activated partial thromboplastin time), offers a useful method for assessing global clotting function.. To investigate the technique of CWA for the hemostatic monitoring of bypass therapy in HA patients with inhibitors.. Ellagic acid (Elg), tissue factor (TF) or both (Elg/TF) were used as trigger reagents in CWA. The standard parameters - clot time (CT), maximum coagulation velocity (|min1|), and acceleration (|min2|) - were recorded. Optimal monitoring was defined as: (i) a significant difference in these parameters between plasma from HA patients with inhibitors and normal plasmas; and (ii) a significant improvement in these indices in HA patients with inhibitors after bypass therapy. Experiments in vitro demonstrated that there were significant differences between plasma from HA patients with inhibitors and normal plasma with various triggers, in the order Elg > Elg/TF >> TF. Addition of therapeutically achievable concentrations of bypassing agents, however, showed significant improvements in the different parameters only with Elg/TF, suggesting that this reagent provided the most appropriate assay. A total of 20 plasmas from HA patients with inhibitors in which bypassing agents were infused were evaluated ex vivo by Elg/TF CWA. The postinfusion parameters CT and |min2| reflected clinical effects, and were close to normal levels. Furthermore, Elg/TF CWA facilitated quantitative evaluation of perioperative hemostatic management of bypass therapy in HA patients with inhibitors.. CWA is a promising method for the quantitative monitoring of bypass therapy during routine automated clotting assays with a modified trigger reagent comprising a well-balanced mixture of Elg and TF.

Topics: Adolescent; Adult; Blood Coagulation; Blood Coagulation Factors; Blood Coagulation Tests; Child; Factor VIII; Hemophilia A; Hemostasis; Humans; Middle Aged; Partial Thromboplastin Time; Prothrombin Time; Thromboplastin; Young Adult

Some hemophilia A patients who have developed inhibitors are poorly responsive to activated prothrombin complex concentrates (aPCC) after daily dosage, but the mechanism(s) underlying this remain unknown. We examined two representative cases. In case 1, we found that changing to recombinant factor VIIa (rFVIIa) therapy was more effective, and the response to aPCC was restored within ~2 weeks. Tissue factor (TF)-triggered thrombin generation demonstrated a prolonged lag-time and decreased peak thrombin, and this impairment was focused on TF pathway inhibitor (TFPI). Plasma-free TFPI was elevated post-infusion of aPCC, while this was unaffected by rFVIIa. TFPI returned to normal range within 2-3 weeks. Plasmas obtained from patients with poor or good response to aPCC (aPCC-poor or aPCC-good), and good response to rFVIIa (FVIIa-good) demonstrated that free TFPI levels are increased in both aPCC groups, but not in FVIIa-good. TFPI levels pre- and post-infusion in aPCC-poor were significantly higher than those in aPCC-good. Addition of anti-TFPI antibody to the reaction samples demonstrated a greater increase of peak thrombin in aPCC-poor compared to aPCC-good, showing the higher TFPI activity in aPCC-poor. Free TFPI contained in aPCC corresponded to the increasing levels in plasma. In conclusion, TFPI in aPCC attenuated thrombin generation, and the reduced effectiveness of therapy in these circumstances appeared to be related to TFPI activity.

Topics: Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Blood Coagulation; Blood Coagulation Factors; Blood Coagulation Tests; Child, Preschool; Factor VIIa; Factor VIII; Hemophilia A; Hemorrhage; Humans; Isoantibodies; Male; Recombinant Proteins; Severity of Illness Index; Thromboplastin; Treatment Outcome; Young Adult

Factor (F)VIIa-based bypassing not always provides sufficient hemostasis in hemophilia.. To investigate the potential of engineered activated factor V (FVa) variants as bypassing agents in hemophilia A.. Activity of FVa variants was studied in vitro using prothrombinase assays with purified components and in FV- and FVIII-deficient plasma using clotting and thrombin generation assays. In vivo bleed reduction after the tail clip was studied in hemophilia A mice.. FVa mutations included a disulfide bond connecting the A2 and A3 domains and ones that rendered FVa resistant to inactivation by activated protein C (APC). '(super) FVa,' a combination of the A2-A3 disulfide (A2-SS-A3) to stabilize FVa and of APC-cleavage site mutations (Arg506/306/679Gln), had enhanced specific activity and complete APC resistance compared with wild-type FVa, FVL eiden (Arg506Gln), or FVaL eiden (A2-SS-A3). Furthermore, (super) FVa potently increased thrombin generation in vitro in FVIII-deficient plasma. In vivo, (super) FVa reduced bleeding in FVIII-deficient mice more effectively than wild-type FVa. Low-dose (super) FVa, but not wild-type FVa, decreased early blood loss during the first 10 min by more than two-fold compared with saline and provided bleed protection for the majority of mice, similar to treatments with FVIII. During the second 10 min after tail cut, (super) FVa at high dose, but not wild-type FVa, effectively reduced bleeding. These findings suggest that (super) FVa enhances not only clot formation but also clot stabilization. Thus, (super) FVa efficiently improved hemostasis in hemophilia in vitro and in vivo and may have potential therapeutic benefits as a novel bypassing agent in hemophilia.

Topics: Animals; Blood Coagulation; Disulfides; Factor Va; Hemophilia A; Hemostasis; Humans; Mice; Mice, Transgenic; Mutation; Partial Thromboplastin Time; Protein C; Protein Engineering; Prothrombin Time; Recombinant Proteins; Thrombin; Thromboplastin

Immune tolerance induction (ITI) is the preferred management of haemophilia A patients who develop high titre inhibitors against factor VIII. However, the optimal ITI regimen, predictors of ITI outcome and definitions of successful and unsuccessful ITI remain unclear. The aim of this project was to develop a consensus on the definition of ITI treatment failure for Australian clinical practice using a modified Delphi approach. Three consecutive surveys were distributed to the directors of 17 haemophilia treatment centres in Australia. Participants were asked to rate their agreement with definitions of ITI treatment failure generated from a literature review. Thirty-five statements regarding ITI achieved consensus (majority agree or strongly agree) during the three survey rounds. After round 3, four statements achieved majority disagreement, and for two statements no consensus was reached. Our study demonstrates that clinicians in Australia necessitate an arbitrary time to assess ITI failure, but that clinical outcomes of ITI are important in assessing response. Assessment over any 3- to 6-month period without a 20% reduction in inhibitor titre is suggestive of failure, but a reduction in bleeding phenotype alone may be sufficient to continue ITI. Overall, a period of 3 or 5 years of ITI may be required to determine response to ITI. Documentation of improvement in clinical measures, supported by the laboratory features of factor VIII inhibitor levels and pharmacokinetics, is essential in assessing the success of failure of ITI in these patients.

Topics: Consensus; Decision Making; Delphi Technique; Hemophilia A; Humans; Immunosuppression Therapy; Thromboplastin; Treatment Failure

Recombinant activated human factor VII (rhFVIIa) is an established hemostatic agent in hemophilia, but its mechanism of action remains unclear. Although tissue factor (TF) is its natural receptor, rhFVIIa also interacts with the endothelial protein C receptor (EPCR) through its γ-carboxyglutamic acid (Gla) domain, with unknown hemostatic consequences in vivo. Here, we study whether EPCR facilitates rhFVIIa hemostasis in hemophilia using a mouse model system. Mouse activated FVII (mFVIIa) is functionally homologous to rhFVIIa, but binds poorly to mouse EPCR (mEPCR). We modified mFVIIa to gain mEPCR binding using 3 amino acid changes in its Gla domain (L4F/L8M/W9R). The resulting molecule mFVIIa-FMR specifically bound mEPCR in vitro and in vivo and was identical to mFVIIa with respect to TF affinity and procoagulant functions. In macrovascular injury models, hemophilic mice administered mFVIIa-FMR exhibited superior hemostatic activity compared with mFVIIa. This was abolished by blocking mEPCR and was absent in ex vivo whole blood coagulation assays, implicating a specific mFVIIa-FMR and endothelial mEPCR interaction. Because mFVIIa-FMR models the TF-dependent and EPCR binding properties of rhFVIIa, our data unmask a novel contribution of EPCR on the action of rhFVIIa administration in hemophilia, prompting the rational design of improved and safer rhFVIIa therapeutics.

Topics: 1-Carboxyglutamic Acid; Amino Acids; Animals; Binding Sites; Binding, Competitive; Blood Coagulation; Blood Coagulation Factors; CHO Cells; Cricetinae; Cricetulus; Factor VIIa; Hemophilia A; Hemostasis; Humans; Kinetics; Male; Mice, Inbred C57BL; Protein Binding; Receptors, Cell Surface; Recombinant Proteins; Thrombelastography; Thromboplastin

Successful competition of activated factor VII (FVIIa) with zymogen factor VII (FVII) for tissue factor (TF) and loading of the platelet surface with FVIIa are plausible driving forces behind the pharmacological effect of recombinant FVIIa (rFVIIa) in hemophilia patients. Thrombin generation measurements in platelet-rich hemophilia A plasma revealed competition for TF, which potentially could reduce the effective (r)FVIIa:TF complex concentration and thereby attenuate factor Xa production. However, (auto)activation of FVII apparently counteracted the negative effect of zymogen binding; a small impact was observed at endogenous concentrations of FVII and FVIIa but was virtually absent at pharmacological amounts of rFVIIa. Moreover, corrections of the propagation phase in hemophilia A required rFVIIa concentrations above the range where a physiological level of FVII was capable to downregulate thrombin generation. These data strongly suggest that rFVIIa acts independently of TF in hemophilia therapy and that FVII displacement by rFVIIa is a negligible mechanistic component.

Topics: Coagulants; Factor VII; Factor VIIa; Factor VIII; Hemophilia A; Humans; Recombinant Proteins; Thrombin; Thromboplastin

Immune responses to therapeutic factor VIII remain a major problem, affecting 30% of patients with severe hemophilia A. The primary factors that drive immune responses in these patients remain elusive. There have been conflicting reports on a role of coagulation (or thrombin) in anti-FVIII immune responses.. To assess the importance of coagulation-associated processes for the onset of the anti-FVIII immune response.. Using FVIII-deficient mice, we compared the immunogenicity of recombinant FVIII or the inactive FVIII(V) (634M) mutant. In parallel, the involvement of tissue factor (TF) activity in the anti-FVIII immune response was investigated upon injection of a neutralizing anti-TF antibody or by the use of chimeric mice that lack TF expression in myeloid cells. The development of the anti-FVIII immune response was also monitored after treatment with warfarin.. The kinetics of the development of antibody responses to FVIII(V) (634M) were indistinguishable from those of wild-type FVIII. Inhibition of TF activity did not modulate immune responses to exogenous FVIII. Additionally, global inhibition of coagulation with warfarin failed to reduce the anti-FVIII immune response.. Thrombin generation or coagulation-associated processes do not modulate the anti-FVIII antibody response in mouse model of severe hemophilia A.

Topics: Animals; Antibodies, Neutralizing; Blood Coagulation; Disease Models, Animal; Factor VIII; Hemophilia A; Immunity, Humoral; Inflammation; Mice; Mutation; Plasmids; Protein Structure, Tertiary; Recombinant Proteins; Thrombin; Thromboplastin; Warfarin

Tissue factor pathway inhibitor (TFPI) is a multidomain protein that negatively regulates the coagulation cascade. TFPI inhibits the tissue factor (TF)-activated factor VII-activated FX (FXa) complex during TF-mediated coagulation initiation. The aptamer BAX 499 binds specifically to TFPI and inhibits its function, mediating a procoagulant effect in both in vitro and in vivo models of hemophilia.. This study sought to identify the regions of TFPI that are critical for BAX 499 binding, and to determine how binding mediates aptamer inhibition of TFPI.. In vitro biochemical methods were used to evaluate the BAX 499 interaction with and inhibition of TFPI. Binding experiments indicated that the full-length TFPI protein is required for tight aptamer binding. Binding-competition experiments implicated the Kunitz 1, Kunitz 3 and C-terminal domains of TFPI in aptamer binding, a finding that is supported by hydrogen-deuterium exchange experiments, and indicated that aptamer and FXa can bind simultaneously to TFPI. In enzymatic assays, BAX 499 inhibited TFPI in a manner that is distinct from domain-specific antibodies, and aptamer inhibitory activity is reduced in the presence of the TFPI cofactor protein S.. These studies demonstrate that BAX 499 binds to TFPI via multiple domains of the protein in a manner that is distinct from other TFPI inhibitors, mediating a mechanism of inhibition that does not involve direct competition with FXa. With this unique inhibitory mechanism, BAX 499 provides a useful tool for studying TFPI biology in health and disease.

Topics: Antibodies; Aptamers, Nucleotide; Blood Coagulation; Coagulants; Deuterium Exchange Measurement; Enzyme-Linked Immunosorbent Assay; Factor Xa; Hemophilia A; Humans; Hydrogen; Inhibitory Concentration 50; Lipoproteins; Peptides; Protein Binding; Protein S; Protein Structure, Tertiary; Thromboplastin

Topics: Blood Coagulation; Factor XI; Factor XI Deficiency; Hemophilia A; Humans; Thrombin; Thromboplastin

Hemophilia A (HA) patients with similar factor VIII levels can demonstrate varying bleeding tendencies. In particular, 10-15% of all severe HA patients (FVIII:C<1IUdL(-1)) do not require regular replacement therapy. Modern global coagulation assays can help to detect and study this "mild" bleeding phenotype. Here, we investigated the coagulation status of different bleeding phenotypes using various types of global coagulation assays.. Ten HA patients with severe phenotype and eleven patients with mild phenotypes were included in the study. For each patient, thromboelastography (TE), thrombodynamics (TD), and kaolin- or tissue factor-induced thrombin generation (TG) were measured. TG in platelet-rich plasma (PRP) was investigated using our original modification when the thrombin generation curve showed two peaks, previously shown to depend on platelet activity. We also utilized TG and TD with the addition of thrombomodulin.. The second peak amplitude and ETP of PRP TG were the only parameters that were significantly higher in mild bleeders (peak 41.6 ± 3.5 nM, ETP 1966 ± 169 nM*min) than in patients with severe bleeding (peak 28.3 ± 3.3 nM, ETP 1359 ± 130 nM*min).. Our results suggest that severe and mild HA phenotypes could be distiguished by TG assay in PRP suggesting that difference in platelet activity can be involved in the phenotype formation. According to our previous results we can suppose that the mechanism of the phenotypic heterogeneity is linked with TG mediated by PS-expressing platelets.

Topics: Adult; Aged; Blood Coagulation Tests; Hemophilia A; Humans; Kaolin; Middle Aged; Platelet-Rich Plasma; Thrombelastography; Thrombin; Thromboplastin; Young Adult

Normal haemostasis is maintained by a controlled balance between coagulation and fibrinolysis, involving thrombin and plasmin the respective key enzymes. Simultaneous evaluation of both enzymes facilitates, therefore, an overall understanding of normal and pathological haemostasis. Combined thrombin and plasmin generation (T/P-G) assays have been recently described, and we have adapted the technique to investigate the interplay between coagulation and fibrinolysis in patients with various haemostatic disorders. Our modified T/P-G was initiated by the addition of a mixture of optimised lower concentrations of tissue factor and tissue-type plasminogen activator. Thrombin generation (TG) and plasmin generation (PG) were monitored simultaneously using individual fluorescent substrates in separate microtitre wells. The relationship between coagulation and fibrinolysis was demonstrated by analysing the effects of thrombin inhibitors, activated protein C and thrombomodulin. The most evident impairments in TG were observed with plasma samples deficient of coagulation factors participating in the prothrombinase complex. Defects in PG were observed with deficiencies of factor (F)V, FX, fibrinogen, and plasminogen. TG appeared to be a prerequisite for the initiation of PG, and overall PG was governed by fibrinogen concentration. TG in patients with haemophilia A correlated with levels of FVIII activity, but there was no significant relationship between PG and FVIII:C, confirming that the abnormal haemostasis in haemophilia A results in a severe imbalance between coagulation and fibrinolysis. The findings demonstrate that global haemostasis depends on a sensitive balance between coagulation and fibrinolysis, and that the modified T/P-G assay could provide an enhanced understanding of haemorrhage and thrombosis in clinical practice.

Topics: Afibrinogenemia; Blood Coagulation; Blood Coagulation Tests; Conjunctivitis; Factor V Deficiency; Factor X Deficiency; Feedback, Physiological; Fibrinolysin; Fibrinolysis; Hemophilia A; Hemostasis; Humans; Plasminogen; Protein C Inhibitor; Skin Diseases, Genetic; Thrombin; Thrombomodulin; Thromboplastin; Tissue Plasminogen Activator

Clinical evidence suggests that individuals with factor VIII (FVIII) deficiency (hemophilia A) are protected against venous thrombosis, but treatment with recombinant proteins can increase their risk for thrombosis. In this study we examined the dynamics of thrombus formation in individuals with hemophilia A and their response to replacement and bypass therapies under venous flow conditions. Fibrin and platelet accumulation were measured in microfluidic flow assays on a TF-rich surface at a shear rate of 100 s⁻¹. Thrombin generation was calculated with a computational spatial-temporal model of thrombus formation. Mild FVIII deficiencies (5-30% normal levels) could support fibrin fiber formation, while severe (<1%) and moderate (1-5%) deficiencies could not. Based on these experimental observations, computational calculations estimate an average thrombin concentration of ∼10 nM is necessary to support fibrin formation under flow. There was no difference in fibrin formation between severe and moderate deficiencies, but platelet aggregate size was significantly larger for moderate deficiencies. Computational calculations estimate that the local thrombin concentration in moderate deficiencies is high enough to induce platelet activation (>1 nM), but too low to support fibrin formation (<10 nM). In the absence of platelets, fibrin formation was not supported even at normal FVIII levels, suggesting platelet adhesion is necessary for fibrin formation. Individuals treated by replacement therapy, recombinant FVIII, showed normalized fibrin formation. Individuals treated with bypass therapy, recombinant FVIIa, had a reduced lag time in fibrin formation, as well as elevated fibrin accumulation compared to healthy controls. Treatment of rFVIIa, but not rFVIII, resulted in significant changes in fibrin dynamics that could lead to a prothrombotic state.

Topics: Adolescent; Biomechanical Phenomena; Blood Coagulation; Child, Preschool; Computer Simulation; Cysteine Endopeptidases; Factor VIIa; Factor VIII; Fibrin; Hemophilia A; Humans; Male; Microfluidics; Models, Biological; Neoplasm Proteins; Phenotype; Platelet Aggregation; Regional Blood Flow; Thromboplastin; Thrombosis; Young Adult

Hemophilia A (HA) is an inherited X-linked recessive bleeding disorder caused by coagulant factor VIII (FVIII) deficiency. Previous studies showed that introduction of mesenchymal stem cells (MSCs) modified by FVIII-expressing retrovirus may result in phenotypic correction of HA animals. This study aimed at the investigation of an alternative gene therapy strategy that may lead to sustained FVIII transgene expression in HA mice. B-domain-deleted human FVIII (hFVIIIBD) vector was microinjected into single-cell embryos of wild-type mice to generate a transgenic mouse line, from which hFVIIIBD-MSCs were isolated, followed by transplantation into HA mice. RT-PCR and real-time PCR analysis demonstrated the expression of hFVIIIBD in multi-organs of recipient HA mice. Immunohistochemistry showed the presence of hFVIIIBD positive staining in multi-organs of recipient HA mice. ELISA indicated that plasma hFVIIIBD level in recipient mice reached its peak (77 ng/mL) at the 3rd week after implantation, and achieved sustained expression during the 5-week observation period. Plasma FVIII activities of recipient HA mice increased from 0% to 32% after hFVIIIBD-MSCs transplantation. APTT (activated partial thromboplastin time) value decreased in hFVIIIBD-MSCs transplanted HA mice compared with untreated HA mice (45.5 s vs. 91.3 s). Our study demonstrated an effective phenotypic correction in HA mice using genetically modified MSCs from hFVIIIBD transgenic mice.

Topics: Animals; Female; Gene Expression Regulation; Genetic Therapy; Hemophilia A; Humans; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Transgenic; Phenotype; Protein Structure, Tertiary; RNA, Messenger; Thromboplastin

Hemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (K(D) = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.

Topics: Animals; Antibodies, Blocking; Antibodies, Monoclonal, Humanized; Antibodies, Neutralizing; Bleeding Time; Blood Coagulation; Cross Reactions; Disease Models, Animal; Epitopes; Factor VIII; Factor Xa; Female; Fibrin; HEK293 Cells; Hemophilia A; Hemostasis; Human Umbilical Vein Endothelial Cells; Humans; Lipoproteins; Models, Molecular; Neutralization Tests; Protein Binding; Protein Structure, Tertiary; Rabbits; Species Specificity; Thromboplastin

Recombinant factor VIIa (rFVIIa) is used for treatment of hemophilia patients with inhibitors, as well for off-label treatment of severe bleeding in trauma and surgery. Effective bleeding control requires supraphysiological doses of rFVIIa, posing both high expense and uncertain thrombotic risk. Two major competing theories offer different explanations for the supraphysiological rFVIIa dosing requirement: (1) the need to overcome competition between FVIIa and FVII zymogen for tissue factor (TF) binding, and (2) a high-dose-requiring phospholipid-related pathway of FVIIa action. In the present study, we found experimental conditions in which both mechanisms contribute simultaneously and independently to rFVIIa-driven thrombin generation in FVII-deficient human plasma. From mathematical simulations of our model of FX activation, which were confirmed by thrombin-generation experiments, we conclude that the action of rFVIIa at pharmacologic doses is dominated by the TF-dependent pathway with a minor contribution from a phospholipid-dependent mechanism. We established a dose-response curve for rFVIIa that is useful to explain dosing strategies. In the present study, we present a pathway to reconcile the 2 major mechanisms of rFVIIa action, a necessary step to understanding future dose optimization and evaluation of new rFVIIa analogs currently under development.

