fibrin and Hemophilia-A

Hemophilia A is caused by decreased or dysfunctional blood coagulation factor VIII (FVIII). Recent developments in the understanding of FVIII biology, in particular the nature of FVIII binding sites on platelets, may provide new insight into the limitations of current assays. Recent data suggest that the phospholipid vesicles, which represent nonphysiologic membranes of high phosphatidylserine (PS) content, poorly reflect functional FVIII binding sites critical to coagulation. This narrative review describes the function of FVIII in clotting and discusses our evolving understanding of FVIII binding sites and their clinical implications. Refined models of FVIII binding sites have the potential to improve FVIII assays, possibly improving bleeding risk stratification for patients with mild and moderate hemophilia A. They may also support earlier and more accurate detection of inhibitors, before they are clinically evident.

Topics: Binding Sites; Evolution, Molecular; Factor VIII; Fibrin; Hemophilia A; Humans; Models, Molecular; Phosphatidylserines; Protein Binding; Protein Conformation; Structure-Activity Relationship

Several different kinds of 'milestone' in the field of blood coagulation are described from the middle decades of the 20th century. Although viewed from the standpoint of clotting per se, attention is also given to implications for innate immunity. The first milestone considered is the protracted saga of clotting dependence on vitamin K, an adventure that spanned more than five decades beginning in the 1920s. The second has to do with the discovery of a half-dozen 'new' clotting factors during the period immediately following World War II. A third pursues a narrower focus and examines the once mysterious transformation of fibrinogen into fibrin. Finally, the clinical treatment of classical hemophilia had a remarkable turning point in the 1960s as the result of simple but sensible measures.

Topics: Blood Coagulation; Cryoglobulins; Fibrin; Fibrinogen; Hemophilia A; Humans; Immunity, Innate; Vitamin K

Fibrin plays an essential role in hemostasis as both the primary product of the coagulation cascade and the ultimate substrate for fibrinolysis. Fibrinolysis efficiency is greatly influenced by clot structure, fibrinogen isoforms and polymorphisms, the rate of thrombin generation, the reactivity of thrombus-associated cells such as platelets, and the overall biochemical environment. Regulation of the fibrinolytic system, like that of the coagulation cascade, is accomplished by a wide array of cofactors, receptors, and inhibitors. Fibrinolytic activity can be generated either on the surface of a fibrin-containing thrombus, or on cells that express profibrinolytic receptors. In a widening spectrum of clinical disorders, acquired and congenital defects in fibrinolysis contribute to disease morbidity, and new assays of global fibrinolysis now have potential predictive value in multiple clinical settings. Here, we summarize the basic elements of the fibrinolytic system, points of interaction with the coagulation pathway, and some recent clinical advances.

Topics: Animals; Blood Coagulation; Blood Coagulation Disorders, Inherited; Fibrin; Fibrin Clot Lysis Time; Fibrinogens, Abnormal; Fibrinolysis; Hemophilia A; Hemostasis; Humans; Thrombelastography

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

Bleeding episodes in patients with hemophilia and inhibitors must be managed using agents that are hemostatically active in the absence of factor VIII or IX. Activated prothrombin complex concentrates have long been used in this context. However, the search for safer and more effective agents has led to the development of recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark). This paper presents an update on the mechanism of action of rFVIIa, and describes how pharmacologic doses of this agent enhance thrombin production and thus contribute to the development of a stable, lysis-resistant fibrin plug at the site of vessel damage. This mechanism explains the reported efficacy of rFVIIa in a range of clinical situations characterized by impaired thrombin generation.

Topics: Blood Coagulation; Dose-Response Relationship, Drug; Factor VII; Factor VIIa; Factor XI Deficiency; Fibrin; Hemophilia A; Hemorrhage; Humans; Recombinant Proteins; Thrombin

Bypassing agents consist of activated prothrombin complex concentrates (aPCC) and recombinant factor VIIa (rFVIIa). Their main utilization is for prevention and treatment of bleeding complications, which may occur in inhibitor-developing haemophiliacs, although new indications for rFVIIa (e.g. trauma-related and cerebral bleeds) are now under evaluation in clinical trials. The mechanisms of action for these agents are still not fully understood. The relative complexity of the composition of aPCC suggests the possibility of multiple modes of action for achieving haemostasis. Among those possibilities, the contributions of activated factor X and prothrombin have been demonstrated in recent years both in vitro and in animal models for the only aPCC which remains on the market. rFVIIa also exhibits a complex mode of action, improving coagulation through both tissue factor-dependent and -independent pathways. The various mechanisms that occur at the cellular surfaces, particularly on the outer leaflet of the platelet membrane, primarily contribute to Xase complex formation and thrombin generation. The ways in which these agents affect the complex kinetics of fibrin formation at the site of vascular damage need further clarification, although significant progress has been achieved in the last 10 years. In addition, the ex vivo monitoring that would reflect achievement of haemostasis in vivo is still not standardized, although several attempts using thromboelastography, thrombin generation and the kinetics of fibrin formation have been initiated.