Topics: Blood Coagulation; Blotting, Western; Enzyme Precursors; Factor VIIa; Hemophilia A; Humans; Models, Theoretical; Phospholipids; Recombinant Proteins; Thrombin; Thromboplastin

Recent studies show that activated factor VII (FVIIa) binds to the endothelial cell protein C receptor (EPCR) on the vascular endothelium; however, the importance of this interaction in hemostasis or pathophysiology is unknown.. The aim of the present study was to investigate the role of the FVIIa interaction with EPCR on the endothelium in mediating FVIIa transport from the circulation to extravascular tissues.. Wild-type, EPCR-deficient or ECPR-over-expressing mice were injected with human recombinant (r)FVIIa (120 μg kg(-1) body weight) via the tail vein. At varying time intervals after rFVIIa administration, blood and various tissues were collected to measure FVIIa antigen and activity levels. Tissue sections were analyzed by immunohistochemistry for FVIIa and EPCR.. The data reveal that, after intravenous (i.v.) injection, rFVIIa rapidly disappears from the blood and associates with the endothelium in an EPCR-dependent manner. Immunohistochemical analyses revealed that the association of FVIIa with the endothelium was maximal at 30 min and thereafter progressively declined. The FVIIa association with the endothelium was undetectable at time points exceeding 24 h post-FVIIa administration. The levels of rFVIIa accumulated in tissue correlate with expression levels of EPCR in mice and FVIIa associated with tissues remained functionally active for periods of at least 7 days.. The observation that an EPCR-dependent association of FVIIa with the endothelium is most pronounced soon after rFVIIa administration and subsequently declines temporally, combined with the retention of functionally active FVIIa in tissue homogenates for extended periods, indicates that FVIIa binding to EPCR on the endothelium facilitates the transport of FVIIa from circulation to extravascular tissues where TF resides.

Topics: Animals; Biological Transport; Blood Coagulation Factors; Endothelial Cells; Endothelial Protein C Receptor; Endothelium, Vascular; Factor VIIa; Hemophilia A; Hemostasis; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Protein Binding; Receptors, Cell Surface; Recombinant Proteins; Signal Transduction; Thromboplastin; Time Factors; Tissue Distribution

Inhibitory antibodies to factors VIII or IX represent a serious complication for hemophilia patients. Treatment involves products that bypass the intrinsic pathway and promote thrombin generation. Direct infusion of factor Xa should also restore hemostasis; however, it has a short half-life in plasma and could activate systemic coagulation in an uncontrolled fashion. Here we show that factor Xa mutants with zymogen-like properties (FXa(I16L) and FXa(V17A)) circumvent these limitations. In the absence of factor Va, the FXa variants are poor enzymes for a range of physiological ligands and are resistant to inactivation by antithrombin III and tissue factor pathway inhibitor. Notably, assembly of FXa(I16L) and FXa(V17A) on activated platelets with factor Va to form prothrombinase completely restores biologic activity. In hemophilic plasma, FXa(I16L) and FXa(V17A) have prolonged half-lives compared with wild-type factor Xa (approximately 60 minutes vs approximately 1 minute) and promote robust thrombin generation that bypasses the intrinsic pathway. The variants require factor Va generated in situ for procoagulant function, and cofactor inactivation by the protein C pathway regulates their activity. The efficacy, extended half-life, and mechanism of action suggest that novel zymogen-like forms of factor Xa might prove useful as new therapeutic procoagulants to treat deficiencies upstream of the common pathway.

Topics: Antithrombin III; Blood Coagulation; Factor Va; Factor Xa; Genetic Variation; Hemophilia A; Humans; Lipoproteins; Mutation; Thrombin; Thromboplastin

Bypass agents are the common treatment for haemophilia patients who develop inhibitory antibodies. Laboratory assessment of the efficacy of bypassing agent therapy is a challenge. In the present work we modified the conditions triggering thrombin generation (TG) assay in order to find the most sensitive assay for detection of rFVIIa and its analogue NN1731 in haemophilic plasma. TG was measured in samples of normal plasma, plasma of haemophilia patient with inhibitors, as well as haemophilia induced plasma. Recalcification-induced TG was compared to tissue factor (TF) -induced TG in the presence and absence of rFVIIa and NN1731. Recalcification-induced TG (without TF) in haemophilic plasma yielded baseline flat curves, with increased TG as a consequence of spiking the plasma rFVIIa. Using our system, we observed both dose-dependence and time-dependence of rFVIIa effect on TG. Elevated concentrations of TF mask the difference between rFVIIa-treated and non-treated haemophilic plasma. NN1731 yielded normalisation of recalcification-induced TG curves (without TF) which may reflect high potency. In conclusion, we suggest that triggering TG by recalcification-only may be the most sensitive assay for determining the impact of bypassing agents in haemophilic plasma, and may serve as a caution surrogate safety marker in future studies.

Topics: Blood Coagulation Factors; Calcium; Dose-Response Relationship, Drug; Factor VIIa; Hemophilia A; Humans; Plasma; Prothrombin Time; Recombinant Proteins; Sensitivity and Specificity; Thrombin; Thromboplastin

Hemophilia A and B are caused by deficiencies in coagulation factor VIII (FVIII) and factor IX, respectively, resulting in deficient blood coagulation via the intrinsic pathway. The extrinsic coagulation pathway, mediated by factor VIIa and tissue factor (TF), remains intact but is negatively regulated by tissue factor pathway inhibitor (TFPI), which inhibits both factor VIIa and its product, factor Xa. This inhibition limits clot initiation via the extrinsic pathway, whereas factor deficiency in hemophilia limits clot propagation via the intrinsic pathway. ARC19499 is an aptamer that inhibits TFPI, thereby enabling clot initiation and propagation via the extrinsic pathway. The core aptamer binds tightly and specifically to TFPI. ARC19499 blocks TFPI inhibition of both factor Xa and the TF/factor VIIa complex. ARC19499 corrects thrombin generation in hemophilia A and B plasma and restores clotting in FVIII-neutralized whole blood. In the present study, using a monkey model of hemophilia, FVIII neutralization resulted in prolonged clotting times as measured by thromboelastography and prolonged saphenous-vein bleeding times, which are consistent with FVIII deficiency. ARC19499 restored thromboelastography clotting times to baseline levels and corrected bleeding times. These results demonstrate that ARC19499 inhibition of TFPI may be an effective alternative to current treatments of bleeding associated with hemophilia.

Topics: Animals; Aptamers, Nucleotide; Bleeding Time; Blood Coagulation; Disease Models, Animal; Factor VIIa; Factor VIII; Factor Xa; Hemophilia A; Hemophilia B; Hemostasis; Humans; In Vitro Techniques; Lipoproteins; Macaca fascicularis; Recombinant Proteins; Thrombin; Thromboplastin

The patient was a 36-year-old man who had been aware of a mild bleeding tendency since childhood, but did not show any history of severe bleeding. After lumbar epidural block was performed for pain caused by lumbar disc herniation, the patient developed paraplegia due to an acute epidural hematoma, and rectovesical disorder. He was admitted to our hospital, and wide fenestration and hematoma evacuation were performed. Because of persistent bleeding, reoperation was performed to achieve hemostasis. Although factor VIII antigen was 138%, its activity was decreased to 18% of normal. A diagnosis of cross-reacting material positive mild hemophilia A was made. Postoperative injection of a factor VIII preparation resulted in complete hemostasis. The activated partial thromboplastin time was within normal range. A thrombin generation test showed reduced endogenous thrombin potential, peak thrombin levels, and prolonged time-to-peak levels. The thrombin generation test, which allows comprehensive assessment of the coagulation profile, was useful for diagnosis and treatment of this case.

Topics: Acute Disease; Adult; Analgesia, Epidural; Biomarkers; Blood Coagulation Tests; Hematoma, Epidural, Spinal; Hemophilia A; Humans; Male; Thrombin; Thromboplastin

The available methods for measuring factor VIII (FVIII) activity suffer reportedly from lack of sensitivity and precision in the < 1 IU dL(-1) range. This precludes correlation of clinical phenotype with FVIII levels.. To study a possible association between clinical phenotype in patients with FVIII levels < 1 IU dL(-1).. The FIXa-driven FVIII assay (FVIII-CAT) has a detection limit of 0.05 IU dL(-1). For the range of 0-2 IU dL(-1) FVIII, the intra-assay coefficient of variation (CV) is around 2% and the inter-assay CV is about 8%. We tested 30 hemophiliacs with FVIII:C between < 1 and 6 IU dL(-1) as measured in the one-stage clotting assay using the FVIII-CAT assay. For genetic defects related to moderate hemophilia, the FVIII-CAT test finds FVIII levels that are in good agreement with those determined with the one-stage assay. Of the 21 hemophilic patients with FVIII < 1 IU dL(-1), four patients exhibited a mild bleeding phenotype. When we applied TF-initiated thrombin generation, patients with a mild clinical phenotype showed significantly higher endogenous thrombin potentials.. The novel developed FVIII assay measures accurately FVIII levels below 1 IU dL(-1). Its application demonstrated that the clinical heterogeneity in individuals with < 1 IU dL(-1) FVIII is not associated with their FVIII level.

Topics: Adult; Aged; Biomarkers; Blood Coagulation Tests; Factor VIII; Factor Xa; France; Hemophilia A; Humans; Kinetics; Middle Aged; Netherlands; Phenotype; Predictive Value of Tests; Reproducibility of Results; Sensitivity and Specificity; Severity of Illness Index; Thrombin; Thromboplastin; Young Adult

Carbon monoxide derived from carbon monoxide releasing molecules (CORMs) has been demonstrated to enhance normal plasma thrombus speed of growth and strength in vitro. We tested the hypothesis that tricarbonyldichlororuthenium (II) dimer (CORM-2) improves the velocity of formation and strength of hemophiliac plasma thrombi as determined by thrombelastography. Plasma deficient (<1% normal activity) in factor VIII (FVIII; n = 11 individuals), factor IX (FIX; n = 5 individuals) or factor VII (FVII; n = 4 individuals) was exposed to 0 or 100 micromol CORM-2, with coagulation initiated with tissue factor. Coagulation kinetics were monitored with thrombelastography for 15 min. Paired t-tests were used to analyze FVIII-deficient plasma results; relative change was used to describe the other plasma types tested. In FVIII-deficient plasma, CORM-2 exposure significantly (P < 0.05) increased the velocity of thrombus formation (84%) and strength (48%) compared with plasma not exposed to CORM-2. FXI-deficient clots demonstrated an increase in velocity of formation (63%) and strength (43%) after CORM-2 exposure. Lastly, CORM-2 exposure increased FVII-deficient plasma velocity of formation (45%) and strength (63%). CORM-2 markedly enhanced the velocity of clot growth and strength in hemophiliac plasma. These findings serve as the rationale to determine whether CORMs could be utilized as hemostatic agents.

Topics: Blood Coagulation; Carbon Monoxide; Drug Evaluation, Preclinical; Factor VII Deficiency; Hemophilia A; Hemophilia B; Hemostatics; Humans; In Vitro Techniques; Organometallic Compounds; Plasma; Prodrugs; Thrombelastography; Thromboplastin

Topics: Animals; Cell Movement; Factor VIIa; Genetic Predisposition to Disease; Hemophilia A; Hemorrhage; Inflammation; Leukocytes; Mice; Mice, Inbred C57BL; Recombinant Proteins; Thrombocytopenia; Thromboplastin; Wound Healing

Haemophilia A displays phenotypic heterogeneity with respect to clinical severity. The aim of this study was to determine if tissue factor (TF)-initiated thrombin generation profiles in whole blood in the presence of corn trypsin inhibitor (CTI) are predictive of bleeding risk in haemophilia A. We studied factor(F) VIII deficient individuals (11 mild, 4 moderate and 12 severe) with a well-characterized 5-year bleeding history that included haemarthrosis, soft tissue haematoma and annual FVIII concentrate usage. This clinical information was used to generate a bleeding score. The bleeding scores (range 0-32) were separated into three groups (bleeding score groupings: 0, 0 and < or = 9.6, >9.6), with the higher bleeding tendency having a higher score. Whole blood collected by phlebotomy and contact pathway suppressed by 100 microg mL(-1) CTI was stimulated to react by the addition of 5 pM TF. Reactions were quenched at 20 min by inhibitors. Thrombin generation, determined by enzyme-linked immunosorbent assay for thrombin-antithrombin was evaluated in terms of clot time (CT), maximum level (MaxL) and maximum rate (MaxR) and compared to the bleeding score. Data are shown as the mean+/-SD. MaxL was significantly different (P < 0.001) between the groups: 504 +/- 114, 315 +/- 117 and 194 +/- 91 nM; with higher thrombin concentrations in the groups with lower bleeding scores. MaxR was higher in the groups with a lower bleeding score; 97 +/- 51, 86 +/- 60 and 39 +/- 16 nM min(-1) (P = 0.09). No significant difference was detected in CT among the groups, 5.6 +/- 1.3, 4.7 +/- 0.7 and 5.6 +/- 1.3 min. Our empirical study in CTI-inhibited whole blood shows that the MaxL of thrombin generation appears to correlate with the bleeding phenotype of haemophilia A.

Topics: Enzyme-Linked Immunosorbent Assay; Hemophilia A; Hemorrhage; Humans; Male; Phenotype; Plant Proteins; Reference Values; Thrombin; Thromboplastin; Whole Blood Coagulation Time

Discrepancies between low levels of FVIII:C and clinical symptoms in severe hemophilia A are well-known. We have recently demonstrated that levels of FVIII:C < 0.2 IU/dl were consistent with clinical phenotype by clot waveform analysis, suggesting that precise measurement of very low levels of FVIII:C was clinically important. Thrombin generation tests (TGTs) triggered by tissue factor (TF) have been recently utilized to monitor coagulation function in hemophilia A. We examined whether TGT was useful for evaluating hemophilia A patients with very low levels of FVIII:C. TGTs in 40 hemophilia A plasmas with FVIII:C < 0.2-17 IU/dl (measured by clot waveform analysis using MDA-II) were performed using TF and/or ellagic acid (ELG). The lagtime in ELG-TGT at very low levels of FVIII:C was shortened dose-dependently, whilst this parameter in TF-TGT was not significantly affected. Other parameters (endogenous thrombin potential, peak thrombin, time to peak) correlated with FVIII:C levels to some extent in both assays (r = 0.4-0.7). Using a TF/ELG mixture in TGT, however, the correlation coefficients increased to ~0.85. TGT parameters correlated well with levels of FVIII:C > 0.2 IU/dl, although the lagtime was not especially informative. We conclude that modified TGT, using a TF/ELG mixture as the trigger, is useful for monitoring coagulation function at very low levels of FVIII:C in hemophilia A.

Topics: Blood Coagulation Tests; Ellagic Acid; Factor VIII; Hemophilia A; Humans; Thrombin; Thromboplastin

Thrombin generation has been suggested as a method to monitor treatment with factor eight inhibitor bypassing activity (FEIBA) or recombinant FVIIa (rFVIIa). The sensitivity of the assay for individual coagulation factors is dependent on the tissue factor (TF) concentration. An inverse relation between the rFVIIa concentration needed to shorten the clotting time and TF concentration has been shown but the data on thrombin generation are inconsistent. Information on TF concentration in measurements with FEIBA is limited. We studied the influence of TF concentration (1 and 5 pM) on thrombin generation through spiking experiments with rFVIIa and/or FEIBA in the plasma of severe haemophilia A patients and after four and three treatment episodes, respectively, using the calibrated automated thrombin generation assay (CAT) in platelet poor plasma. Spiking with FEIBA showed a linear relation with the endogenous thrombin potential (ETP)/peak at 1 pM but substrate depletion at 5 pM. Spiking with rFVIIa showed a near linear dose-response relation with the ETP/peak at 1 pm but only a shortening of the initiation phase at 5 pM. Similar effects were present in post-treatment samples. FEIBA acted synergistically with rFVIIa. This suggest a role for CAT in monitoring inhibitor bypass treatment but low TF concentrations are required to avoid substrate depletion with FEIBA and to demonstrate the effect of rFVIIa.

Topics: Blood Coagulation Factors; Blood Coagulation Tests; Drug Monitoring; Factor VIIa; Hemophilia A; Humans; Recombinant Proteins; Thrombin; Thromboplastin

Supraphysiological concentrations of recombinant activated factor VII (rVIIa, NovoSeven) are used to control bleeding in hemophilia. Current experimental evidence suggests that rVIIa may increase thrombin generation via two pathways: one being tissue factor (TF)-dependent and another being activated platelet-dependent. Contribution of TF to the rVIIa action may justify different administration profiles of rVIIa. In the present study, thrombin and fibrin generation and spatial clot formation assays in platelet-free hemophilia A and normal plasma were used to investigate this contribution. By varying the concentration of TF and the way it becomes available to plasma, we obtained the following results. Activation of clotting with less than 5 pmol/l of TF facilitates thrombin and fibrin generation at low, but not at supraphysiological rVIIa concentrations. Activation with more than 13 pmol/l of TF saturates thrombin and fibrin generation kinetics, making it insensitive to rVIIa. rVIIa minimally modulates clot growth on the surface of TF-expressing fibroblasts. On the contrary, rVIIa produces spontaneous clot formation in nonflowing platelet-free plasma far away from fibroblasts via plasma lipid particles. Therefore, both the concentration and the distribution of TF determine relevance of a particular experimental system for the studies of rVIIa action. The results indicate that 300-1600 nmol/l (megadoses) of rVIIa may deliver coagulation outside of the TF-rich areas of blood vessel damage via the platelet-derived microparticles. Therefore, rate and extent of platelet-derived microparticles generation might be important with regard to rVIIa treatment safety.

Topics: Blood Coagulation; Blood Platelets; Cell-Derived Microparticles; Cells, Cultured; Dose-Response Relationship, Drug; Factor VIIa; Fibrin; Hemophilia A; Humans; Kinetics; Recombinant Proteins; Thrombin; Thromboplastin

The function of tissue factor (Tf)-initiated coagulation is hemorrhage control through the formation and maintenance of an impermeable platelet-fibrin barrier. The catalytic processes involved in the clot maintenance function are not well defined, although the rebleeding problems characteristic of individuals with hemophilias A and B suggest a link between specific defects in the Tf-initiated process and defects in the maintenance function. We have previously demonstrated, using a methodology of "flow replacement" (or resupply) of ongoing Tf-initiated reactions with fresh reactants, that procoagulant complexes are produced during Tf-initiated coagulation, which are capable of reinitiating coagulation without input from extrinsic factor Xase activity (Orfeo, T., Butenas, S., Brummel-Ziedins, K. E., and Mann, K. G. (2005) J. Biol. Chem. 280, 42887-42896). Here we used Tf-initiated reactions in normal and hemophilia blood or in their corresponding proteome mixtures as sources of procoagulant end products and then varied the resupplying material to determine the identity of the catalysts that drive the new cycle of thrombin formation. The central findings are as follows: 1) the prothrombinase complex (fVa-fXa-Ca(2+)-membrane) accumulated during the episode of Tf-initiated coagulation is the primary catalyst responsible for the observed pattern of prothrombin activation after resupply; 2) impairments in intrinsic factor Xase function, i.e. hemophilias A and B, result in an impaired capacity to mount a resupply response; and 3) in normal hemostasis the intrinsic factor Xase function contributes to the durability of the resupply response.

Topics: Adult; Blood Coagulation; Factor V; Factor X; Factor Xa; Hemophilia A; Hemophilia B; Hemorrhage; Hemostasis; Humans; Male; Proteome; Thromboplastin

For the treatment of haemophilia patients with inhibitors, recombinant factor VIIa (rFVIIa) is available as a therapeutic option to control bleeding episodes with a good balance of safety and efficacy. However, the short in-vivo half-life of approximately 2.5 hours makes multiple injections necessary, which is inconvenient for both physicians and patients. Here we describe the generation of a recombinant FVIIa molecule with an extended half-life based on genetic fusion to human albumin. The recombinant FVII albumin fusion protein (rVII-FP) was expressed in mammalian cells and upon activation displayed a FVII activity close to that of wild type FVIIa. Pharmacokinetic studies in rats demonstrated that the half-life of the activated recombinant FVII albumin fusion protein (rVIIa-FP) was extended six- to seven-fold compared with wild type rFVIIa. The in-vitro and in-vivo efficacy was evaluated and was found to be comparable to a commercially available rFVIIa (NovoSeven((R))). The results of this study demonstrate that it is feasible to develop a half-life extended FVIIa molecule with haemostatic properties very similar to the wild-type factor.

Topics: Animals; Base Sequence; Coagulants; DNA, Recombinant; Factor VIIa; Half-Life; Hemophilia A; Hemostasis; Humans; In Vitro Techniques; Protein Binding; Protein Engineering; Rats; Recombinant Fusion Proteins; Recombinant Proteins; Serum Albumin; Surface Plasmon Resonance; Thrombelastography; Thromboplastin

In some mild haemophilia A patients (discrepant haemophilia), factor VIII coagulant activity (FVIII:C) levels, by one-stage assay are more than double than those by two-stage assay. This may be due to the longer incubation times (10-12 min) in the two-stage assay. This study aimed to determine the time course of the activation phase of the two-stage assay, using both classical coagulation and chromogenic detection methods. In both systems, for equivalent patients (equivalent FVIII:C levels by one-stage and two-stage assays, n = 6, all different mutations), similar FVIII:C results were obtained with short- or long-incubation times. In contrast, plasma from discrepant patients (n = 8, five different mutations) showed higher FVIII:C at shorter incubation times than after longer incubation times. In the chromogenic assay, FVIII:C levels were higher after incubation for 2 min (23-56%, mean 41%) than after 10 min (19-41%, mean 29%). In the classical coagulation assay, FVIII:C levels were higher at shorter incubation times (21-64%, mean 37%) than with the longer incubation times usually used (13-29%, mean 23%). These time-course experiments have verified that the longer incubation time used in the two-stage assay is at least partly responsible for the lower FVIII:C measured by that assay in discrepant haemophilia.

Topics: Blood Coagulation Tests; Chromogenic Compounds; Factor VIII; Hemophilia A; Humans; Mutation; Predictive Value of Tests; Thromboplastin; Time Factors

Activation of platelets with a combination of collagen and thrombin generates a subpopulation of highly procoagulant 'coated' platelets characterized by high surface expression of fibrinogen and other procoagulant proteins.. To analyze the interaction of recombinant factor VIIa (rFVIIa) with coated platelets.. rFVIIa localized to the coated platelets in flow cytometry experiments, while minimal rFVIIa was found on platelets activated with adenosine diphosphate, thrombin or via glycoprotein VI individually, and essentially no rFVIIa was found on non-stimulated platelets. Removal of the gamma-carboxyglutamic acid (Gla) domain of rFVIIa, and addition of EDTA, annexin V or excess prothrombin inhibited rFVIIa localization to the coated platelets, indicating that the interaction was mediated by the calcium-dependent conformation of the Gla domain and platelet exposure of negatively charged phospholipids. A reduced level of platelet fibrinogen exposure was observed at hemophilia A-like conditions in a model system of cell-based coagulation, indicating that coated platelet formation in hemophilia may be diminished. Addition of rFVIIa dose-dependently enhanced thrombin generation and partly restored platelet fibrinogen exposure.. The data suggest that rFVIIa localized preferentially on platelets activated with dual agonists, thereby ensuring enhanced thrombin generation localized at the site of injury where both collagen and tissue factor are exposed, the latter ensuring the formation of thrombin necessary for coated platelet formation.

Topics: 1-Carboxyglutamic Acid; Binding Sites; Blood Coagulation; Blood Platelets; Coagulants; Collagen; Factor VIIa; Flow Cytometry; Hemophilia A; Humans; Phospholipids; Platelet Membrane Glycoproteins; Protein Structure, Tertiary; Thrombin; Thromboplastin

Two assays [coagulant activity of factor VII (FVII:C) and activated factor VII (FVIIa) activity] are currently available for the assessment of factor VII and FVIIa pharmacokinetics. This article presents the results of a comparison of the two assays when applied both in vitro as well as during clinical pharmacokinetic trials of recombinant FVIIa (rFVIIa) administered to healthy individuals and haemophilia patients. The in-vitro data showed that, for the FVII:C assay, plasma samples do not dilute in parallel. For the FVIIa activity assay, dilutions of samples are both parallel and linear with different dilutions of the calibrator. Moreover, intra-assay variation was found to be smaller for the FVIIa activity assay than for the FVII:C assay. When adding different amounts of rFVIIa (0-6 microg/ml) to normal plasma, a mean specific activity of rFVIIa of 48.6 U/mug was observed on applying the FVII:C assay; however, the specific activity decreased with increasing levels of rFVIIa. For the FVIIa activity assay, the mean specific activity was 45.4 IU/mug. Direct comparison of the two activity assays showed that no simple conversion between FVII:C and FVIIa activity measurements are possible. When applying the two assays for pharmacokinetic assessments in two clinical trials, statistically significant different estimates for the area under the curve, half-life, clearance and volume of distribution were obtained. In conclusion, for evaluation of rFVIIa pharmacokinetic properties, activity should be measured with the FVIIa activity assay - which is a more specific and reliable assay of the two available factor VII activity assays, especially when assessing low activity levels.

Topics: Adolescent; Adult; Blood Coagulation; Blood Coagulation Tests; Child; Child, Preschool; Coagulants; Factor VII; Factor VII Deficiency; Factor VIIa; Hemophilia A; Humans; Male; Middle Aged; Plasma; Recombinant Proteins; Reference Values; Thromboplastin

Topics: Blood Coagulation; Clinical Trials as Topic; Cohort Studies; Factor VII; Factor VIIa; Fibrinolysis; Hemophilia A; Humans; Recombinant Proteins; Risk; Thromboplastin; Time Factors; Tranexamic Acid

Recombinant factor VIIa (rFVIIa) used for the treatment of hemophilia A or B patients with an inhibitor is hemostatically effective because it induces thrombin generation (TG), despite grossly impaired FVIII- and FIX-dependent amplification of FX activation. Tissue factor (TF) and or activated platelets were shown to be essential for the rFVIIa activity.. To evaluate the relative effects of TF and phospholipids on rFVIIa-induced TG in FVIII-, FIX- and FXI-deficient plasmas.. Phospholipids had an independent effect that was augmented by TF. The contribution of blood-borne TF in FVIII-, FIX- and FXI-deficient plasma to rFVIIa-induced TG was demonstrated by removing microparticles and use of anti-TF antibodies.. At increasing concentrations of rFVIIa, the dependence of rFVIIa-induced TG on TF declined, but the presence of phospholipids was essential. rFVIIa was also shown to activate purified FIX and FX in the presence of phospholipids and absence of TF. rFVIIa-induced TG was dramatically augmented in FVIII- or FIX-deficient plasma in which the level of FVIII or FIX was increased to 1 or 2 U dL(-1).. The data indicate that rFVIIa-induced TG is affected by TF, phospholipids, rFVIIa concentration, and the presence of FVIII and FIX.

Topics: Blood Platelets; Coagulation Protein Disorders; Dose-Response Relationship, Drug; Factor IX; Factor VII; Factor VIIa; Factor X; Factor XI Deficiency; Hemophilia A; Hemophilia B; Humans; Kinetics; Phospholipids; Plasma; Recombinant Proteins; Thrombin; Thromboplastin

Clinical reports suggest that treatment regimens employing both activated prothrombin complex concentrates (aPCCs) and recombinant activated factor VII (rFVIIa) may control the bleeding in patients with haemophilia who fail to respond to either agent alone. We hypothesised that increased concentrations of prothrombin, as may be observed after the infusion of aPCCs, favourably influence parameters of thrombin generation in haemophilia treated with high-dose rFVIIa. We examined the effect of varied prothrombin and rFVIIa concentrations on thrombin generation in a model of haemophilia. At all concentrations of rFVIIa, increased prothrombin concentrations led to increases in the peak and rate of thrombin generation. In assays with the highest concentrations of prothrombin and rFVIIa, peak thrombin actually equalled that measured in the model of normal haemostasis. The significant impact of prothrombin concentration on the effect of rFVIIa in vitro may explain the improved haemostasis reported with concurrent use of aPCCs and rFVIIa. These results imply that persons with plasma prothrombin levels at either end of the 'normal' range could have significantly different responses to similar rFVIIa doses. Furthermore, these results suggest that increasing plasma prothrombin concentration prior to rFVIIa administration may offer advantages over the use of rFVIIa alone in the treatment of haemophilic bleeding.

Topics: Blood Platelets; Cells, Cultured; Drug Administration Schedule; Factor VII; Factor VIIa; Hemophilia A; Hemostasis; Humans; Models, Biological; Monocytes; Prothrombin; Recombinant Proteins; Thrombin; Thromboplastin

The thrombin-platelet feedback loop was examined at low concentration tissue factor using calibrated automated thrombography in combination with the elimination of contact factor activation by corn trypsin inhibitor. The results indicated that, when contact factor activation was eliminated, the thrombin-platelet feedback loop was a major determinant of thrombin generating capacity and that platelets had a greater role in regulating the propagation of thrombin generation than its initiation. This method has potential application to the measurement of platelet-dependent thrombin generation in clinical diagnostic laboratories and hence the investigation of patients with apparent hypo- or hypercoagulable phenotypes.

Topics: Adult; Area Under Curve; Blood Platelets; Case-Control Studies; Feedback, Physiological; Female; Hemophilia A; Humans; Male; Middle Aged; Platelet Activation; Thrombin; Thromboplastin; Time Factors

The clinical phenotype of patients with haemophilia A (HA) often differs between individuals with the same factor VIII (FVIII) gene defect (e.g. within the same family) or the same coagulant activity of FVIII (FVIII:C). We proposed that because the thrombin generation assay in platelet-poor plasma of HA patients provides more information [peak thrombin concentration, endogenous thrombin potential (ETP), rate of thrombin generation and lag-time] than a clot-based FVIII assay it might provide insight into these differences. We therefore investigated the relation between the results of the thrombin generation assay and the clinical severity in nine families with HA (23 patients with different phenotypes). We also examined the contribution of prothrombotic risk factors: (FV Leiden G1691A and prothrombin G20210A), the coagulant activity of FVIII and tissue factor (5'UTR) polymorphisms. Our data detect marked differences between individuals but these did not correlate with the reported clinical phenotype. These differences were also reflected in a marked difference in response to the therapeutic amounts of FVIII. This might account for differences in amounts of treatment consumption. Reduced peak and possibly rate of thrombin generation, rather than FVIII:C or ETP appear to represent the critical defects in FVIII-deficient plasma. We suggest that the analysis of parameters in thrombin generation is a useful tool to detect bleeding tendency in HA but not to predict the modulation of the haemorrhagic tendency in patients within families. However the presence of the other factors such as vessel wall components, protein C and platelets might need to be incorporated into this system.

Topics: Blood Coagulation; Factor V; Factor VIII; Family Health; Hemophilia A; Humans; Phenotype; Polymorphism, Genetic; Prothrombin; Severity of Illness Index; Thrombin; Thromboplastin

A variety of factor concentrates are currently available for replacement therapy for patients with hemophilia. These differ by several parameters, including source (pooled from pooled blood vs recombinant), purity, pathogen inactivation, and by the presence or absence of extraneous proteins such as albumin. The choice of replacement product reflects both safety issues of pathogen transmission or inhibitor development, and personal preferences of the patient and the physician. In general, currently available products are viral pathogen-free, although there is debate about the risk of transmission of parvovirus B19 and prion pathogens. Because of this very small risk, recombinant factor is the treatment of choice in previously untreated patients. In addition, a subset of concentrates contain factor that is activated during manufacture, yielding activated products that can be used in the treatment of patients with inhibitors. Such activated products, especially recombinant factor VIIa (rFVIIa), have also acquired several off-label indications in the management of bleeding in non-hemophiliac patients. The management of hemophilia patients with inhibitors is an ongoing challenge. Immune tolerance induction using a desensitization technique is successful in up to 90% of patients with alloantibodies against factor VIII, with greatest success seen in patients with low titer inhibitors who are treated soon after detection of an alloantibody and in whom treatment includes administration of immunosuppression along with repeated infusions of high titer concentrates. Such therapy is less successful in patients with factor IX alloantibodies. Non-hemophiliac patients with acquired inhibitors represent a unique patient population that requires special management. These patients have a mortality rate that approaches 25% because of the association of acquired inhibitors with severe bleeding complications, occurrence in a largely elderly population, and the frequent presence of an underlying, often serious, primary medical condition. Treatment consists of immunosuppression with steroids, chemotherapy, or intravenous immunoglobulin. Recent studies using rituximab for selective B-cell depletion in these patients have been very promising, although prospective controlled studies have not yet been performed. Finally, although hemophilia A and B appear to be ideal diseases to target with gene therapy approaches, the promise of this therapy remains to be realized.

Topics: Animals; B-Lymphocytes; Blood Coagulation; Blood Coagulation Factors; Consumer Product Safety; Factor VIIa; Hemophilia A; Humans; Lymphocyte Depletion; Parvoviridae Infections; Prion Diseases; Recombinant Proteins; Thromboplastin

Inhibition of blood coagulation appears to be an important therapeutic strategy to improve the outcome in sepsis. However, the beneficial effect of anticoagulant treatment in sepsis is solely based on experimental data using inhibitors of the extrinsic coagulant pathway. The role of the intrinsic pathway of coagulation in the pathogenesis of sepsis has not been explored yet.. In the current study, we contribute to determine the role of factor (F)VIII, the key player of the intrinsic coagulant pathway, on host defense against peritonitis.. Hemizygous FVIII-deficient mice and their wild-type littermates were challenged with 1 x 10(4) bacteria in a septic peritonitis model.. The intraperitoneal injection of Escherichia coli led to growth and dissemination of bacteria and provoked an inflammatory response as evident from elevated cytokine levels, increased cell influx into tissues, liver necrosis, and endothelialitis resulting in mortality. The FVIII-deficient genotype slightly reduced bacterial outgrowth but had no effect on markers of inflammation and/or survival. In addition, FVIII-deficient mice showed profound activation of coagulation, thereby improving the hemophilic phenotype of FVIII-deficient mice.. FVIII deficiency slightly modifies host defense in septic peritonitis in mice, but does not influence the final outcome of peritonitis. Therefore, we question the importance of the intrinsic coagulant pathway during sepsis.

Topics: Animals; Blood Coagulation; Colony Count, Microbial; Escherichia coli; Factor VIII; Hemophilia A; Immunity; Inflammation; Lipoproteins; Mice; Peritonitis; RNA, Messenger; Sepsis; Survival Rate; Thromboplastin

Measurement of thrombin generation by calibrated automated thrombography (CAT) could fulfill the requirements of a global test of coagulability and is potentially applicable to routine clinical laboratory practice. The purpose of this study was to determine if corn trypsin inhibitor (CTI) could be used to abolish contact factor activation in this assay, thus allowing accurate measurement of low tissue factor (TF) concentration-triggered thrombin generation on samples taken in a routine clinical setting.. The endogenous thrombin potential (ETP) was measured by CAT.. The study demonstrated that addition of CTI after plasma separation is not sufficient and blood must be drawn into tubes containing CTI if in-vitro contact factor-activated thrombin generation is to be abolished. Contact factor-activated thrombin generation is completely inhibited at a CTI concentration of 18.3 microg mL(-1) whole blood. Increasing the CTI concentration above this level does not lead to suppression of the TF-triggered ETP. At a TF concentration of 2 pmol, ETPs were significantly lower in the presence of CTI (P < 0.001). The difference (no CTI minus CTI) between results ranged from - 1 to 2159 nM min(-1) (median - 754). Whilst the low concentration TF-ETP assay was not optimized to distinguish degrees of coagulability between patient samples, there was a significant difference in ETP between normal and hemophilia samples and samples from patients with a clinical prothrombotic tendency.. CTI can be applied to ETP measurement by CAT. This permits the use of CAT in a low TF-triggered thrombin generation assay without concern for the effect of interference from in-vitro contact factor activation and the optimum reagent conditions for using CAT as a global test of coagulability in clinical practice can now be defined.

Topics: Adult; Aged; Aged, 80 and over; Automation; Blood Coagulation Tests; Calibration; Clinical Laboratory Techniques; Hemophilia A; Humans; Middle Aged; Plant Proteins; Reproducibility of Results; Thrombin; Thrombophilia; Thromboplastin

The endogenous thrombin potential (ETP) represents the balance between pro- and anti-coagulant forces operating in plasma and can be used to investigate hyper- and hypo-coagulability. As a preliminary step to larger clinical studies we investigated the effect on ETP of phospholipids, tissue factor (TF) and residual platelets in frozen plasma.. We investigated platelet-poor and platelet-rich plasmas from healthy subjects, patients on oral anticoagulants (OA), or with hemophilia and women on oral contraceptives (OC), chosen as examples of the normal, hypo- and hyper-coagulable states in which ETP has been reported to be impaired.. Phospholipids had only a slight effect on ETP in all conditions except in women on OC, in whom the best diagnostic efficacy was observed at 0.5 microM. TF had only a slight effect in all conditions except hemophilia, in which an ETP impairment was observed only at low (1 pM) concentration. Residual platelets had considerable effects on ETP in frozen plasmas, but this was abrogated by filtration before freezing. ETP in platelet-rich plasma at 150x103/mm3 was similar to that obtained in filtered-plasma with 1.5 microM phospholipids in healthy subjects, patients on OA and patients with severe hemophilia, but not in those with mild- or moderate-hemophilia, where the ETP was higher in platelet-rich plasma.. The results suggest that the method can be used for investigations on the clinical value of ETP. Platelet-rich and platelet-poor plasma are suitable testing materials. The latter should be filtered before freezing to minimize the effect of residual platelets.

Topics: Anticoagulants; Blood Coagulation Disorders; Blood Coagulation Tests; Blood Platelets; Contraceptives, Oral; Dose-Response Relationship, Drug; Female; Freezing; Hemophilia A; Humans; Phospholipids; Reproducibility of Results; Thrombin; Thrombophilia; Thromboplastin

To improve the standardization of the factor VII clotting activity (FVII:C) assay in patients treated with recombinant activated factor VII (rFVIIa), we conducted a multicentre study on plasma samples from four patients with haemophilia A, before and at various times after injection of a single dose of rFVIIa. FVII:C and prothrombin time were measured with the methods and reagents routinely used in each laboratory. Strong inter-laboratory variability of FVII:C values was found. The main source of variability was the type of thromboplastin. FVII:C values measured using rabbit thromboplastin were very close to activated factor VII clotting activity values (FVIIa:C) measured with a commercial assay (Staclot VIIa-TF). FVII:C values obtained with human placental thromboplastin were about three times lower than those obtained with rabbit and recombinant thromboplastins, and with the FVIIa:C assay. There was a good relationship between FVIIa:C and activated factor VII antigen values measured using a commercial immunoassay (Imubind FVIIa ELISA). In conclusion, rFVIIa at pharmacological concentrations can be easily monitored on the basis of FVII:C, using rabbit and probably also recombinant thromboplastin; equivalent results are obtained with a specific activated factor VII bioassay.

Topics: Animals; Antigens; Factor VII; Factor VIIa; Female; Hemophilia A; Humans; Indicators and Reagents; Isoantibodies; Placenta; Plasma; Prothrombin Time; Rabbits; Recombinant Proteins; Reference Standards; Thromboplastin

Factor VIII (FVIII)-bypassing agents have complex modes of action but all control bleeding in inhibitor patients by triggering the generation of thrombin. No routine test is available for monitoring this therapy in patients with inhibitors against FVIII. We present an assay that records FEIBA- or FVIIa-mediated changes in thrombin generation (TG) in FVIII inhibitor plasma samples. In plasma samples spiked with FEIBA TG was normalized above 0.4 U/ml, while for recombinant FVIIa (rFVIIa) more than 12.5 microg/ml were required to induce TG in the absence of tissue factor (TF). Addition of TF increased the TG potential of rFVIIa in vitro. This assay seems suitable for monitoring the pharmacokinetics of inhibitor bypassing agents during treatment and possibly for predicting responses to treatment.

Topics: Autoantibodies; Blood Coagulation Factors; Computer Systems; Dose-Response Relationship, Drug; Factor VII; Factor VIIa; Factor VIII; Hemophilia A; Humans; Isoantibodies; Models, Biological; Recombinant Proteins; Thrombin; Thromboplastin

We investigated whole blood coagulation by thrombelastography (TEG) employing activation with minute amounts of tissue factor (TF). Continuous raw data captured were transformed into novel parameters, such as the maximum velocity (MaxVel) and the time to maximum velocity (t,MaxVel) of whole blood clot formation. The courses of the whole blood clot development were very similar to thrombin generation curves reported in plasma. In this assay healthy women (n = 30) showed an earlier onset and an increased coagulation velocity compared to healthy men (n = 30). In patients with severe hemophilia, and persons undergoing thromboprophylaxis, distinctly abnormal coagulation profiles were observed with a decrease in the MaxVel, as well as a prolonged t,MaxVel. Changes appeared to be dependent on the nature and severity of the hemostatic deficit. Preliminary studies in patients substituted with recombinant factor VIIa demonstrated a marked change in the coagulation profile, in which the MaxVel and t,MaxVel shifted towards normal in a dose-dependent way. Data suggest that the whole blood coagulation TEG profile, following activation with minute amounts of TF, may reflect the hemostatic potential in patients suspected of impaired hemostasis.