Topics: Blood Coagulation; Blood Coagulation Factors; Factor VIIa; Fibrin; Hemophilia A; Hemostatics; Humans; Thrombin

Fibrin glues, a kind of biological sealant, have been used for enhancement of local haemostasis, tissue sealing and wound healing for haemophilia and similar disorders. Commercial viral-inactivated fibrin glue products are available in Europe and have recently been approved in the USA. Using fibrin glue, hospitalization, medical cost, viral transmission risk and factor supplementation will be reduced. In the near future, the role of fibrin glues will be increased in the haemophilia field.

Topics: Biocompatible Materials; Fibrin; Hemophilia A; Hemostasis; Humans; Tissue Adhesives

Activation and inhibition of the haemostatic system was reviewed including the interaction between the four biological systems involved in haemostasis: the vessel wall, the platelets, the coagulation system and the fibrinolytic system. The haemostatic mechanism is initiated at the site of injury through local activation of surfaces and release of tissue thromboplastin, resulting in formation and deposition of fibrin. The coagulation process is regulated by physiological anticoagulants. Activation of fibrinolysis is triggered by the presence of fibrin, and the role of tissue-type plasminogen activators (t-PA) at the site of fibrin formation in particular is emphasized. The process is regulated by physiological inhibitors, of which alpha 2-antiplasmin, histidine-rich glycoprotein and plasminogen activator inhibitor are reported to be of major physiological significance. The role of fibrinolysis in the regulation of the dynamic haemostatic balance is discussed, elucidated through examples of congenital deficiencies of the coagulation and the fibrinoytic system. Pharmacological inhibitors of fibrinolysis (i.e. epsilon-aminocaproic acid and tranexamic acid) and their possible effect on the haemostatic system are described. The systemic effects on the fibrinolytic system of surgery and oral surgery is reviewed, and it is concluded, that oral surgery has insignificant effects on blood fibrinolysis. In contrast, oral surgery induces changes of fibrinolysis in the oral environment; initially the fibrinolytic activity of saliva is reduced, due to the presence of inhibitors of fibrinolysis originating from the blood and the wound exudate. When bleeding and exudation cease, the fibrinolytic activity of the saliva will increase. Plasminogen and plasminogen activator, identified as t-PA are present in the oral environment under physiological conditions. Plasminogen is secreted in the saliva and the sources of t-PA include oral epithelial cells and gingival crevicular fluid. The presence of plasminogen and t-PA in the oral environment implies that when fibrin is present (i.e. after surgery), fibrinolysis is triggered. Haemorrhagic complications to oral surgery in patients without known defects of the coagulation system is reviewed. It is concluded that the investigations conducted to the present day do not permit final conclusions with respect to the pathophysiological role of defects in the coagulation and the fibrinolytic systems for the development of bleeding after

Topics: Anticoagulants; Blood Coagulation; Blood Platelets; Deamino Arginine Vasopressin; Fibrin; Fibrinolysis; Gingiva; Hemophilia A; Hemorrhage; Hemostasis; Hemostasis, Surgical; Humans; Mouth Mucosa; Saliva; Surgery, Oral; Tissue Plasminogen Activator; von Willebrand Diseases

The use of clotting substances from blood for hemostasis dates back to 1909. In 1972, modern fibrin sealing (FS) was developed in Vienna. For application, the two components, i.e., a sealer protein solution (mainly fibrinogen) and a thrombin solution, are mixed to produce the fibrin clot. The sealant may be applied with a needle, as a spray, or by premixing (e.g., with antibiotics, bone chips) for subsequent sealant application in cavities. While the positive effect of FS in normal wound healing has been conclusively demonstrated, its influence on bone healing remains controversial. The positive experimental results mostly refer to the early phase of bone healing in rabbits (cortical drill hole, autologous and heterologous (Kiel) cancellous transplants, osteotomies, osteochondral fractures) and dogs (cortical bone and spongiosa defects). Some authors observed no effects (in dogs, osteotomy) or delayed healing in artificial bone growth chambers with the use of heterologous sealant. FS was also applied in combination with implantation material (tricalcium phosphate and bone gelatin) to facilitate application. Clinical results are especially convincing as to osteochondral fractures, repair of the Achilles tendon, and in hemophiliacs. Fibrin sealant facilitates hemostasis, permits tissue fixation, enhances plasticity of granular implant material, and stimulates fibroblast growth. Although its direct osteogenic effect remains questionable, fibrin sealant is known to be an excellent tool in orthopedic and trauma surgery.