Topics: Adult; Blood Coagulation; Factor VII; Factor VIIa; Female; Fibrinolytic Agents; Hemophilia A; Hemostasis; Humans; Kinetics; Male; Recombinant Proteins; Sex Factors; Thrombelastography; Thromboplastin; Thrombosis

Recombinant coagulation factor VIIa (rFVIIa) is generally accepted for treatment of patients with inhibitor-complicated hemophilia. Recently, rFVIIa variants with a specific enhancement of the tissue factor (TF)-independent proteolytic activity have been described.. The procoagulant and [thrombin-activatable fibrinolysis inhibitor (TAFI)-dependent] antifibrinolytic potentials of two superactive rFVIIa variants were compared with those of wild-type rFVIIa in a hemophilic setting.. Clot lysis assays were performed in plasma from six patients with inhibitor-complicated hemophilia A or in antibody-induced factor VIII-deficient platelet-rich plasma in the presence of different concentrations of the rFVIIa variants.. In the plasma model, M298Q-rFVIIa had a moderately increased procoagulant and antifibrinolytic potential, whereas V158D/E296V/M298Q/K337A-rFVIIa had a strongly increased procoagulant and antifibrinolytic activity compared with wild-type rFVIIa. The increased antifibrinolytic potential of the rFVIIa variants was completely dependent on enhancement of TAFI activation. In the platelet-rich plasma model similar results were obtained. The presence of TF was mandatory for clot formation in the absence of exogenous rFVIIa. At lower concentrations of rFVIIa (wild-type or variants), clot formation did occur but was significantly slower when TF activity was blocked. At increasing concentrations of rFVIIa, clotting times were no longer dependent on TF. In conclusion, should a TF-independent mechanism be involved in the efficacy of rFVIIa in patients with hemophilia, the superactive rFVIIa variants studied here might be clinically advantageous, as both procoagulant and antifibrinolytic potencies are significantly enhanced compared with those of wild-type rFVIIa. This ought to result in more efficient cessation of bleeding episodes and reduced risk of rebleeding.

Topics: Antifibrinolytic Agents; Blood Coagulation; Blood Coagulation Disorders; Blood Coagulation Tests; Blood Platelets; Carboxypeptidase B2; Coagulants; Dose-Response Relationship, Drug; Factor VII; Factor VIIa; Hemophilia A; Hemorrhage; Humans; In Vitro Techniques; Plasma; Recombinant Proteins; Thromboplastin

Recombinant factor VIIa (rFVIIa) is a novel prohemostatic drug for patients with hemophilia who have developed inhibitory antibodies. The postulation has been made that hemophilia is not only a disorder of coagulation, but that hyperfibrinolysis due to a defective activation of thrombin activatable fibrinolysis inhibitor (TAFI) might also play a role. In this in vitro study, the potential of rFVIIa to down-regulate fibrinolysis via activation of TAFI was investigated. rFVIIa was able to prolong clot lysis time in plasmas from 17 patients with severe hemophilia A. The prolongation of clot lysis time by rFVIIa was completely abolished by addition of an inhibitor of activated TAFI. The concentration of rFVIIa required for half maximal prolongation of clot lysis time (C(lys 1/2)-VIIa) varied widely between patients (median, 73.0 U/mL; range, 10.8-250 U/mL). The concentration of rFVIIa required for half maximal reduction of clotting time (C(clot 1/2)-VIIa) was approximately 10-fold lower than the C(lys 1/2)-VIIa value (median, 8.4 U/mL; range, 1.7-22.5 U/mL). Inhibition of TFPI with a polyclonal antibody significantly decreased C(lys 1/2)-VIIa values (median, 2.6 U/mL; range, 0-86.9 U/mL), whereas C(clot 1/2)-VIIa values did not change (median, 7.2 U/mL; range, 2.2-22.5 U/mL). On addition of 100 ng/mL recombinant full-length TFPI, a nonsignificant increase of C(lys 1/2)-VIIa values was observed (median, 119.2 U/mL; range, 12.3-375.0 U/mL), whereas C(clot 1/2)-VIIa values did not change (median, 8.8 U/mL; range, 2.6-34.6 U/mL). In conclusion, this study shows that rFVIIa both accelerates clot formation and inhibits fibrinolysis by activation of TAFI in factor VIII-deficient plasma. However, a large variability in antifibrinolytic potential of rFVIIa exists between patients.

Topics: Antibodies; Blood Coagulation; Blood Proteins; Carboxypeptidase B2; Factor VIIa; Factor VIII; Fibrinolysis; Fibrinolytic Agents; Hemophilia A; Humans; Lipoproteins; Recombinant Proteins; Thromboplastin; Time Factors

The ability of factor VIIa to initiate thrombin generation and clot formation in blood from healthy donors, blood from patients with hemophilia A, and in anti-factor IX antibody-induced ("acquired") hemophilia B blood was investigated. In normal blood, both factor VIIa-tissue factor (TF) complex and factor VIIa alone initiated thrombin generation. The efficiency of factor VIIa was about 0.0001 that of the factor VIIa-TF complex. In congenital hemophilia A blood and "acquired" hemophilia B blood in vitro, addition of 10 to 50 nM factor VIIa (pharmacologic concentrations) corrected the clotting time at all TF concentrations tested (0-100 pM) but had little effect on thrombin generation. Fibrinopeptide release and insoluble clot formation were only marginally influenced by addition of factor VIIa. TF alone had a more pronounced effect on thrombin generation; an increase in TF from 0 to 100 pM increased the maximum thrombin level in "acquired" hemophilia B blood from 120 to 480 nM. Platelet activation was considerably enhanced by addition of factor VIIa to both hemophilia A blood and "acquired" hemophilia B blood. Thus, pharmacologic concentrations of factor VIIa cannot restore normal thrombin generation in hemophilia A and hemophilia B blood in vitro. The efficacy of factor VIIa (10-50 nM) in hemophilia blood is dependent on TF.

Topics: Adult; Blood Coagulation; Blood Coagulation Tests; Case-Control Studies; Dose-Response Relationship, Drug; Factor VIIa; Fibrinogen; Hemophilia A; Hemophilia B; Humans; Kinetics; Male; Models, Biological; Thrombin; Thromboplastin

Topics: Blood Platelets; Dose-Response Relationship, Drug; Factor VIIa; Hemophilia A; Humans; Platelet Activation; Thromboplastin

Recombinant factor VIII and factor IX concentrates, human-plasma-derived albumin, and samples from previously untreated patients with hemophilia were examined for the presence of TT virus (TTV) by using polymerase chain reaction testing. Blood samples from the patients were obtained prospectively before and every 3 to 6 months after therapy was begun. TTV was detected in 23.5% of the recombinant-product lots and 55.5% of the albumin lots tested. Only first-generation factor VIII recombinant concentrates stabilized with human albumin were positive for TTV, whereas all second-generation (human protein-free) concentrates were negative for the virus. In 59% of patients treated with either first- or second-generation recombinant factor concentrates, TTV infection developed at some point after the initial infusion. Infection with TTV in these patients before and after treatment did not appear to be clinically important. Thus, first-generation recombinant factor VIII concentrates may contain TTV and the source of the viral contamination may be human albumin.

Topics: Blood Transfusion; DNA, Viral; Drug Contamination; Hemophilia A; Hemophilia B; Humans; Recombinant Proteins; Thromboplastin; Torque teno virus

TAFI (thrombin activatable fibrinolysis inhibitor) down regulates fibrinolysis after activation by relatively high concentrations of thrombin generated during coagulation via thrombin mediated factor XI activation and subsequent activation of the intrinsic pathway. It is this secondary burst of thrombin that is severely diminished in haemophilia A, a deficiency of coagulation factor VIII. We therefore investigated the role of TAFI in haemophilia A by measuring the clot lysis times of tissue factor induced fibrin formation and tPA mediated fibrinolysis. In haemophilia A plasma clot lysis times were normal at relatively high tissue factor concentrations but severely decreased at moderate to low tissue factor concentrations, indicating that the thrombin generation via the extrinsic pathway was insufficient to activate TAFI. Addition of factor VIII, TAFI or thrombomodulin restored the clot lysis times at low tissue factor concentrations. This confirms the hypothesis that the bleeding disorder in haemophilia A is not merely a defect in the initial clot formation but is in fact a triple defect: reduced thrombin formation via the extrinsic pathway at low tissue factor concentrations, a reduced secondary burst of thrombin generation via the intrinsic pathway and a defective down regulation of the fibrinolytic system by the intrinsic pathway.

Topics: Adolescent; Adult; Biomarkers; Blood Coagulation; Blood Coagulation Tests; Carboxypeptidase B2; Child; Factor VIII; Fibrinolysis; Hemophilia A; Humans; Male; Peptide Fragments; Prothrombin; Thrombin; Thrombomodulin; Thromboplastin; Time Factors

Factor VII circulates as a single chain inactive zymogen (10 nmol/L) and a trace ( approximately 10-100 pmol/L) circulates as the 2-chain form, factor VIIa. Factor VII and factor VIIa were studied in a coagulation model using plasma concentrations of purified coagulation factors with reactions initiated with relipidated tissue factor (TF). Factor VII (10 nmol/L) extended the lag phase of thrombin generation initiated by 100 pmol/L factor VIIa and low TF. With the coagulation inhibitors TFPI and AT-III present, factor VII both extended the lag phase of the reaction and depressed the rate of thrombin generation. The inhibition of factor Xa generation by factor VII is consistent with its competition with factor VIIa for TF. Thrombin generation with TF concentrations >100 pmol/L was not inhibited by factor VII. At low tissue factor concentrations (<25 pmol/L) thrombin generation becomes sensitive to the absence of factor VIII. In the absence of factor VIII, factor VII significantly inhibits TF-initiated thrombin generation by 100 pmol/L factor VIIa. In this hemophilia A model, approximately 2 nmol/L factor VIIa is needed to overcome the inhibition of physiologic (10 nmol/L) factor VII. At 10 nmol/L, factor VIIa provided a thrombin generation response in the hemophilia model (0% factor VIII, 10 nmol/L factor VII) equivalent to that observed with normal plasma, (100% factor VIII, 10 nmol/L factor VII, 100 pmol/L factor VIIa). These results suggest that the therapeutic efficacy of factor VIIa in the medical treatment of hemophiliacs with inhibitors is, in part, based on overcoming the factor VII inhibitory effect. (Blood. 2000;95:1330-1335)

Topics: Anticoagulants; Antithrombin III; Factor VII; Factor VIIa; Factor Xa; Hemophilia A; Humans; Kinetics; Lipoproteins; Thrombin; Thromboplastin

Recombinant factor VIIa (rVIIa) has been reported to be clinically effective and safe in haemophilic patients with inhibitor antibodies. Compared to activated prothrombin complex concentrates the risk of thrombotic complications seems to be very low after rVIIa administration. Determination of free thrombin generation has been shown to identify hypercoagulability. Therefore, free thrombin and prothrombinase activity (Xa generation) were assessed after extrinsic activation of rVIIa supplemented factor VIII and factor IX deficient plasma. Free thrombin generation was also determined after supplementation of (activated) prothrombin complex concentrates. Addition of 150 U rVIIa/ml shortened the clotting times markedly in control, factor VIII, and factor IX deficient plasma. In contrast, free thrombin and Xa generation were not different in the absence or presence of 150 U rVIIa/ml. Addition of (activated) prothrombin complex concentrates resulted in a marked increase of free thrombin generation in all investigated plasmas. Although in vitro studies cannot reflect specific clinical circumstances our results support the notion that rVIIa does not induce a hypercoagulable state as sporadically observed after administration of (activated) prothrombin complex concentrates.

Topics: Blood Coagulation; Blood Coagulation Factors; Factor VIIa; Factor Xa; Hemophilia A; Hemophilia B; Humans; Platelet Activation; Prothrombin; Recombinant Fusion Proteins; Thrombin; Thromboplastin

The rate of conversion of fibrinogen (Fg) to the insoluble product fibrin (Fn) is a key factor in hemostasis. We have developed methods to quantitate fibrinopeptides (FPs) and soluble and insoluble Fg/Fn products during the tissue factor induced clotting of whole blood. Significant FPA generation (>50%) occurs prior to visible clotting (4 +/- 0.2 min) coincident with factor XIII activation. At this time Fg is mostly in solution along with high molecular weight cross-linked products. Cross-linking of gamma-chains is virtually complete (5 min) prior to the release of FPB, a process that does not occur until after clot formation. FPB is detected still attached to the beta-chain throughout the time course demonstrating release of only low levels of FPB from the clot. After release of FPB a carboxypeptidase-B-like enzyme removes the carboxyl-terminal arginine resulting exclusively in des-Arg FPB by the 20-min time point. This process is inhibited by epsilon-aminocaproic acid. These results demonstrate that transglutaminase and carboxypeptidase enzymes are activated simultaneously with Fn formation. The initial clot is a composite of Fn I and Fg already displaying gamma-gamma cross-linking prior to the formation of Fn II with Bbeta-chain remaining mostly intact followed by the selective degradation of FPB to des-Arg FPB.

Topics: Adolescent; Adult; Amino Acid Sequence; Aminocaproic Acid; Blood Coagulation; Carboxypeptidase B; Carboxypeptidases; Enzyme Activation; Factor XIII; Fibrin; Fibrinogen; Fibrinopeptide A; Fibrinopeptide B; Hemophilia A; Humans; Models, Biological; Molecular Sequence Data; Protease Inhibitors; Protein Conformation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thromboplastin; Transglutaminases

Tissue factor (TF)-induced coagulation was compared in contact pathway suppressed human blood from normal, factor VIII-deficient, and factor XI-deficient donors. The progress of the reaction was analyzed in quenched samples by immunoassay and immunoblotting for fibrinopeptide A (FPA), thrombin-antithrombin (TAT), factor V activation, and osteonectin. In hemophilia A blood (factor VIII:C <1%) treated with 25 pmol/L TF, clotting was significantly delayed versus normal, whereas replacement with recombinant factor VIII (1 U/mL) restored the clot time near normal values. Fibrinopeptide A release was slower over the course of the experiment than in normal blood or hemophilic blood with factor VIII replaced, but significant release was observed by the end of the experiment. Factor V activation was significantly impaired, with both the heavy and light chains presenting more slowly than in the normal or replacement cases. Differences in platelet activation (osteonectin release) between normal and factor VIII-deficient blood were small, with the midpoint of the profiles observed within 1 minute of each other. Thrombin generation during the propagation phase (subsequent to clotting) was greatly impaired in factor VIII deficiency, being depressed to less than 1/29 (<1.9 nmol TAT/L/min) the rate in normal blood (55 nmol TAT/L/min). Replacement with recombinant factor VIII normalized the rate of TAT generation. Thus, coagulation in hemophilia A blood at 25 pmol/L TF is impaired, with significantly slower thrombin generation than normal during the propagation phase; this reduced thrombin appears to affect FPA production and factor V activation more profoundly than platelet activation. At the same level of TF in factor XI-deficient blood (XI:C <2%), only minor differences in clotting or product formation (FPA, osteonectin, and factor Va) were observed. Using reduced levels of initiator (5 pmol/L TF), the reaction was more strongly influenced by factor XI deficiency. Clot formation was delayed from 11.1 to 15.7 minutes, which shortened to 9.7 minutes with factor XI replacement. The maximum thrombin generation rate observed ( approximately 37 nmol TAT/L/min) was approximately one third that for normal (110 nmol/L TAT/min) or with factor XI replacement (119 nmol TAT/L/min). FPA release, factor V activation, and release of platelet osteonectin were slower in factor XI-deficient blood than in normal blood. The data demonstrate that factor XI deficiency results in significantl

Topics: Adolescent; Adult; Blood Coagulation; Factor XI Deficiency; Hemophilia A; Humans; Immunoblotting; Male; Middle Aged; Thromboplastin

With the use of the principal neutralizing determinant (PND) peptide-based ELISA to measure anti-PND antibodies that specifically bound synthetic peptides derived from HIVIIIB, HIVMN, HIVRF, HIVSC, HIVWJM-2, HIVAf1l.con, or HIVAf2.con, type-specific antibodies to the HIVMN peptide were studied in 350 serum specimens from Japanese with hemophilia A who had been injected with known unheated factor VIII concentrates until 1985 and had been infected with HIV-1 subtype B. These antibodies were not found in any of the seronegative sera of hemophiliacs, patients with autoimmune diseases, or normal healthy controls. Further, all hemophiliacs rapidly progressing to AIDS and death among the 95 hemophiliacs in a restricted Nara area had antibody titers of less than 20 and their low levels preceded the rapid progression to the disease state. In contrast, slowly progressing hemophiliacs maintained an antibody titer of more than 100 from the initial stages of viral infection and remained asymptomatic. Sequence analysis of the V3 regions of HIV-1 indicated that the hemophiliacs who maintained a high anti-PNDMN antibody level showed a conserved MN sequence. In contrast, the HIV-infected hemophiliacs with nonreactivity in the ELISA showed sequence changes in the neutralizing epitopes of HIVMN. The dynamic of the serum anti-PNDMN antibody titer appear to be a characteristic indicator of the progression of the HIV-infected status in Japanese hemophiliacs seropositive for HIV-1.

Topics: Adult; Amino Acid Sequence; Consensus Sequence; Disease Progression; Enzyme-Linked Immunosorbent Assay; Female; Hemophilia A; HIV Antibodies; HIV Envelope Protein gp120; HIV Infections; HIV-1; Humans; Japan; Male; Middle Aged; Molecular Sequence Data; Neutralization Tests; Peptide Fragments; Prevalence; Thromboplastin

The classification of factor VIII deficiency, generally used based on plasma levels of factor VIII, consists of severe (<1% normal factor VIII activity), moderate (1% to 4% factor VIII activity), or mild (5% to 25% factor VIII activity). A recent communication described four individuals bearing identical factor VIII mutations. This resulted in a severe bleeding disorder in two patients who carried a normal factor V gene, whereas the two patients who did not display severe hemophilia were heterozygous for the factor V(LEIDEN) mutation, which leads to the substitution of Arg506 --> Gln mutation in the factor V molecule. Based on the factor VIII level measured using factor VIII-deficient plasma, these two patients were classified as mild/moderate hemophiliacs. We studied the condition of moderate to severe hemophilia A combined with the factor V(LEIDEN) mutation in vitro in a reconstituted model of the tissue factor pathway to thrombin. In the model, thrombin generation was initiated by relipidated tissue factor and factor VIIa in the presence of the coagulation factors X, IX, II, V, and VIII and the inhibitors tissue factor pathway inhibitor, antithrombin-III, and protein C. At 5 pmol/L initiating factor VIIa x tissue factor, a 10-fold higher peak level of thrombin formation (350 nmol/L), was observed in the system in the presence of plasma levels of factor VIII compared with reactions without factor VIII. Significant increase in thrombin formation was observed at factor VIII concentrations less than 42 pmol/L (approximately 6% of the normal factor VIII plasma concentration). In reactions without factor VIII, in which thrombin generation was downregulated by the addition of protein C and thrombomodulin, an increase of thrombin formation was observed with the factor V(LEIDEN) mutation. The level of increase in thrombin generation in the hemophilia A situation was found to be dependent on the factor V(LEIDEN) concentration. When the factor V(LEIDEN) concentration was varied from 50% to 150% of the normal plasma concentration, the increase in thrombin generation ranged from threefold to sevenfold. The data suggested that the analysis of the factor V genotype should be accompanied by a quantitative analysis of the plasma factor V(LEIDEN) level to understand the effect of factor V(LEIDEN) in hemophilia A patients. The presented data support the hypothesis that the factor V(LEIDEN) mutation can increase thrombin formation in severe hemophilia A.

Topics: Factor V; Factor VIIa; Factor VIII; Hemophilia A; Humans; In Vitro Techniques; Models, Biological; Mutation; Protein C; Thrombin; Thromboplastin

Current therapies for treatment of hemophilia A involve infusion of factor VIII, but are ineffective for patients who develop inhibitory antibodies. We have previously proposed that bypassing the intrinsic pathway (VIIIa/IXa) with reversibly acylated factor Xa offers an improvement on existing therapies as it provides a time-dependent release of procoagulant activity without the addition of factors VIII or IX. The present study was designed to determine the effect of substituted 4-amidinophenyl benzoates on the acylation of factor Xa, as well as the subsequent deacylation rates of the resulting acyl Xa. A subset of this series of acyl Xa's were incorporated into the prothrombinase complex and recovery of catalytic activity was measured by activation of prothrombin to thrombin. Similarly, some acyl Xa's were also evaluated for their capacity to enhance clotting times of human plasma. Our study indicates that by choosing the appropriate acyl Xa, the time course of factor Xa regeneration can be modulated extensively. Animal studies will be required to show that the use of acyl Xa as a procoagulant agent is feasible in an in vivo system.

Topics: Acylation; Animals; Benzoates; Blood Coagulation; Blood Coagulation Factors; Factor Xa; Hemophilia A; Humans; In Vitro Techniques; Indicators and Reagents; Kinetics; Thromboplastin

Topics: Animals; Factor VIIa; Hemophilia A; Hemostasis; Humans; Recombinant Proteins; Thromboplastin

Thrombin activation of the soluble plasma protein fibrinogen is vital for successful haemostasis. Thrombin is generated from prothrombin by the prothrombinase complex which also includes factor Xa, factor Va, Ca2+ and a procoagulant membrane surface. Factor X activation is catalysed in a complex including either factor VIIa and tissue factor, or factor IXa and factor VIIIa. Factor IXa can be generated either by the factor VIIa/tissue factor complex or by factor XIa which is in turn produced by the contact phase reactions in vitro. Once activated, fibrinogen develops into the fibrin polymeric matrix at the site of injury. It is not known to what extent the properties of this haemostatic plug are sensitive to the pathway leading up to thrombin generation. Here static human plasma is studied in vitro using magnetically induced birefringence. It is shown that the contact phase/factor XIa pathway gives rise to linear fibrin assembly process curves whereas the factor VIIa/ tissue factor activation of factor X provokes largely sigmoid assembly. The latter pathway also causes the formation of significantly thicker fibres even though assembly is more rapid. This result is the inverse of that anticipated from the study of simple model systems. Whilst the streptokinase activated lysis both types of clot exhibits similar biphasic kinetics, an exponential main phase followed by a sigmoidal tailing off, the data suggest that clots produced by the contact phase/factor XIa pathway are more recalcitrant to lysis. These results demonstrate that the profile of thrombin generation not only determines the kinetics of assembly but also influences the rate of lysis and structure of the haemostatic plug.

Topics: Birefringence; Blood Coagulation; Blood Coagulation Factors; Blood Coagulation Tests; Enzyme Activation; Fibrin; Fibrinogen; Fibrinolysis; Gels; Hemophilia A; Humans; Myocardial Infarction; Plasma; Prothrombin; Thrombin; Thromboplastin

Topics: Animals; Aprotinin; Blood Coagulation Tests; Factor VIIa; Factor X; Hemophilia A; Humans; Reference Values; Thromboplastin

Several enzymes can activate factor VII in vitro, but the protease responsible for generating factor VIIa in vivo has not been determined. Using recombinant tissue factor that has undergone a COOH-terminal truncation, a sensitive functional assay has been established for measuring plasma factor VIIa levels. To evaluate the mechanism responsible for the generation of factor VIIa in vivo, we measured the levels of this enzyme after administering purified concentrates of factor IX and factor VIII to patients with severe deficiencies of these clotting factors. In patients with hemophilia B, factor VIIa levels were initially reduced to 0.5 +/- 0.1 ng/mL and gradually increased to normal after infusing 100 U/kg of body weight (BW) of factor IX. Despite these increases, there were no significant changes in the generation of factor Xa or thrombin. In patients with hemophilia A, only a slight reduction in factor VIIa levels (2.5 +/- 1.3 ng/mL) was observed as compared with controls (3.3 +/- 1.1 ng/mL) and no significant changes were observed after factor VIII levels were normalized. The administration of recombinant factor VIIa (10 micrograms/kg BW) to patients with factor VII deficiency increased the mean circulating level of the enzyme to 118 ng/mL, but this only resulted in normalization of the levels of the activation peptides of factor IX and factor X. The above data indicate that factor IXa is primarily responsible for the basal levels of free factor VIIa generated in vivo (ie, in the absence of thrombosis or provocative stimuli) and that changes in the plasma concentrations of free factor VIIa in the blood do not necessarily lead to alterations in the extent of factor X activation.

Topics: Adolescent; Adult; Enzyme Activation; Factor IX; Factor IXa; Factor VIIa; Factor VIII; Factor Xa; Hemophilia A; Hemophilia B; Humans; Middle Aged; Recombinant Proteins; Thrombin; Thromboplastin

Elevation of cytoplasmic Ca2+ levels in human erythrocytes induces a progressive loss of membrane phospholipid asymmetry, a process that is impaired in erythrocytes from a patient with Scott syndrome. We show here that porcine erythrocytes are similarly incapable of Ca2+-induced redistribution of membrane phospholipids. Because a complex of phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ has been proposed as the mediator of enhanced transbilayer movement of lipids (J Biol Chem 269:6347,1994), these cell systems offer a unique opportunity for testing this mechanism. Analysis of both total PIP2 content and the metabolic-resistant pool of PIP2 that remains after incubation with Ca2+ ionophore showed no appreciable differences between normal and Scott erythrocytes. Moreover, porcine erythrocytes were found to have slightly higher levels of both total and metabolic-resistant PIP2 in comparison with normal human erythrocytes. Although loading of normal erythrocytes with exogenously added PIP2 gave rise to a Ca2+-induced increase in prothrombinase activity and apparent transbilayer movement of nitrobenzoxadiazolyl (NBD)-phospholipids, these PIP2-loaded cells were also found to undergo progressive Ca2+-dependent cell lysis, which seriously hampers interpretation of these data. Moreover, loading Scott cells with PIP2 did not abolish their impaired lipid scrambling, even in the presence of a Ca2+-ionophore. Finally, artificial lipid vesicles containing no PIP2 or 1 mole percent of PIP2 were indistinguishable with respect to transbilayer movement of NBD-phosphatidylcholine in the presence of Ca2+. Our findings suggest that Ca2+-induced redistribution of membrane phospholipids cannot simply be attributed to the steady-state concentration of PIP2, and imply that such lipid movement is regulated by other cellular processes.

Topics: 4-Chloro-7-nitrobenzofurazan; Calcium; Erythrocyte Membrane; Erythrocytes; Hemophilia A; Humans; In Vitro Techniques; Kinetics; Lipid Bilayers; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phospholipids; Reference Values; Syndrome; Thromboplastin

Tissue factor (TF)/FVIIa initiates coagulation by activating factor IX (FIX) and factor X (FX). Tissue factor pathway inhibitor (TFPI)-FXa complexes form and inhibit TF/FVIIa. Blocking of TFPI may facilitate haemostasis initiated by FVIIa/TF thereby compensating for impaired FIX/FVIII-dependent coagulation. This hypothesis was tested in a study using rabbits made temporarily haemophilic by the injection of antibodies against FVIII. These rabbits were given i.v. injections of anti-TFPI IgG antibodies and 40 min later bleeding was initiated by cutting the nail including the apex of the cuticle. Injection of anti-TFPI IgG shortened the bleeding time significantly from 26 min to 11 min (normal mean bleeding time in non-haemophilia rabbits: 5 min). Treatment with anti-TFPI IgG also resulted in a shortening of the haemophilic aPTT to a level slightly longer than the normal aPTT. In addition, the prolonged dilute TF clotting time was shortened by the anti-TFPI IgG treatment. Thus, both bleeding and coagulation parameters indicated that blocking of TFPI may be potentially haemostatic in haemophilia.

Topics: Animals; Antibodies; Antibody Specificity; Bleeding Time; Blood Coagulation; Disease Models, Animal; Factor VIII; Female; Hemophilia A; Immunoglobulin G; Lipoproteins; Partial Thromboplastin Time; Rabbits; Thromboplastin

The synthesis of tissue factor pathway inhibitor (TFPI) was investigated in cloned human synovial cells and human chondrocytes. TFPI-specific DNA transcription products of these cells were isolated, and a full-length cDNA of about 1000 base pairs was amplified by reverse transcription and polymerase chain reaction. The amplified DNA was cloned into the vector pUC 18. The TFPI coding sequence was confirmed by double stranded sequencing and was identical with that previously published for human TFPI coding nucleotide sequence from human placental cDNA (1). The inhibitory activity of TFPI in the cell medium of cultivated human chondrocytes and cloned human synovial cells was determined by a specific chromogenic substrate assay of factor Xa activity. The inhibitory activity of TFPI in the medium of human chondrocytes and cloned human synovial cells was 630-720 mU/10(8) cells and 1080-1665 mU/10(8) cells, respectively. In addition, TFPI activity in cell culture media of human chondrocytes and cloned human synovial cell was suppressed by a polyclonal goat anti-TFPI antibody directed against the inhibitory domain I and domain II. In the chromogenic substrate assay, the anti-TFPI antibody completely suppressed the inhibitory activity of TFPI in the samples.

Topics: Amino Acid Sequence; Antibodies; Antibody Specificity; Base Sequence; Cartilage; Cells, Cultured; DNA, Complementary; Factor VII; Hemophilia A; Hemorrhage; Humans; Lipoproteins; Molecular Sequence Data; RNA; Synovial Membrane; Thromboplastin; Transcription, Genetic

Topics: Animals; Aprotinin; Blood Coagulation Tests; Factor VIIa; Hemophilia A; Humans; Reference Values; Thromboplastin

Procoagulant albumin (P-Al) is present in normal human plasma and increases monocyte and endothelial cell expression of tissue factor activity. To develop a bioassay for P-Al, we partially purified plasma from healthy volunteers and several patient groups using BaCl2 and (NH4)2SO4 precipitation. The samples were assayed for tissue factor (TF) inducing activity, expressed as a percentage increase compared to a serum-free media control. Over six months, the assay was reproducible in stored samples and in serial samples from normal volunteers. The plasma P-Al activities of 35 volunteers averaged 141 +/- 8.2% (SEM). There was no diurnal variation. There was no difference in the P-Al activity after a 12 hour fast and 2 hours after a large meal in 4 healthy volunteers. There was no increase in activity (r = 0.16) with the subject's age. The average activity from 16 poorly-controlled diabetics was 131 +/- 11% (SEM). No alteration in activity was seen with samples from patients with uremia, liver dysfunction, hemophilia, thrombotic events, or adenocarcinoma. These results indicate that P-Al activity can be bioassayed in individual patient samples; however, pathologic states associated with abnormal P-Al-induced tissue factor activity presently remain unidentified.

Topics: Adult; Aged; Aged, 80 and over; Blood Coagulation; Blood Coagulation Factors; Cells, Cultured; Diabetes Mellitus; Endothelium, Vascular; Hemophilia A; Humans; Middle Aged; Pulmonary Edema; Serum Albumin; Thromboplastin; Thrombosis

A seventeen-year-old man with hemophilia A developed nausea, vomiting, and unsteady gait after mild head trauma. Magnetic resonance imaging clearly demonstrated localized bleeding in cerebellar vermis. Quick administration of factor VIII concentrates prevented further extension of the bleeding and the patient completely recovered without neurologic impairment. In hemophiliac patients, careful evaluation of intracranial lesions is desired after head trauma even if they show only nonspecific symptoms.

Topics: Adolescent; Cerebellar Diseases; Craniocerebral Trauma; Hemophilia A; Hemorrhage; Humans; Magnetic Resonance Imaging; Male; Thromboplastin; Tomography, X-Ray Computed

High doses of recombinant factor VIIa are useful in managing bleeding in hemophiliacs with inhibitors. Whether this therapeutic effect of factor VIIa is dependent on tissue factor (TF) is a matter of debate. We examined the ability of freshly isolated human monocytes (which lack TF) to support the activation of coagulation-factor X by factor VIIa. The rate of factor-X activation by factor VIIa was accelerated in the presence of monocytes compared with the rate of X activation in solution. This activation of factor X on monocytes was saturable with a K1/2 of about 400 to 600 pmol/L factor VIIa. The rate of activation was not inhibited by an excess of inhibitory anti-TF antibody or a Gla-containing fragment of prothrombin. In contrast to monocytes, an endothelial cell line did not support activation of factor X by factor VIIa. Our findings suggest that at least one cell type can accelerate activation for factor X by factor VIIa in the absence of TF. This activity requires higher concentrations of factor VIIa than does the TF mechanism. The concentrations of VIIa required are of a similar order of magnitude to those required for a therapeutic effect of VIIa in bleeding hemophiliacs with inhibitors.

Topics: Cells, Cultured; Factor VIIa; Factor X; Hemophilia A; Humans; Monocytes; Thromboplastin

An increasing amount of evidence suggests that coagulation factors VIII and IX play a role not only in the intrinsic but also in the extrinsic pathway of coagulation. In this context the influence of the Extrinsic Pathway Inhibitor (EPI) on the coagulation time of hemophilia plasma lacking FVIII or FIX has been investigated. The coagulation time was measured in a dilute thromboplastin assay. Addition of recombinant EPI (rEPI) prolonged the coagulation time of normal plasma while the addition of an inhibitory antibody against EPI shortened the coagulation time. At low concentrations of thromboplastin the coagulation time of hemophilia plasma was prolonged and at all dilutions of thromboplastin, addition of anti-EPI IgG normalized the coagulation time of a hemophilia plasma. Analysis of 10 individual donor plasma samples and 8 individual hemophilia samples showed that addition of anti-EPI IgG shortened the coagulation time more in hemophilia plasma than in normal plasma. This illustrates the importance of a powerful extrinsic FVII dependent pathway to achieve hemostasis in the case of FVIII or FIX deficiency (hemophilia A and B).

Topics: Antibody Specificity; Blood Coagulation; Factor VII; Hemophilia A; Humans; Immunoglobulin G; Lipoproteins; Partial Thromboplastin Time; Protease Inhibitors; Reference Values; Thromboplastin; Time Factors

Lipoprotein-associated coagulation inhibitor (LACI) is a plasma-derived protein that inhibits the tissue factor/factor VIIa/calcium/phospholipid complex in a factor Xa-dependent manner. Recombinant LACI (rLACI) added exogenously to plasma prolonged the activated partial thromboplastin time (APTT) and prothrombin time (PT) as a function of rLACI concentration in linear and curvilinear manners, respectively. Under these standard assay conditions, the amounts of rLACI required to double the APTT and PT were approximately 350- and 90-fold the plasma concentration of LACI, respectively. Likewise, addition of antibodies against LACI to pooled normal, factor VIII-deficient, or factor IX-deficient plasma had no effect on their respective APTTs and PTs, demonstrating the insensitivity of these assays to endogenous LACI. The prothrombin time assay was modified by using dilute thromboplastin. Unlike the standard prothrombin time assay, the clotting times were prolonged for factors VIII- or IX-deficient plasma relative to pooled normal plasma in this modified PT assay. Additionally, the degree of factor deficiency, as determined by the APTT assay, was correlated with that determined by the modified PT assay using dilute thromboplastin. When antibodies against LACI were added to pooled normal, factor VIII-deficient, or factor IX-deficient plasma and the prothrombin time assay initiated using dilute thromboplastin, the clotting times for antibody-treated plasma were shorter than for the corresponding plasma in the absence of antibodies. Moreover, the clotting times for factors VIII- and IX-deficient plasmas treated with antibodies raised against LACI were at least as fast as pooled normal plasma in the absence of LACI antibodies when dilute thromboplastin was used to initiate clotting. These results suggest that the prothrombin time assay using dilute thromboplastin may more accurately reflect what occurs in vivo and that LACI may play an important role in the prolonged bleeding of those with hemophilia A or B.

Topics: Antibodies, Monoclonal; Blood Coagulation; Factor VII; Hemophilia A; Humans; Lipoproteins; Partial Thromboplastin Time; Protease Inhibitors; Prothrombin Time; Recombinant Proteins; Reference Values; Thromboplastin

Factor IX Amagasaki (AMG) is a naturally occurring mutant of factor IX having essentially no coagulant activity, even though normal levels of antigen are detected in plasma. Factor IX AMG was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Factor IX AMG was cleaved normally by factor VIIa-tissue factor complex, yielding a two-chain factor IXa. Amino acid composition and sequence analysis of one of the tryptic peptides isolated from factor IX AMG revealed that Gly-311 had been replaced by Glu. We identified a one-base substitution of guanine to adenine in exon VIII by amplifying exon VIII using the polymerase chain reaction method and sequencing the product. This base mutation also supported the replacement of Gly-311 by Glu. In the purified system, factor IXa AMG did not activate factor X in the presence of factor VIII, phospholipids, and Ca2+, and no esterase activity toward Z-Arg-p-nitrobenzyl ester was observed. The model building of the serine protease domain of factor IXa suggests that the Gly-311----Glu exchange would disrupt the specific conformational state in the active site environment, resulting in the substrate binding site not forming properly. This is the first report to show the experimental evidence for importance of a highly conserved Gly-142 (chymotrypsinogen numbering) located in the catalytic site of mammalian serine proteases so far known.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blood Coagulation; Cattle; DNA; Exons; Factor IXa; Factor VIII; Factor X; Hemophilia A; Humans; Male; Models, Molecular; Molecular Sequence Data; Mutation; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Protein Conformation; Sequence Homology, Nucleic Acid; Serine Endopeptidases; Thromboplastin; Trypsin

Factor VIII heavy chain (FVIII HC) polypeptides have been studied in both normal plasma and FVIII concentrates on exposure to three coagulation proteases. FVIII samples were incubated with labelled affinity-purified anti-FVIII Fab' fragments, immunocomplexes formed were visualized by autoradiography after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and apparent relative molecular masses (Mr) of each band assigned. FVIII HC polypeptides were detected in all types of samples, including plasma, without further purification. Normal plasma contained a range of polypeptides with the largest dominant band at a net apparent Mr of 250-300 kD, and the smallest at 80-90 kD: the bands visualized correspond to the 90-210 kD HC species seen on conventional analysis of purified FVIII. No bands were produced from samples of haemophilic plasma. Treatment of plasma or FVIII concentrate with low concentrations (1 IU/ml) of thrombin removed the 250-300 kD and other intermediate bands, intensified then removed the 80-90 kD polypeptide and produced a band at 40-50 kD. Thrombin-associated rise and fall in FVIII clotting activity by one-stage assay correlated with intensity of the 80-90 kD polypeptide. A polypeptide of Mr 40-50 kD was also produced after incubation with activated factor X: activated factor VII plus thromboplastin had no effect on HC structure. FVIII polypeptides were visualized in prothrombin complex concentrates, with a more degraded profile seen in a deliberately 'activated' product.

Topics: Biological Products; Factor VIIa; Factor Xa; Hemophilia A; Humans; Peptide Hydrolases; Peptides; Thrombin; Thromboplastin

The effect of factors VIII and IX on the ability of the tissue factor-factor VIIa complex to activate factor X was studied in a continuous-flow tubular enzyme reactor. Tissue factor immobilized in a phospholipid bilayer on the inner surface of the tube was exposed to a perfusate containing factors VIIa, VIII, IX, and X flowing at a shear rate of 57, 300, or 1130 sec-1. Factor Xa in the effluent was determined by chromogenic assay. The flux of factor Xa (moles formed per unit surface area per unit time) was strongly dependent on wall shear rate, increasing about 3-fold as wall shear rate increased from 57 to 1130 sec-1. The addition of factors VIII and IX at their respective plasma concentrations resulted in a further 2- to 3-fold increase. The direct activation of factor X by tissue factor-factor VIIa could be virtually eliminated by the lipoprotein-associated coagulation inhibitor; however, when factors VIII and IX were present at their approximate plasma concentrations, factor Xa production rates were enhanced 15- to 20-fold. These results suggest that the tissue factor pathway, mediated through factors VIII and IX, produces significant levels of factor Xa even in the presence of an inhibitor of the tissue factor-factor VIIa complex; moreover, the activation is dependent on local shear conditions. These findings are consistent both with a model of blood coagulation in which initiation of the system results from tissue factor and with the bleeding observed in hemophilia.

Topics: Blood Coagulation; Factor IX; Factor VIIa; Factor VIII; Factor X; Factor Xa; Hemophilia A; Humans; Kinetics; Lipid Bilayers; Models, Biological; Recombinant Proteins; Thromboplastin

Hemophilia Bm is characterized by a strikingly prolonged plasma ox brain prothrombin time. In an attempt to find an explanation for this phenomenon we have analyzed various aspects of the Bm variants factor IX Deventer, factor IX Milano, factor IX Novara, and factor IX Bergamo. Proteolytic cleavage by factor XIa was normal in two Bm variants, but absent at the Arg180-Val bond in the other two. In the latter variants Arg180 was replaced by either Trp or Gln, whereas Val181----Phe and Pro368----Thr replacements have occurred in the variants that were normally cleaved by factor XIa. In all four variants the Bm effect could be neutralized with a single monoclonal antibody against factor IX. Also, after treatment with factor XIa, none of the Bm variants reacted with antithrombin III (in contrast to normal factor IXa). Purified factor IX Deventer (one of the variants with a replacement of Arg181), either with or without pretreatment with factor XIa, was found to be a more effective competitive inhibitor of the factor VIIa-tissue factor-induced factor X activation than similarly treated normal factor IX. In addition, this inhibitory effect was much more pronounced when bovine tissue factor was used instead of human tissue factor. We propose that the normal activation of factor IX not only produces a conformational change around the active site serine that allows efficient substrate binding and catalysis, but that the same conformational change is instrumental in effectively dissociating factor IXa from the activating factor VIIa-tissue factor complex. Amino acid replacements that disrupt this conformational transition directly (e.g. Pro368----Thr near the catalytic center) or indirectly (mutations at the Arg180-Val activation site) therefore lead to a combination of 1) the loss of coagulant activity and 2) an inhibitory effect in the ox brain prothrombin time assay.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Arginine; Binding Sites; Binding, Competitive; Chymotrypsin; DNA Restriction Enzymes; Factor IX; Factor VIIa; Factor X; Factor XIa; Hemophilia A; Humans; Molecular Sequence Data; Molecular Weight; Mutation; Protein Conformation; Prothrombin Time; Thromboplastin; Valine

Infusing factor VIIa (FVIIa) has been reported to control bleeding in hemophilic patients with factor VIII (FVIII) inhibitors. This is difficult to attribute to an enhanced FVIIa/tissue factor (TF) activation of factor X, since in vitro studies suggest that infusion of FVIIa should neither increase substantially the rate of formation of FVIIa/TF complexes during hemostasis (Proc Natl Acad Sci USA 85:6687, 1988) nor bypass the dampening of TF-dependent coagulation by the extrinsic pathway inhibitor (EPI) (Blood 73:359, 1989). Partial thromboplastin times have also been reported to shorten after infusion of FVIIa. The experiments reported herein establish that shortening of partial thromboplastin times after adding FVIIa to hemophilic plasma in vitro stems from an FVIIa-catalyzed activation of factor X independent of possible trace contamination of reagents with TF. Experiments in purified systems confirmed that FVIIa can slowly activate factor X in a reaction mixture containing Ca2+ and phospholipid but no source of TF. The rate of activation was sufficient to account for the shortening of partial thromboplastin times observed. EPI, which turned off continuing FVIIa/TF activation of factor X, was unable to prevent continuing FVIIa/phospholipid activation of factor X. Because circulating plasma contains only a trace, if any, free FVIIa, such a reaction could never occur physiologically. However, infusing FVIIa creates a nonphysiologic circumstance in which a continuing slow FVIIa/phospholipid catalyzed activation of factor X could conceivably proceed in vivo unimpeded by EPI. Such a mechanism of factor X activation might compensate for an impaired factor IXa/FVIIIa/phospholipid activation of factor X during hemostatis, and therefore control bleeding in a hemophilic patient.