Topics: Animals; Aprotinin; Bone and Bones; Cartilage; Dogs; Drug Combinations; Factor XIII; Fibrin; Fibrin Tissue Adhesive; Fibrinogen; Fractures, Bone; Hemophilia A; Hemorrhage; Hemostasis; Humans; Rabbits; Tendon Injuries; Thrombin; Tissue Adhesives; Wound Healing

The hemostatic mechanism has evolved to provide efficient protection from traumatic blood loss and yet maintain the blood in a fluid state in the circulation as a whole. Recent advances in biochemistry have provided both detailed understanding of hemostasis and clinically useful coagulation assays to exploit this understanding. Clinicians now have the means to delineate most of the hemostatic problems of clinical significance.

Topics: Blood Coagulation; Blood Coagulation Factors; Blood Coagulation Tests; Factor VIII; Factor X; Factor Xa; Fibrin; Fibrin Fibrinogen Degradation Products; Fibrinogen; Fibrinolysis; Hemophilia A; Humans; Prothrombin Time; Thrombin; Thrombin Time; Vitamin K Deficiency; von Willebrand Diseases

Disturbances of haemostasis caused immunologically and non-immunologically were observed after transfusion of blood and blood derivatives. Transfusion of heparin blood increased the bleeding susceptibility only in case of pre-existing high-degree defects of haemostasis or if they were performed as massive or exchange transfusions. Massive transfusions with blood stored for a long time will induce complex defects. Under intensive substitution therapy of haemophilia A the so-called paradoxical bleeding will occur in spite of a high factor VIII level. These bleedings are supposed to be disturbances of the thrombocyte function and are caused by fibrin(ogen) derivatives. Post-transfusional thrombocytopenias may be brought to remission by repeated plasmapheresis. Factor specific inhibitory bodies will appear after substitution in a small percentage of haemophilic patients. 5 to 7 days after the onset of therapy an anamnestic reaction can be observed as a titre increase by leaps. Usually, the inhibitory titre will decrease to a mostly low basal value in the course of three to five months. The therapy with cyclophosphamide simultaneously started with the substitution will more frequently prevent the anamnestic reaction or reduce it. Titres with more than 5 units cannot be overcome at the beginning even by higher concentrations of preparations. The substitution therapy should be preceded by exchange transfusions or plasmapheresis of up to 25 units. With still higher titres only procedures of inhibitor-bypassing are possible with factor VIII preparations of animal origin or better with activated prothrombin complex preparations, such as FEIBA. Recent reports give evidence that permanent substitution with factor VIII concentrates at a highest dosage can eliminate the production of inhibitors completely.

Topics: Blood Coagulation Disorders; Blood Transfusion; Cyclophosphamide; Factor IX; Factor IXa; Factor XIII; Fibrin; Fibrinogen; Hemophilia A; Hemostasis; Heparin; Humans; Platelet Aggregation; Thrombocytopenia; von Willebrand Diseases

The evidence for intravascular coagulation in liver diseases is critically reviewed. Alternative mechanisms for hypofibrinogenemia and the accelerated disappearance of fibrinogen from blood are proposed, such as loss into extravascular compartments (e.g., ascites, areas of liver necrosis, etc.). Possible mechanisms other than DIC for the elevation of serum FDP are also considered, such as extravascular fibrinogen proteolysis with subsequent transfer of FDP to blood. Therapy is discussed with reference to the current knowledge on pathophysiology of the coagulation defect in liver diseases.

Topics: Afibrinogenemia; Blood Cell Count; Blood Platelets; Disseminated Intravascular Coagulation; Factor V Deficiency; Fibrin; Fibrin Fibrinogen Degradation Products; Fibrinogen; Half-Life; Hemophilia A; Heparin; Humans; Liver Diseases; Peptide Hydrolases; Thrombocytopenia; Thrombosis

Topics: Adrenal Cortex Hormones; Adrenal Glands; Adult; Afibrinogenemia; Age Factors; Aged; Anticoagulants; Antineoplastic Agents; Blood Proteins; Child; Cicatrix; Fibrin; Growth Hormone; Hemophilia A; Humans; Insulin; Mineralocorticoids; Pancreas; Postoperative Complications; Vitamins; Wound Healing