Topics: Blood Coagulation; Enzyme Activation; Factor VIIa; Factor X; Hemophilia A; Humans; In Vitro Techniques; Partial Thromboplastin Time; Phospholipids; Thromboplastin

We report a study on the importance of factor IX activation in thromboplastin-dependent coagulation in plasma. Diluted, CaCl2-containing thromboplastin solutions at constant phospholipid concentration were used to trigger the coagulation in plasma from patients with congenital factor IX and factor VIII deficiency in the presence and absence of added factors IX and VIII, and the generation of thrombin activity was monitored. When coagulation is triggered with the high thromboplastin concentrations normally used in clinical routine tests, the generation of thrombin activity in plasma of patients with congenital factor IX deficiency before and after reconstitution with purified factor IX appears identical. When, however, coagulation is triggered with low thromboplastin concentrations, a clear dependency of the generation of thrombin activity on the concentration of factor IX becomes evident at factor IX concentrations lower than 30 nM (about 40% clotting factor activity). Factor VIII is a compulsory cofactor for this factor IX activity because the prothrombinase activity at optimal factor IX concentration is still critically dependent upon the amount of factor VIII present. The lower the amount of thromboplastin, the higher the importance of factor IX and factor VIII activation in thromboplastin-dependent coagulation. This suggests a role of this pathway in pathophysiological thrombin formation.

Topics: Blood Coagulation; Hemophilia A; Hemophilia B; Humans; Phospholipids; Thrombin; Thromboplastin

The bleeding disorder of hemophilia A currently treated by replacement therapy of the missing coagulation factor, factor VIII, is frequently complicated by the development of neutralizing antibodies. The therapeutic potential of attenuated forms of the lipid-associated glycoprotein tissue factor, a known initiator of coagulation, was investigated as a factor VIII-by-passing activity. The protein moiety of tissue factor (Apo-TF) was partially purified and exhibited minimal procoagulant activity before relipidation in vitro. In pilot studies, Apo-TF injection into rabbits previously anticoagulated with an antibody to factor VIII was found to have a procoagulant effect. The efficacy of the material was further demonstrated when injection of Apo-TF in hemophilic dogs resulted in a normalization of the cuticle bleeding time. Little or no change in the blood parameters associated with disseminated intravascular coagulation was observed at lower doses, although mild to moderate effects were seen at higher doses. These data suggest a novel role for Apo-TF preparations as a potential therapeutic agent for hemophiliacs with antibodies to factor VIII once the potential thrombogenicity of such materials is evaluated.

Topics: Animals; Blood Coagulation Tests; Cattle; Disease Models, Animal; Dogs; Factor IXa; Factor VIII; Hemophilia A; Phospholipids; Rabbits; Serine Endopeptidases; Thromboplastin

Topics: Freeze Drying; Hemophilia A; Humans; Oroantral Fistula; Placenta; Thromboplastin; Tooth Extraction

Disseminated intravascular coagulation invariably accompanies placement of peritoneovenous (LeVeen) shunts, which suggests that ascitic fluid contains procoagulant material capable of activating blood coagulation. In this study, we identified thrombogenic activity in human ascites and the hemostatic pathway by which it acts. Peritoneal fluid was removed percutaneously from patients with ascites due to various causes. Four fractions were prepared by centrifugation: cells, a low-speed, cell-free fluid, a high-speed supernatant, and the precipitate from the high-speed centrifugation. Cellular fractions from all ascitic fluids shortened a one-stage clotting time of normal pooled plasma by 68% in comparison with saline solution and endotoxin controls. Similarly, the cell-free fluids also shortened the clotting time of normal pooled plasma by 41%. The cellular and cell-free fractions shortened the clotting time of factor VIII-deficient plasma but failed to demonstrate procoagulant activity in factor VII-deficient plasma. These fractions had no effect on platelet aggregation or the platelet release reaction. The high-speed precipitate was dissociated by ethylenediaminetetra-acetate (EDTA) into fluid phase and precipitate, both of which demonstrated procoagulant activity. Furthermore, high-speed precipitate contained protein, phospholipid, and sterol in proportions similar to those of plasma membranes and contained membrane-bound vesicles as identified by means of electron microscopy. This material could be rendered inactive by heating to 100 degrees C for 2 minutes or by incubation with phospholipase C for 15 minutes. Finally, the ability of the high-speed precipitate to shorten the clotting time was prevented by preincubation with a monoclonal antibody, which is known to inhibit the procoagulant activity of human tissue factor. We suggest that several entities contribute to the procoagulant properties of human ascites, with procoagulant material deriving at least in part from peritoneal cells. The sedimentable procoagulant factor appears to be associated with cellular membranes or membrane fragments and is thromboplastin-like in its chemical composition, immunoreactivity, and substrate specificity.

Topics: Adult; Aged; Ascitic Fluid; Blood Coagulation Factors; Blood Coagulation Tests; Centrifugation; Factor VII Deficiency; Female; Hemophilia A; Humans; Leukocytes; Male; Microscopy, Electron; Middle Aged; Platelet Aggregation; Thromboplastin

Fibrin deposition and platelet thrombus dimensions on subendothelium were studied in four groups of patients with coagulation factor deficiencies. Five patients with factor VIII deficiency (APTT 120 +/- 8 sec) and three patients with factor IX deficiency (APTT 125 +/- 11 sec) were severe bleeders, whereas four patients with factor XII deficiency and seven with factor XI deficiency were either asymptomatic or only mild bleeders despite APTT values of 439 +/- 49 and 153 +/- 13 sec, respectively. Everted segments of deendothelialized rabbit aorta were exposed at a shear rate of 650 sec(-1) for 5 and 10 min to directly sampled venous blood in an annular chamber. Blood coagulation was evaluated by measuring fibrin deposition (percent surface coverage) on the subendothelium and post-chamber fibrinopeptide A levels; platelet thrombus dimensions on the subendothelium were evaluated by determining the total thrombus volume per surface area (using an optical scanning technique) and the average height of the three tallest thrombi. Consistent differences were observed among the patient groups for both the 5-min and 10-min exposure times. The larger of the 5- and 10-min exposure-time values was used to calculate group averages. Fibrin deposition in normal subjects was 81% +/- 5% surface coverage, and post-chamber fibrinopeptide A values were 712 +/- 64 ng/ml. Markedly decreased fibrin deposition and fibrinopeptide A levels were observed in factor VIII deficiency (2% +/- 1% and 102 +/- 19 ng/ml) and factor IX deficiency (11% +/- 7% and 69 +/- 11 ng/ml). In contrast, significantly higher values were obtained in patients deficient in factor XI (33% +/- 5% and 201 +/- 57 ng/ml) and factor XII (66% +/- 12% and 306 +/- 72 ng/ml). Differences in thrombus dimensions were also observed. In normal subjects, the value for thrombus volume and average height of the tallest thrombi were 8.3 +/- 1.3 cu micron/sq micron and 145 +/- 11 micron, respectively, and in patients were as follows: FVIII, 2.7 +/- 0.6 and 71 +/- 7; FIX, 4.5 +/- 1.8 and 88 +/- 14; FXI, 11.8 +/- 1.9 and 125 +/- 10; and FXII, 7.9 +/- 3.1 and 130 +/- 25. Platelet thrombus dimensions were normal in a patient with fibrinogen deficiency, indicating that the smaller thrombi in factor VIII and factor IX deficiencies were probably due to impaired evolution of thrombin rather than diminished fibrin formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Adolescent; Adult; Aged; Blood Coagulation Disorders; Blood Platelets; Endothelium; Factor XI Deficiency; Factor XII Deficiency; Female; Fibrin; Fibrinogen; Fibrinopeptide A; Hemophilia A; Hemophilia B; Humans; In Vitro Techniques; Male; Middle Aged; Partial Thromboplastin Time; Platelet Adhesiveness; Prothrombin Time; Thromboplastin

To study the interrelationships of the major human coagulation pathways, factor X activation in normal and various deficient human plasmas was evaluated when clotting was triggered by dilute rabbit or human thromboplastin. Various dilutions of thromboplastin were added to plasma samples containing 3H-labeled factor X, and the time course of factor X activation was determined. At a 1/250 dilution of rabbit brain thromboplastin the rate of factor X activation in factor VIII or factor IX deficient plasma was only 10% of the activation rate seen for normal or factor XI deficient plasma. Reconstitution of the deficient plasmas with factors VIII or IX, respectively, restored normal factor X activation. Similar results were obtained when various dilutions of human thromboplastin replaced the rabbit thromboplastin. From these experiments, it is inferred that normal activation of factor X in plasma due to dilute thromboplastin requires factors VII, IX and VIII. An alternative extrinsic pathway that involves factors VII, IX, and VIII may be a major physiologic extrinsic pathway, and this pathway may help to explain the clinical observations of bleeding diatheses in patients deficient in factors IX or VIII.

Topics: Animals; Blood Coagulation; Factor VII Deficiency; Factor X; Factor Xa; Factor XI Deficiency; Hemophilia A; Humans; Kinetics; Prothrombin Time; Rabbits; Thromboplastin

Topics: Blood Coagulation; Factor IX; Factor VII; Factor VIII; Hemophilia A; Humans; Thromboplastin

Topics: Blood Coagulation Tests; Diagnosis, Differential; Factor IX; Factor VIII; Hemophilia A; Hemophilia B; Humans; In Vitro Techniques; Thromboplastin

Topics: Adolescent; Adult; Antibodies; Child; Child, Preschool; Drug Therapy, Combination; Factor IX; Factor IXa; Factor VIII; Hemophilia A; Humans; Infant; Middle Aged; Prothrombin; Thromboplastin

Major surgical procedures are now performed with acceptable risk on patients with hemophilia A with pre- and postoperative anti-hemopilic Factor (AHF) infusions. However, there is almost no literature on care of the burned hemophiliac. We recently treated a patient with Factor VIII levels of less than 2% of normal and 45% TBSA burns. A forearm escharotomy was done with hemostatic protection by AHF infusion, but burn therapy, which included operative debridement and successful split-thickness skin grafting, was accomplished without the use of AHF. It is concluded that after early loading with cryoprecipitate, burned hemophiliacs do not require continued AHF, because repair and restoration of vascular integrity in small vessels may occur due to platelet plugging and vessel retraction. Tissue thromboplastin may also contribute to clotting in burned hemophiliacs.

Topics: Adult; Blood Coagulation; Burns; Cryoglobulins; Debridement; Factor VIII; Hemophilia A; Humans; Male; Microcirculation; Risk; Skin Transplantation; Thromboplastin; Transplantation, Autologous

Topics: Adolescent; Adult; Blood Coagulation Tests; Child; Child, Preschool; Factor VIII; Female; Hemarthrosis; Hematoma; Hemophilia A; Hemophilia B; Humans; Infant; Male; Oral Hemorrhage; Thromboplastin; von Willebrand Diseases

Topics: Afibrinogenemia; Blood Coagulation Disorders; Blood Coagulation Tests; Factor V Deficiency; Factor VII Deficiency; Factor X Deficiency; Factor XI Deficiency; Factor XII Deficiency; Factor XIII Deficiency; Hemophilia A; Hemophilia B; Humans; Hypoprothrombinemias; Kaolin; Phosphatidylethanolamines; Prekallikrein; Prothrombin Time; Thrombin; Thromboplastin; von Willebrand Diseases

The coagulant of normal human saliva has been identified as tissue factor (thromboplastin, TF) by virtue of its ability to cause rapid coagulation in plasmas deficient in first-stage coagulation factors and to activate factor x in the presence of factor VII and by virtue of the fact that its activity is expressed only in the presence of factor VII and is inhibited by an antibody to TF. The TF is related to cells and cell fragments in saliva. Salivary TF activity has been found to be significantly reduced in patients taking warfarin. The decline in TF activity during induction of warfarin anticoagulation occurs during the warfarin-induced decline in vitamin-K-dependent clotting factor activity, as judged by the prothrombin time. The decrease in TF activity is not related to a reduction in salivary cell count or total protein content or to a direct effect of warfarin on the assay. It is hypothesized that the mechanism by which warfarin inhibits TF activity may be related to the mechanism by which it inhibits expression of the activity of the vitamin-K-dependent clotting factors. Inhibition of the TF activity may be involved in the antithrombotic effect of warfarin.

Topics: Factor VII; Factor VII Deficiency; Factor X; Factor XI Deficiency; Factor XII Deficiency; Freezing; Hemophilia A; Hemophilia B; Humans; Hyaluronoglucosaminidase; Prothrombin; Thromboplastin; Warfarin

The influence of different temperatures between 13 degrees C and 45 degrees C on coagulation factors in vitro was studied by measuring clotting time with the recalcification time, partial thromboplastin time (PTT), and thromboplastin time test. In all three tests the shortest clotting times were measured at a temperature of 40 degrees C. The relation between temperature and clotting time was similar in fresh plasma and in plasma which had been stored at a temperature of --20 degrees C before examination. However, in all tests stored plasma showed shorter coagulation times. Prolongation of coagulation time at 45 degrees C is caused by irreversible reduction of coagulation activity in the plasma. At the same time thromboplastin- and PTT-reagent are imparied in their coagulation acitvity by a temperature of 45 decrees C. In comparison to plasma obtained from healthy persons plasma from patients with hemophilia A or B or with v. Willebrand's disease reacted more sensitive to changes in temperature in the PTT test. The coagulation defect was definitely more pronounced at 27 degrees and 17 degrees C than at 37 degrees C. It was not possible to differentiate these three coagulopathies with the PTT test at 27 degrees and 17 degrees C.

Topics: Blood Coagulation; Blood Coagulation Tests; Freezing; Hemophilia A; Hemophilia B; Humans; Temperature; Thromboplastin; von Willebrand Diseases

Some problems involved in grading the severity of hemophilia were discussed. No definite correlation existed between the clinical severity of hemophilia and the results of coagulation tests (activity of deficient factors, whole blood clotting time, thromboplastin screening test). There was also no correlation between the presence or absence of oral bleeding or hemarthrosis and the test results. It was concluded that the clinical severity of the disease can be graded more reasonably on the basis of clinical hemorrhagic symptoms than according to the activity of deficient factors.

Topics: Blood Coagulation Tests; Factor IX; Factor VIII; Hemarthrosis; Hemophilia A; Hemophilia B; Humans; Oral Hemorrhage; Thromboplastin

Six commercially available reagents for the determination of the activated partial thromboplastin time have been evaluated and compared with respect to their sensitivity to the coagulation factors VIII, IX and XI and to their response to heparin. Some variation was observed among the reagents regarding their sensitivity to factor XI and even greater differences were obtained with factors VIII and IX. It was also clear that none of the reagents was sensitive to the same extent to the factors tested. The sensitivity to heparin shows considerable variation, in terms of time as well as mode of response to increasing heparin levels. In four reagents this response is linear, it is logarithmic in one and the remaining one is yet again different. It seems unlikely that any standardization of the APTT determination is at present possible with the reagents studied.

Topics: Factor XI Deficiency; Hemophilia A; Hemophilia B; Heparin; Humans; Indicators and Reagents; Thromboplastin; Time Factors

Nine patients with severe classic hemophilia and inhibitors against factor VIII were treated for 156 bleeding episodes with 503 infusions of Proplex, Konyne, or Auto-Factor IX, three preparations of prothrombin complex concentrates (PCCs). Approximately two thirds of the bleeding episodes were managed successfully. Although the prothrombin time (PT) and partial thromboplastin time (PTT) were shortened after most PCC infusions, there was no evidence of disseminated intravascular coagulation. The degree of shortening of PT or PTT was not related to the particular PCC preparation used, dose, or cessation of hemorrhage. All PCC preparations contained activated clotting factors, as manifested by their ability to shorten the PTT of normal plasma, factor-VIII-deficient plasma, and factor-IX-deficient plasma. Shortening, which was greater with Auto-Factor IX than with the other products, was inhibited partially by a factor IX antibody and blocked completely by prolonged incubation with plasma. Although the nature of the procoagulant material in PCCs is uncertain, these products are of proven benefit to hemophilic patients with high-titer inhibitors. Side effects have been minimal and inhibitor titers have not risen.

Topics: Adolescent; Adult; Blood Coagulation Factors; Blood Coagulation Tests; Child; Factor IX; Factor VIII; Hemophilia A; Humans; In Vitro Techniques; Male; Prothrombin; Prothrombin Time; Thromboplastin

Three children with haemophilia and antibodies to factor VIII were treated with a non-activated prothrombin concentrate (Prothrombinex) for 12 bleeding episodes. There was clear clinical response and joint aspirations were performed after infusions of phothrombinex in a dose of 30--50 factor IX units/kg body weight and there was no clinical evidence of thrombosis or febrile reactions. There was a significant shortening of the activated partial thromboplastin time (PTT) at one and four hours after the initial infusion with a return to pre-infusion levels 9--24 hours after infusion. The shortening in the PTT was less marked in subsequent infusions. There were no changes in the level of factor VIII procoagulant activity, factor VIII related antigen or factor VIII antibodies after the infusion. In two patients platelet function studies were unaltered by the infusion and in one patient procoagulant levels of factor II, IX and X were no greater than expected from the infusion. We conclude that infusions of non-activated prothrombin concentrates are clinically effective in the treatment of children with haemophilia and factor VIII antibodies but the mechanism of action is unknown.

Topics: Adolescent; Antibodies; Blood Coagulation Tests; Child; Child, Preschool; Factor VIII; Hemophilia A; Humans; Prothrombin; Thromboplastin

Topics: Adolescent; Blood Coagulation Tests; Child; Hemophilia A; Hemophilia B; Humans; Male; Thromboplastin

A few neonates born to mothers receiving anticonvulsant drugs during pregnancy have shown defects in vitamin K dependent clotting factors with or without clinical bleeding. Experimentally, phenytoin (diphenyl hydantoin, DPH) has induced clotting defects in cats and inhibited production of clotting factors in rat liver slices. Phenobarbital has produced similar but milder defects. Anticonvulsants have been observed to produce clotting defects in 9 children, 2 weeks to 8 years in age. Elevated levels of phenytoin or other anticonvulsants, or a combination of anticonvulsants were measured in the children. Six patients were on drug combination including two or more of the following: phenytoin, phenobarbital, primidone, carbamazepine, diazepam, ethosuximide. Clotting defects included: elevated prothrombin time, elevated partial thromboplastin time, diminished factors V, VII or X. All children had neurologic symptoms of anticonvulsant toxicity, but the only hematologic problems were oozing from venipuncture sites and increased bruising in 3. All patients were on normal diets and had normal liver function tests. By lowering the level of anticonvulsants, clotting factors returned toward normal. Elevated levels of anticonvulsants can potentially produce clotting defects in neonates and young children.

Topics: Anticonvulsants; Blood Coagulation; Blood Coagulation Disorders; Blood Coagulation Tests; Child; Child, Preschool; Factor V Deficiency; Factor VII Deficiency; Factor X Deficiency; Female; Hemophilia A; Humans; Infant; Infant, Newborn; Male; Maternal-Fetal Exchange; Pregnancy; Prothrombin Time; Thromboplastin

A case of acquired hemophilia is presented. Widespread blisters and then ecchymoses developed in a 79-year-old woman who was severely demented. Laboratory studies revealed specific complete inhibition of factor VIII in the blood. In any elderly patient with a bleeding diathesis and an abnormality in intrinsic coagulation, the presence of inhibitors to factor VIII should be suspected.

Topics: Aged; Dementia; Factor VIII; Female; Hemophilia A; Humans; Male; Thromboplastin

Variations of the partial thromboplastin time (PTT) were tested to determine the best screening method for detection of inhibitors of factor VIII. Variables tested included the duration of preincubation of a mixture of patient plasma and factor VIII source (normal plasma), the ratio of the patient plasma to the normal plasma, and the duration of incubation of the normal plasma-patient plasma mixture with kaolin-cephalin suspension prior to recalcification. The following conclusions were reached: (1) The PTT performed on a mixture of equal amounts of patient and normal plasma without preincubation of the mixture was inadequate to detect many factor VIII inhibitors. (2) Factor VIII inhibitors of more than 0.5 Bethesda units could be detected if the PTT was performed on a mixture of four parts patient plasma and one part normal plasma, with preincubation of the mixture for 60 min at 37 degrees C. (3) Factor VIII inhibitors as weak as 0.1 Bethesda units could be detected if the PTT was performed on a mixture of four parts patient plasma and one part normal plasma incubated with kaolin-cephalin suspension for 120 min at 37 degrees C before recalcification. The last method may make detection of mild factor VIII inhibitors possible in routine clinical laboratories not equipped to perform the more technically difficult Bethesda inhibitor assays.

Topics: Factor VIII; Hemophilia A; Prothrombin Time; Thromboplastin

Plasma from a patient factor VIII inhibitor was mixed at several low concentrations with fresh normal plasma. The factor VIII deficient plasmas obtained after a short incubation period were used as substrate plasmas in the normal one-stage factor VIII assay method. Results obtained using 0.5-1% of the inhibitor plasma in normal plasma compared favourably with those obtained using normal haemophilia A factor VIII deficient plasma provided that tests were carried out without delay. A single plasma exchange of this patient provided enough antibody to prepare more than 500 litres of factor VIII deficient plasma by simple mixing with fresh normal plasma.

Topics: Adult; Antibodies; Blood Coagulation Tests; Factor VIII; Hemophilia A; Humans; Male; Plasma; Thromboplastin; Time Factors

A report is presented on the performance of the correction of PTT by means of factor VIII and IX deficiency plasma, which may be used at least one year, when preserved in liquid nitrogen. The method allows reliable, qualitative statements to be made about disturbances in the area of the coagulation factors VIII, IX, XI and XII; the small amount of time required for preparing and carrying out these works representing an essential advantage.

Topics: Blood Coagulation Tests; Diagnosis, Differential; Hemophilia A; Hemophilia B; Humans; Thromboplastin

Topics: Adsorption; Anemia, Aplastic; Anticoagulants; Blood Coagulation; Blood Coagulation Factors; Blood Preservation; Blood Specimen Collection; Citrates; Decalcification Technique; Dose-Response Relationship, Drug; Factor V; Hemophilia A; Humans; Oxalates; Prothrombin Time; Thromboplastin

Topics: Blood Coagulation; Calcium; Dose-Response Relationship, Drug; Factor IX; Factor X; Hemophilia A; Hemophilia B; Humans; Oxalates; Phospholipids; Prothrombin Time; Thromboplastin

The course and treatment of a life-threatening hemorrhagic episode in a patient with hemophilia A whose plasma contained a high concentration of an inhibitor of factor VIII activity is presented. The inhibitor of factor VIII was localized to the most anodal fractions of immunoglobulin G on electrophoresis, and was thus presumed to be an antibody directed against factor VIII. No therapeutic benefit occurred with infusions of massive amounts of fresh blood and factor VIII concentrates, or with a brief course of immunosuppressive therapy. Administration of standard and activated prothrombin complex concentrates resulted in reduction of the partial thromboplastin time to almost normal values and control of hemorrhage. Eight months later, another hemorrhagic episode occurred. Although a higher titer of inhibitor of factor VIII activity was still present in the patient's plasma, a beneficial therapeutic response was again achieved with standard prothrombin complex infusions.

Topics: Adult; Blood Coagulation Tests; Factor VIII; Hemophilia A; Humans; Male; Prothrombin; Thromboplastin

Activated partial thromboplastin times (APTT's) performed with a semi-automated electrical-conductivity type of clot timer on plasmas from patients with hepatic disease and intravascular coagulation, and on warfarin or heparin therapy, were significantly lower than when done on the same plasmas with either a manual optical method or an automated optical-endpoint instrument. Results of APTT's done on normal plasmas by the three methods were not significantly different. Substitution of different activator-phospholipid reagents resulted in some variability in results, but these differences were less than those between the different done with both the electrical clot timer and the automated optical instrument on prepared plasmas containing 5.0 or 1.0% of factor II, V, VIII, IX, OR X revealed shorter times with the electrical clot timer only in the case of factor II- and factor V-deficient plasmas. APTT's done on normal plasmas to which 0.1 or 0.3 units per ml. of heparin had been added vitro also were shorter with the electrical clot itmer than the automatic optical instrument. Prothrombin times done on normal and abnormal control plasmas and on a series of plasmas from patients on warfarin therapy showed no significant difference between the two methods.

Topics: Autoanalysis; Blood Chemical Analysis; Blood Coagulation Tests; Erythrocyte Aggregation; Factor V Deficiency; Factor X Deficiency; Hemophilia A; Hemophilia B; Heparin; Hydrogen-Ion Concentration; Hypoprothrombinemias; Liver Diseases; Optics and Photonics; Phospholipids; Prothrombin Time; Thromboplastin; Time Factors; Warfarin

Reagents may be prepared from normal plasma and used with the prothrombin time and partial thromboplastin time tests to distinguish isolated defects of factors I, II, VII, VIII, IX, X, XI, or XII.

Topics: Adolescent; Adult; Aluminum Hydroxide; Blood Coagulation Disorders; Blood Coagulation Tests; Child; Child, Preschool; Factor XI Deficiency; Female; Hemophilia A; Hemophilia B; Humans; In Vitro Techniques; Kaolin; Male; Middle Aged; Phospholipids; Plasma; Prothrombin Time; Thromboplastin; Tromethamine; von Willebrand Diseases

Analysis of the data for the 1969 to 1973 CAP Surveys of proficiency in partial thromboplastin time (PTT) determination indicates more than desirable variability in this measurement. Non-activated procedures show greater interlaboratory variability than activated methods; therefore, they may be preferable for routine use. It is likely that many laboratories have not determined their own upper limit of normal for their PTT system and thus have received unacceptable evaluations in the Surveys. It also was determined that many laboratories do not closely follow the manufacturer's directions, especially in regard to incubation times and calcium concentration of the recalcification solution.

Topics: Blood Coagulation Tests; Factor VIII; Hemophilia A; Humans; Laboratories; Pathology; Societies, Medical; Thromboplastin; Time Factors; United States

The maximal rate of change in optical density (Vmax-deltaOD)of plasma clots forming in the activated partial thromboplastin time test (APTT) may be significantly influenced by reductions in factor VIII activity insufficient to also cause a distinctly abnormal timed clotting endpoint. Analysis of relationships between Vmax-deltaOD of clotting plasma in the APTT, prothrombin time, and thrombin time tests provides a basis for increasing the screening value of the APTT in suggesting intrinsic system abnormalities. Three illustrative case reports support the added benefit of thrombokinetics in the detection of mild factor VIII deficiency in hemophilia A and in von Willebrand's disease.

Topics: Adult; Blood Coagulation Disorders; Blood Coagulation Tests; Factor VIII; Fibrin; Hemophilia A; Humans; Kinetics; Male; Middle Aged; Prothrombin; Prothrombin Time; Thrombin; Thromboplastin

Topics: Blood Coagulation Tests; Freeze Drying; Hemophilia A; Humans; Prothrombin Time; Quality Control; Thromboplastin

Topics: Adult; Afibrinogenemia; Animals; Blood Cell Count; Blood Coagulation; Blood Coagulation Tests; Blood Platelets; Cattle; Citrates; Collagen; Crystallization; Dogs; Edetic Acid; Factor VII Deficiency; Factor X Deficiency; Factor XII; Female; Fibrinogen; Hemophilia A; Hemostasis; Heparin; Humans; Male; Platelet Adhesiveness; Platelet Aggregation; Purpura, Thrombocytopenic; Thrombin; Thromboplastin; von Willebrand Diseases

Topics: Blood Cell Count; Blood Coagulation; Blood Coagulation Tests; Blood Platelets; Capillary Resistance; Child; Factor VIII; Fibrinogen; Hemolytic-Uremic Syndrome; Hemophilia A; Heparin; Hot Temperature; Humans; In Vitro Techniques; Infant; Kidney Failure, Chronic; Male; Nephrotic Syndrome; Plasminogen; Platelet Adhesiveness; Platelet Aggregation; Prothrombin Time; Serum Globulins; Thromboplastin

Topics: Adult; Blood Coagulation Tests; Factor VIII; Hemophilia A; Humans; Male; Prothrombin; Prothrombin Time; Thromboplastin

Topics: Blood Coagulation Disorders; Blood Coagulation Tests; Factor VII Deficiency; Hemophilia A; Humans; Prothrombin Time; Thrombocytopenia; Thromboplastin; Vitamin K Deficiency

Topics: Automation; Blood Coagulation Disorders; Blood Coagulation Tests; Evaluation Studies as Topic; Hemophilia A; Humans; Indicators and Reagents; Male; Thromboplastin; Time Factors

Topics: Calcium; Factor VIII; Hemophilia A; Heparin; Humans; Thrombelastography; Thromboplastin

Topics: Animals; Blood Coagulation Tests; Brain; Calcium Chloride; Concanavalin A; Culture Techniques; Fibroblasts; Hemophilia A; Humans; Mannose; Methylglycosides; Rabbits; Thromboplastin

Topics: Adenosine Diphosphate; Antigen-Antibody Reactions; Blood Coagulation; Blood Platelets; Blood Transfusion; Chromosome Aberrations; Chromosome Disorders; Cryoglobulins; Diagnosis, Differential; Factor VIII; Female; Hemophilia A; Humans; Male; Models, Biological; Platelet Adhesiveness; Serotonin; Sex Chromosome Aberrations; Thromboplastin; von Willebrand Diseases

Topics: Adolescent; Adult; Blood Coagulation Disorders; Child, Preschool; Culture Techniques; Factor VII; Factor X; Factor XI; Factor XI Deficiency; Factor XII; Female; Fibroblasts; Hemophilia A; Hemophilia B; Humans; Male; Middle Aged; Skin; Thromboplastin; von Willebrand Diseases

Topics: Blood Coagulation Tests; Hemophilia A; Hemophilia B; Humans; Indicators and Reagents; Methods; Thromboplastin

Topics: Animals; Blood Coagulation Tests; Cattle; Costs and Cost Analysis; Drug Stability; Evaluation Studies as Topic; Factor VIII; Hemophilia A; Humans; Indicators and Reagents; Plasma; Thromboplastin

Topics: Adolescent; Antibody Formation; Antigen-Antibody Reactions; Autoantibodies; Cyclophosphamide; Epitopes; Factor VIII; Hemarthrosis; Hemophilia A; Hemostasis; Humans; Immunosuppression Therapy; Male; Prednisone; Thromboplastin

Topics: Blood Cell Count; Blood Coagulation Tests; Blood Platelets; Densitometry; Factor IX; Factor VIII; Fibrinogen; Hemophilia A; Hemophilia B; Humans; Nitrogen; Photometry; Prothrombin; Thromboplastin; Time Factors

Human factor VIII from normals and hemophiliacs was partially purified by ethanol and polyethylene glycol precipitations. Final purification was achieved by gel filtration on 2 or 4% agarose or ion exchange chromatography on diethylaminoethyl cellulose. Comparable amounts of highly purified protein were obtained from normal and hemophilic plasma following the agarose chromatography step. Highly purified factor VIII was not dissociated by 6 M guanidine hydrochloride or 1% sodium dodecyl sulfate. However, when reduced by beta-mercaptoethanol and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, a single subunit species with an estimated 195,000 molecular weight was found for both normal and hemophilic factor VIII. By sedimentation equilibrium analysis, the normal factor VIII subunit was homogeneous and had an estimated molecular weight of 202,000. The subunit polypeptides from normal or hemophilic factor VIII contained carbohydrate. Each was homogeneous by isoelectric focusing. Immunodiffusion of purified normal and hemophilic factor VIII against rabbit antiserum to purified normal human factor VIII showed a single line of precipitation. Very low concentrations of purified human thrombin initially increased the activity of normal factor VIII about threefold and then progressively destroyed activity by 3 h. Only minimal activation occurred with hemophilic factor VIII. Both the activation and inactivation of normal and hemophilic factor VIII were unaccompanied by detectable changes in subunit molecular weight. These findings may have implications for the definition of the molecular defect in hemophilic factor VIII.

Topics: Amino Acids; Blood Coagulation Tests; Chromatography, DEAE-Cellulose; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Factor VIII; Hemophilia A; Humans; Immune Sera; Immunodiffusion; Isoelectric Focusing; Mercaptoethanol; Molecular Weight; Sodium Dodecyl Sulfate; Thrombin; Thromboplastin; Urea

Topics: Blood Coagulation Disorders; Blood Coagulation Tests; Factor VIII; Hemophilia A; Hemophilia B; Humans; Hypoprothrombinemias; Methods; Thromboplastin; Time Factors

Topics: Adult; Blood Coagulation Tests; Blood Protein Electrophoresis; Blood Transfusion; Factor IX; Factor VIII; Freeze Drying; Hemophilia A; Hemophilia B; Humans; Kaolin; Male; Thromboplastin

Topics: Adolescent; Adult; Aged; Animals; Blood Coagulation Tests; Cattle; Factor IX; Factor VII; Factor X; Female; Hemophilia A; Hemophilia B; Humans; Male; Middle Aged; Prothrombin Time; Rabbits; Rats; Thromboplastin

Topics: Blood Coagulation Tests; Factor VIII; Hemophilia A; Humans; Methods; Thromboplastin

Topics: Blood Cell Count; Blood Coagulation; Blood Coagulation Disorders; Blood Coagulation Factors; Blood Platelet Disorders; Blood Platelets; Diagnosis, Differential; Disseminated Intravascular Coagulation; Female; Hemoglobinometry; Hemophilia A; Hemophilia B; Hemorrhage; Humans; Male; Prothrombin Time; Purpura; Purpura, Thrombocytopenic; Purpura, Thrombotic Thrombocytopenic; Rheumatic Diseases; Telangiectasia, Hereditary Hemorrhagic; Thromboplastin; von Willebrand Diseases

Topics: Blood Coagulation; Blood Coagulation Factors; Blood Platelets; Fibrin; Fibrinogen; Fibrinolysis; Hematology; Hemophilia A; Hemostasis; History, 15th Century; History, 16th Century; History, 17th Century; History, 18th Century; History, 19th Century; History, 20th Century; History, Ancient; History, Medieval; History, Modern 1601-; Humans; Prothrombin; Prothrombin Time; Purpura, Thrombocytopenic; Thrombin; Thromboplastin

Topics: Blood Coagulation; Clot Retraction; Factor VIII; Fibrin; Hemophilia A; Humans; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Thromboplastin

Topics: Blood Coagulation Disorders; Blood Coagulation Tests; Hemophilia A; Hemophilia B; Humans; Kaolin; Thromboplastin

Nine partial thromboplastin (cephalin) reagents have been compared in a parallel investigation of groups of patients on ;long-term' anticoagulants, a group with moderate haemophilia, and patients on heparin infusion. Results with the seven commercial reagents and a human cephalin extract have been correlated with those of a specially prepared and standardized reference preparation of human brain origin. The comparison was similar in principle to that of the prothrombin time thromboplastin standardization using the British Comparative Thromboplastin (BCT). Results, which for comparative purposes were expressed as ratio of patients' cephalin times to control cephalin times, varied greatly in all three groups. In the oral anticoagulant group some of the commercial reagents were particularly insensitive to the ;intrinsic' clotting defect. The correlation between the ;standardized preparation' and the other reagents was not good and the use of a reference cephalin material for quality control of cephalin time tests does not appear promising. In moderate haemophilia the commercial reagents were either relatively poor at picking out the clotting defect compared with the ;standardized preparation' or gave such a bad endpoint that the results were not dependable. The poor endpoint also limited the dependability of the results of all but the ;standardized preparation' and two of the commercial reagents in controlling heparin administration. In view of these standardization difficulties, which cannot apparently be resolved by the use of reference material, there is need for bulk, routine supplies of a sensitive, standardized cephalin reagent giving good reproducible endpoints. The method for the provision of such material in a recently introduced national supply scheme is described.

Topics: Blood Coagulation Tests; Hemophilia A; Heparin; Humans; Methods; Phosphatidylethanolamines; Prothrombin Time; Thromboplastin; Time Factors

Topics: Blood Coagulation Disorders; Diagnosis, Differential; Factor VIII; Fibrinogen; Hemophilia A; Humans; Prothrombin Time; Thromboplastin

Topics: Anticoagulants; Blood Coagulation; Blood Coagulation Disorders; Blood Coagulation Tests; Chemical Phenomena; Chemistry; Chromatography, DEAE-Cellulose; Factor V Deficiency; Factor VII Deficiency; Factor XII; Hemophilia A; Humans; Hypoprothrombinemias; Phospholipids; Prothrombin; Prothrombin Time; Thrombin; Thromboplastin

Topics: Aged; Anticoagulants; Antithrombins; Blood Coagulation Disorders; Factor VIII; Female; Hemophilia A; Hemorrhage; Hemorrhagic Disorders; Humans; Male; Middle Aged; Pregnancy; Pregnancy Complications, Hematologic; Thromboplastin

Topics: Animals; Blood Coagulation Tests; Brain Chemistry; Factor IX; Hemophilia A; Humans; Male; Phospholipids; Prothrombin; Prothrombin Time; Rabbits; Snakes; Thromboplastin; Tissue Extracts; Venoms

Topics: Blood Coagulation Tests; Evaluation Studies as Topic; Factor IX; Factor VIII; Hemophilia A; Humans; Thromboplastin

Topics: Blood Coagulation Tests; Hemophilia A; Humans; Plasma; Thrombelastography; Thromboplastin

Topics: Anticoagulants; Blood Coagulation Disorders; Consanguinity; Enzyme Precursors; Factor IX; Factor VIII; Hemophilia A; Humans; Pedigree; Thromboplastin; von Willebrand Diseases

Topics: Animals; Blood Coagulation Tests; Blood Platelets; Factor IX; Factor V; Factor VIII; Female; Fibrinogen; Guinea Pigs; Hemophilia A; Male; Prothrombin; Prothrombin Time; Serum Globulins; Thromboplastin

Topics: Blood Coagulation Tests; Hemophilia A; Humans; Mass Screening; Prothrombin Time; Thromboplastin

Topics: Animals; Azathioprine; Blood Coagulation; Dogs; Factor VIII; Hemophilia A; Homeostasis; Intestine, Small; Male; Methylprednisolone; Prothrombin Time; Radionuclide Imaging; Spleen; Splenectomy; Thromboplastin; Time Factors; Transplantation Immunology; Transplantation, Autologous; Transplantation, Homologous

Topics: Blood Coagulation Disorders; Blood Coagulation Tests; Hematocrit; Hemophilia A; Humans; Methods; Thromboplastin

Topics: Hemophilia A; Humans; Methods; Phospholipids; Thromboplastin

The concentration of factor VIII and partial thromboplastin times became normal and have remained normal for 140 days after orthotopic tranplantation of a normal liver to a hemophilic dog. Transplantation of a normal spleen into a hemophilic recipient did not result in a significant increase in factor VIII although the splenic graft was viable for at least 47 days. Transplantation of normal marrow to a lethally irradiated hemophilic dog did not result in an increase in factor VIII during 34 days of observation.

Topics: Animals; Azathioprine; Blood Coagulation Tests; Bone Marrow Transplantation; Dogs; Factor VIII; Hemophilia A; Liver Transplantation; Methylprednisolone; Parabiosis; Radiation Injuries, Experimental; Radionuclide Imaging; Spleen; Technetium; Thromboplastin; Transplantation, Homologous

Topics: Animals; Blood Coagulation Tests; Culture Techniques; Factor IX; Factor VIII; Fibroblasts; Haplorhini; HeLa Cells; Hemophilia A; Humans; Kidney; Liver; Thromboplastin; Time Factors

Topics: Animals; Factor VIII; Hemophilia A; Perfusion; Puromycin; Spleen; Swine; Thromboplastin

Topics: Blood Coagulation Factors; Blood Coagulation Tests; Diagnosis, Differential; Factor IX; Factor XIII; Hemophilia A; Humans; Methods; Thromboplastin

Topics: Adolescent; Blood Coagulation; Blood Coagulation Tests; Blood Platelets; Blood Transfusion; Chromosomes; Female; Hemophilia A; Heterozygote; Humans; Infant, Newborn; Male; Pedigree; Sex Factors; Thromboplastin; Time Factors

Topics: Adolescent; Adult; Blood Coagulation Disorders; Child; Clinical Laboratory Techniques; Consanguinity; Exercise Test; Factor V Deficiency; Factor VIII; Female; Hemophilia A; Homozygote; Humans; Hypoprothrombinemias; Male; Prothrombin Time; Thromboplastin

Topics: Blood Coagulation Disorders; Blood Coagulation Tests; Centrifugation; Hemophilia A; Humans; Methods; Thromboplastin; Uremia

Topics: Blood Coagulation Tests; Diagnosis, Differential; Factor IX; Factor VIII; Hemophilia A; Hemophilia B; Humans; Male; Methods; Prothrombin Time; Thromboplastin; von Willebrand Diseases

Topics: Adolescent; Factor IX; Female; Hemophilia A; Humans; Male; Prothrombin Time; Thromboplastin

Topics: Adult; Blood Coagulation Tests; Capillaries; Citrates; Factor VIII; Female; Hemophilia A; Humans; Infant, Newborn; Male; Methods; Middle Aged; Sodium Chloride; Thromboplastin; Veins

Topics: Blood Coagulation Disorders; Blood Coagulation Tests; Hemophilia A; Humans; Methods; Thromboplastin; Time Factors

Topics: Blood Coagulation Tests; Hemophilia A; Humans; Male; Prothrombin Time; Thromboplastin

Topics: Blood Coagulation Factors; Hemophilia A; Hemophilia B; Humans; Hypoprothrombinemias; Thromboplastin; Wiskott-Aldrich Syndrome

Topics: Blood Coagulation Disorders; Blood Coagulation Tests; Hemophilia A; Humans; Phosphatidylethanolamines; Prothrombin Time; Thromboplastin

Topics: Factor VIII; Hemophilia A; Humans; Methods; Thromboplastin

Topics: Anticoagulants; Blood Coagulation Tests; Factor VIII; Hemophilia A; Hemorrhage; Humans; Male; Middle Aged; Thromboplastin

Topics: Child; Child, Preschool; Diagnosis, Differential; Hemophilia A; Hemophilia B; Humans; Male; Singapore; Thromboplastin

Topics: Adipose Tissue; Adolescent; Adult; Aged; Blood Coagulation; Bone Marrow; Brain Chemistry; Factor VIII; Female; Fracture Fixation; Fractures, Bone; Fractures, Spontaneous; Hematoma; Hemophilia A; Hemophilia B; Hemorrhage; Humans; Male; Middle Aged; Muscles; Plasma; Radiography; Thromboplastin

Topics: Absenteeism; Adolescent; Adult; Aminocaproates; Blood Coagulation Tests; Blood Transfusion; Child; Child, Preschool; Hemophilia A; Hemorrhage; Humans; Thromboplastin; Time Factors

Topics: Blood Coagulation Disorders; Blood Coagulation Tests; Diagnosis, Differential; Hemophilia A; Hemophilia B; Humans; Prothrombin Time; Thromboplastin

Topics: Animals; Cattle; Dicumarol; Dogs; Factor VII Deficiency; Factor XI Deficiency; Fibrin; Hemophilia A; Hemophilia B; In Vitro Techniques; Prothrombin; Snakes; Thrombin; Thromboplastin; Time Factors; Tissue Extracts; Venoms; Vitamin K

Topics: Blood Coagulation Tests; Hemophilia A; Humans; Male; Methods; Thromboplastin

Topics: Child; Child, Preschool; Factor VIII; Female; Hematoma; Hemophilia A; Hemophilia B; Hemorrhage; Humans; Infant; Infant, Newborn; Italy; Male; Thromboplastin; von Willebrand Diseases

Topics: Blood Coagulation Tests; Cataract; Cataract Extraction; Diagnosis, Differential; Hemophilia A; Hemorrhage; Humans; Male; Methods; Middle Aged; Postoperative Complications; Thromboplastin

Topics: Blood Coagulation Tests; Blood Platelets; Hemolysin Proteins; Hemophilia A; Humans; Thrombocytopenia; Thromboplastin

Deficiencies of factor VIII (in haemophilia) and factor IX (in Christmas disease) prolong the partial thromboplastin time. If normal plasma is treated with alumina, the factor VIII remains but the factor IX is removed and can subsequently be recovered by elution of the alumina. If a long partial thromboplastin time is found on investigating a male patient whose history suggests a life-long bleeding disorder, the plasma may be retested after adding either alumina-adsorbed normal plasma or eluate. If the patient's partial thromboplastin time is shortened (relative to the control) by adding adsorbed normal plasma the patient is likely to be a haemophiliac; but if it is shortened by adding eluate then he is likely to have Christmas disease. Practical details for carrying out these manoeuvres are given and experiments on the validity of the test described.

Topics: Blood Coagulation Tests; Hemophilia A; Hemophilia B; Kaolin; Thromboplastin; Time Factors

Topics: Aminocaproates; Hemophilia A; Thrombelastography; Thromboplastin

Topics: Blood Coagulation Tests; Hemophilia A; Humans; Thromboplastin

Topics: Animals; Antibodies, Anti-Idiotypic; Antigen-Antibody Reactions; Factor VIII; Hemophilia A; Humans; Rabbits; Thrombelastography; Thromboplastin

Topics: Adult; Factor IX; Factor VIII; Female; Greece; Health Surveys; Hemophilia A; Hemophilia B; Humans; Prothrombin Time; Thromboplastin

Topics: Blood Coagulation Disorders; Blood Coagulation Tests; Child; Child, Preschool; Hemophilia A; Humans; Thromboplastin

Topics: Adolescent; Adult; Blood Platelets; Child; Factor VIII; Hemophilia A; Humans; Male; Prothrombin Time; Thrombin; Thromboplastin

Topics: Adult; Factor V Deficiency; Factor VIII; Hemophilia A; Humans; Hypoprothrombinemias; Male; Prothrombin Time; Thromboplastin

Topics: Animals; Blood Coagulation; Blood Coagulation Factors; Cattle; Chromatography, Gel; Dogs; Electrophoresis; Female; Hemophilia A; Humans; Iodine Isotopes; Male; Methods; Prothrombin; Prothrombin Time; Rabbits; Thromboplastin

Topics: Autografts; Blood Coagulation Tests; Burns; Hemophilia A; Hemostatics; Humans; Skin Transplantation; Thromboplastin; Transplantation, Autologous

Topics: Anticoagulants; Antithrombin III; Blood Coagulation; Blood Coagulation Disorders; Blood Platelets; Calcium; Chemical Phenomena; Chemistry; Drug Therapy; Enzyme Inhibitors; Factor VII Deficiency; Glucose; Glycine; Hemophilia A; Heparin; Humans; Hypoprothrombinemias; Mannitol; Phospholipids; Sucrose; Thiourea; Thrombin; Thromboplastin

Topics: Blood Coagulation Tests; Counseling; Diagnosis; Factor VIII; Factor XI; Genetics, Medical; Hemophilia A; Hemophilia B; Humans; Statistics as Topic; Thromboplastin

Topics: Factor VIII; Hemophilia A; Hemophilia B; Humans; Medicine; Surgical Procedures, Operative; Thromboplastin

The partial thromboplastin time test provides a convenient and sensitive screening procedure for deficiencies of thromboplastic factors, especially factors VIII and IX. The test is carried out after preincubating the plasma for 10 minutes with kaolin, and Inosithin is used as a platelet substitute. The ;normal range' of the test has been estimated in terms of the differences encountered between random normal plasmas tested in pairs, because individual patients are usually tested against single control subjects. A patient's partial thromboplastin time should be regarded as abnormal if it is more than six seconds longer than the control time. In the diagnosis of haemophilia, patients' plasmas with concentrations of factor VIII as low as about 20% might be regarded as being within the range of normal, if the selected control subject's factor VIII happened to lie near the lower end of the normal range. When mild haemophilia is suspected, discrimination may be improved by diluting both the patient's and the control plasmas 1 in 20 in haemophilic plasma. With the test modified in this way the clotting time is prolonged, though the range of differences among normal subjects is unaltered, and plasmas with factor VIII concentrations below about 30%, i.e., in undiluted plasma, would be unlikely to be regarded as normal. The partial thromboplastin time may be similarly modified as a screening test for factor IX deficiency.Some clinical examples are reported.

Topics: Aged; Blood Coagulation Tests; Diagnosis; Factor VIII; Hemophilia A; Hemophilia B; Humans; Indicators and Reagents; Kaolin; Partial Thromboplastin Time; Plasma; Reference Values; Thromboplastin

Topics: Blood Coagulation; Deoxyribonuclease I; Drug Therapy; Geriatrics; Hemophilia A; Humans; Liver Cirrhosis; Streptodornase and Streptokinase; Streptokinase; Thromboplastin

Topics: Blood Coagulation Tests; Blood Platelet Disorders; Blood Transfusion; Factor VIII; Gastrointestinal Hemorrhage; Hemophilia A; Humans; Male; Thromboplastin; von Willebrand Diseases

Topics: Blood Chemical Analysis; Blood Coagulation Factors; Factor XI; Hemophilia A; Hemorrhagic Disorders; Humans; Medicine; Thromboplastin

Topics: Biomedical Research; Blood Coagulation Tests; Blood Volume Determination; Chromium Isotopes; Factor VIII; Fibrinogen; Hematocrit; Hemophilia A; Hemostasis; Humans; Longevity; Surgical Procedures, Operative; Thromboplastin; Tooth Extraction

Topics: Animals; Cattle; Factor V; Factor VIII; Freeze Drying; Hemophilia A; Prothrombin Time; Rabbits; Research; Thromboplastin

Topics: Blood Coagulation; Blood Coagulation Disorders; Blood Platelet Disorders; Blood Platelets; Classification; Fibrin; Genetics, Medical; Hemophilia A; Humans; Purpura; Surgery, Oral; Thrombin; Thromboplastin

Topics: Blood Coagulation Tests; Blood Platelet Disorders; Blood Transfusion; Factor VIII; Genetics, Medical; Hemophilia A; Hot Temperature; Humans; Hydrogen-Ion Concentration; Refrigeration; Thromboplastin; von Willebrand Diseases

Topics: Animals; Blood Coagulation Tests; Blood Transfusion; Dogs; Factor VIII; Genetics; Hemophilia A; Parabiosis; Pathology; Physiology; Splenectomy; Thromboplastin

Topics: Biomedical Research; Blood Coagulation Factors; Catechols; Dihydroxyphenylalanine; Dopamine; Ellagic Acid; Factor VII Deficiency; Factor XII; Flavonoids; Gallic Acid; Hemophilia A; Hemophilia B; Humans; Lactones; Pharmacology; Prothrombin; Pyrogallol; Research; Serum Globulins; Tannins; Thrombin; Thromboplastin

Topics: Animals; Blood Coagulation Factors; Blood Coagulation Tests; Calcium; Factor VII; Fibrinogen; Fibrinolysin; Hemophilia A; Magnesium; Nitrogen; Prothrombin Time; Research; Swine; Thromboplastin

Topics: Blood Coagulation Tests; Factor V; Factor VII; Factor X; Factor XI; Genetics, Medical; Hemophilia A; Humans; Prothrombin Time; Thromboplastin

Topics: Blood Coagulation Tests; Hemophilia A; Humans; Partial Thromboplastin Time; Prothrombin; Thromboplastin

Topics: Aminocaproates; Aminocaproic Acid; Anticoagulants; Biomedical Research; Chloroform; Dicumarol; Dogs; Hemophilia A; Hemorrhage; Pharmacology; Research; Thromboplastin

Topics: Autoimmune Diseases; Female; Hemophilia A; Hemorrhagic Disorders; Humans; Medicine; Pregnancy; Pregnancy Complications, Hematologic; Thromboplastin

Topics: Blood; Blood Coagulation Tests; Blood Platelets; Calcium; Calcium, Dietary; Hemophilia A; Hemophilia B; Humans; Pharmacology; Phosphatidylethanolamines; Research; Thrombin; Thromboplastin

Topics: Blood Platelet Disorders; Dicumarol; Drug Therapy; Factor V Deficiency; Factor VII Deficiency; Factor VIII; Factor XII; Hemophilia A; Hemophilia B; Hemorrhagic Disorders; Heparin; Humans; Hypoprothrombinemias; Prothrombin; Prothrombin Time; Serum Globulins; Thrombocytopenia; Thromboplastin

Topics: Animals; Dog Diseases; Dogs; Factor VIII; Female; Genetics; Hemophilia A; Heterozygote; Humans; Research; Sex; Thromboplastin

Topics: Aluminum; Blood Coagulation Factors; Brain; Chemical Phenomena; Chemistry; Electrophoresis; Factor IX; Factor VIII; Hemophilia A; Imidazoles; In Vitro Techniques; Phosphatidylethanolamines; Physiology; Spectrophotometry; Thromboplastin; Tissue Extracts

Topics: Blood Coagulation Tests; Diagnosis, Differential; Factor VIII; Hemophilia A; Hemophilia B; Humans; Methods; Thromboplastin

Topics: Blood Coagulation Tests; Cadmium; Cadmium Compounds; Hemophilia A; Humans; Medicine; Sulfates; Thromboplastin

Topics: Blood Coagulation Factors; Hemophilia A; Humans; Prothrombin Time; Thrombelastography; Thromboplastin; Urine

Topics: Anticoagulants; Blood Coagulation Factors; Hemophilia A; Humans; Pathology; Proteins; Thromboplastin

Topics: Blood Coagulation; Blood Coagulation Tests; Blood Platelets; Electrons; Factor IX; Factor VIII; Factor X; Hemophilia A; Humans; Microscopy; Microscopy, Electron; Thromboplastin; Ultrasonics

Topics: Diagnosis, Differential; Factor XI Deficiency; Hemophilia A; Humans; Medicine; Prothrombin Time; Thromboplastin

Topics: Blood Coagulation Tests; Child; Hemophilia A; Hemorrhage; Humans; Research; Thromboplastin

Topics: Hemophilia A; Hemorrhagic Disorders; Humans; Medicine; Sex Chromosome Disorders; Thrombocytopenia; Thromboplastin

Topics: Adrenal Cortex Hormones; Blood Chemical Analysis; Child; Factor IX; Hemophilia A; Hemophilia B; Hepatitis; Hepatitis A; Humans; Infant; Infant, Newborn; Infant, Premature; Jaundice; Jaundice, Obstructive; Leukemia; Pharmacology; Physiology; Purpura; Thromboplastin

Topics: Blood Coagulation Tests; Ether; Ethers; Factor IX; Hemophilia A; Hemophilia B; Humans; Plasma; Thromboplastin

Topics: Adolescent; Blood Coagulation Tests; Hemophilia A; Humans; Research; Thromboplastin; von Willebrand Diseases

Topics: Adolescent; Blood Coagulation Tests; Blood Platelets; Child; Cholecystitis; Coronary Disease; Duodenal Ulcer; Geriatrics; Glycine max; Hemophilia A; Humans; Lecithins; Myocardial Infarction; Phosphatidylcholines; Polycythemia Vera; Thrombophlebitis; Thromboplastin

Topics: Adolescent; Aminocaproates; Aminocaproic Acid; Child; Chromatography; Factor IX; Factor VIII; Fibrinolysin; Fibrinolysis; Hemophilia A; Hemorrhagic Disorders; Humans; Thromboplastin

Topics: Hemophilia A; Humans; Thromboplastin

Topics: Diagnosis, Differential; Hemophilia A; Hemorrhagic Disorders; Humans; Medicine; Syndrome; Thromboplastin

Topics: Globulins; Hemophilia A; Serum Globulins; Thromboplastin; von Willebrand Diseases

Topics: Blood Coagulation Disorders; Hemophilia A; Humans; Thrombocytopenia; Thromboplastin; Thrombosis; von Willebrand Diseases

Topics: Adsorption; Blood Coagulation; Factor VIII; Globulins; Hemophilia A; Humans; Thromboplastin; von Willebrand Diseases

Topics: Blood Coagulation; Globulins; Hemophilia A; Plasma; Thromboplastin; von Willebrand Diseases

Topics: Hemophilia A; Hemophilia B; Humans; Medical Records; Medicine; Thromboplastin

Topics: Glycine max; Hemophilia A; Hemorrhagic Disorders; Hemostatics; Phospholipids; Prothrombin; Thromboplastin

Topics: Blood Transfusion; Hemophilia A; Humans; Purpura; Purpura, Thrombocytopenic; Thrombocytopenia; Thromboplastin

Topics: Anticoagulants; Blood Coagulation; Hemophilia A; Humans; Thromboplastin; von Willebrand Diseases

Topics: Hemophilia A; Hemorrhagic Disorders; Humans; Medical Records; Medicine; Plasma; Sex Chromosome Disorders; Thromboplastin

Topics: Blood Platelets; Factor V Deficiency; Hemophilia A; Hemorrhagic Disorders; Humans; Medical Records; Medicine; Thromboplastin

Topics: Complement Factor D; Hemophilia A; Hereditary Complement Deficiency Diseases; Immunologic Deficiency Syndromes; Plasma; Sequence Deletion; Thromboplastin

Topics: Hemophilia A; Humans; Ligaments; Plasminogen; Plasminogen Activators; Synovial Membrane; Thromboplastin

Topics: Blood Coagulation Factors; Factor XI; Hemophilia A; Hemorrhage; Humans; Medical Records; Thromboplastin; Tooth Extraction

Topics: Aged; Blood Coagulation Disorders; Factor XI Deficiency; Hemophilia A; Hemorrhagic Disorders; Humans; Medical Records; Medicine; Thromboplastin

Topics: Hemophilia A; Humans; Medicine; Thromboplastin

Topics: Hemophilia A; Humans; Syndrome; Thromboplastin; von Willebrand Diseases

Topics: Blood Platelets; Hemophilia A; Medicine; Plasma; Platelet Factor 3; Thromboplastin

Topics: Freezing; Hemophilia A; Humans; Thromboplastin

Topics: Hemophilia A; Medicine; Prostheses and Implants; Tablets; Thromboplastin

Topics: Blood Coagulation Disorders; Hemophilia A; Hemophilia B; Hemorrhage; Hemorrhagic Disorders; Medicine; Thromboplastin

Topics: Anemia; Factor XI Deficiency; Hemophilia A; Medicine; Sex Chromosome Disorders; Thromboplastin

Topics: Blood Coagulation Factors; Blood Platelets; Coagulants; Factor V Deficiency; Factor Xa; Hemophilia A; Humans; Medicine; Thromboplastin

Topics: Factor Xa; Hemophilia A; Medicine; Thromboplastin

Topics: Blood Coagulation; Blood Platelets; Hemophilia A; Humans; Thromboplastin; von Willebrand Diseases

Topics: Hemophilia A; Humans; Medicine; Sex Chromosome Disorders; Thromboplastin

Topics: Blood Coagulation; Hemophilia A; Humans; Medicine; Thromboplastin

Topics: Complement Factor B; Factor IX; Hemophilia A; Hemophilia B; Humans; Immunologic Deficiency Syndromes; Metabolism, Inborn Errors; Plasma; Thromboplastin

Topics: Blood Platelets; Hemophilia A; Humans; Medicine; Sex Chromosome Disorders; Thromboplastin

Topics: Factor IX; Factor XI; Globulins; Hemophilia A; Medicine; Thromboplastin

Topics: Factor VIII; Hemophilia A; Humans; Syndrome; Thromboplastin

Topics: Hemophilia A; Humans; Thromboplastin; von Willebrand Diseases

Topics: Globulins; Hemophilia A; Hemorrhagic Disorders; Plasma; Thromboplastin

Topics: Factor IX; Hemophilia A; Hemophilia B; Hemorrhagic Disorders; Medicine; Sex Chromosome Disorders; Thromboplastin

Topics: Globulins; Hemophilia A; Medicine; Serum Globulins; Thromboplastin

Topics: Hemophilia A; Hemorrhagic Disorders; Medicine; Plasma; Thromboplastin

Topics: Blood Coagulation Tests; Factor VIII; Hemophilia A; Humans; Sex Chromosome Disorders; Thromboplastin

Topics: Factor VIII; Hemophilia A; Medicine; Thromboplastin

Topics: Blood; Factor XI; Hemophilia A; Hemorrhagic Disorders; Humans; Medicine; Sex Chromosome Disorders; Thromboplastin

Topics: Diagnosis, Differential; Hematologic Diseases; Hemophilia A; Hemophilia B; Hemorrhagic Disorders; Medicine; Thromboplastin

Topics: Anticoagulants; Hemophilia A; Medicine; Thromboplastin

Topics: Hemophilia A; Hemophilia B; Humans; Medicine; Sex Chromosome Disorders; Thromboplastin; von Willebrand Diseases

Topics: Hemophilia A; Hemophilia B; Thromboplastin; von Willebrand Diseases

Topics: Factor V; Factor Xa; Hemophilia A; Hemorrhage; Humans; Thromboplastin

Topics: Hemophilia A; Humans; Plasma; Thromboplastin

Topics: Hemophilia A; Humans; Medicine; Thromboplastin

A study was made of the clotting defect and the course of the malady in a group of male dogs with an inherited, sex-linked bleeding disease. The clotting defect is characterized by a prolonged clotting time and a delayed prothrombin utilization, and is corrected by the addition either of thromboplastin or of normal plasma. A plasma protein fraction, fraction I, also corrects the defect. The defect appears to be due to a deficiency of a plasma factor, which normally, in the presence of platelets, makes thromboplastin available in shed blood. The clotting anomaly appears to be identical with that found in human hemophilia. The hemostatic defect is characterized by repeated hemorrhages, usually without obvious relationship to trauma. Hemarthroses occur frequently and may result in permanent joint deformity. The animals usually die early in life from massive hemorrhage. Transfusions with normal blood or plasma correct the clotting defect and readily control the hemorrhagic phenomena. By the use of transfusions, these dogs have been reared to maturity.

Topics: Animals; Blood Transfusion; Dogs; Hemophilia A; Hemorrhage; Humans; Male; Thromboplastin

Topics: Blood Coagulation; Body Fluids; Hemophilia A; Humans; Kidney; Male; Medicine; Thromboplastin

Topics: Blood Coagulation Disorders; Blood Platelets; Hemophilia A; Humans; Plasma; Thromboplastin; von Willebrand Diseases