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: Abruptio Placentae; Blood Coagulation; Blood Coagulation Disorders; Blood Coagulation Factors; Blood Coagulation Tests; Blood Platelet Disorders; Blood Transfusion; Disseminated Intravascular Coagulation; Embolism, Amniotic Fluid; Factor XIII Deficiency; Female; Fetal Death; Fetal Diseases; Fibrin; Fibrinogen; Fibrinolysis; Hemophilia A; Humans; Liver Diseases; Pre-Eclampsia; Pregnancy; Pregnancy Complications, Hematologic; Sepsis; Uterine Hemorrhage; Vitamin K

Topics: Amino Acids; Animals; Binding Sites; Blood Coagulation; Blood Coagulation Disorders; Blood Coagulation Factors; Cattle; Crystallization; Factor V; Factor VII; Factor VIII; Factor X; Factor XI; Factor XII; Fibrin; Fibrinogen; Hemophilia A; Humans; Isoelectric Focusing; Isoflurophate; Macromolecular Substances; Molecular Weight; Protein Binding; Protein Conformation; Prothrombin; Species Specificity; Thrombin; Tosyl Compounds

Topics: Blood Coagulation Factors; Blood Platelets; Diagnosis, Differential; Factor VII Deficiency; Fibrin; Fibrinolysis; Hemophilia A; Hemophilia B; Hemorrhage; Humans; Hypoprothrombinemias; Prothrombin Time; Thrombin; Vitamin K Deficiency

Recombinant activated factor VII (rFVIIa) is a haemostatic agent that is used for the treatment of haemophilia A patients with inhibitors. However, clinical response to rFVIIa is variable and unpredictable with currently available assays. We investigated the anti-fibrinolytic effects of rFVIIa in relation to thrombin generation (TG) and other haemostatic parameters in haemophilia A patients with inhibitors. After addition of rFVIIa to plasma, the clot-lysis assay, TF-dependent TG, TF-independent TG and parameters involved in coagulation, anticoagulation and fibrinolysis were assessed. The clot-lysis test distinguished two groups of patients: a group with a normal and a group with impaired anti-fibrinolytic response to rFVIIa. Our results showed a dose-dependent increase in TF-dependent TG and TF-independent TG in all individuals. There was a significant difference in TF-independent TG parameters between the normal and impaired response groups. In addition, there was a difference between the normal and impaired response group in prothrombin time, which could be explained by significantly higher levels of coagulation factors in the normal response group, and soluble thrombomodulin. In conclusion, we observed different in vitro responses following rFVIIa addition in plasma of patients with haemophilia A and inhibitors, which could be partially attributed to levels of procoagulant proteins and soluble thrombomodulin.

Topics: Adolescent; Adult; Aged; Blood Coagulation Factor Inhibitors; Child; Factor VIIa; Factor VIII; Female; Fibrin; Fibrin Clot Lysis Time; Fibrinolysis; Hemophilia A; Humans; Male; Middle Aged; Phenotype; Recombinant Proteins; Thrombin; Young Adult

The clot waveform analysis (CWA) provide valuable information beyond clotting time. The present study was planned to assess whether the activated partial thromboplastin time (aPTT)-CWA can differentiate between hemophilia A (HA), hemophilia B (HB), or hemophilia A with inhibitors (HAWI).. The aPTT-CWA was generated by an optical detection system (ACL-TOP™ 500 coagulation analyzer) and the other tests were performed as per instructions from the manufacturer in the kit.. A total of 75 samples (47-HA, 16-HAWI, and 12-HB) with prolonged aPTT were recruited. On analyzing the quantitative aPTT-CWA data of HA (non-inhibitors) and HB samples, the width of acceleration 1 [+] peak was the differentiating finding. Among the significant parameters, the second derivative [+] peak was lower in both mild and moderate HA, equating to HB. The time for the height of 1/2 fibrin formation and width of velocity was significantly higher in mild, moderate and severe HA. The study did not show any significant differentiating finding while comparing HAWI and hemophilia A non-inhibitors (HANI). In the subgroups of HAWI and HANI with aPTT <70 s and 70-100 s, the second derivative [+] peak (2A) was higher and the time for the height of 1/2 fibrin formation (1C) was lesser in HAWI.. The aPTT-CWA parameters may be supportive for the differentiation of hemophilia including its severity and the existence of inhibitors.

Topics: Blood Coagulation Tests; Fibrin; Hemophilia A; Hemophilia B; Humans; Partial Thromboplastin Time; Thrombosis

 Emicizumab is a bispecific antibody mimicking coagulation factor VIII (FVIII) employed to treat patients with hemophilia A (PwHA) regardless of FVIII inhibitor status. The identification of biological markers reflecting the hemostatic competence of patients under emicizumab therapy would have a great clinical value. Unfortunately, emicizumab over-corrects standard coagulation assays, precluding their use for evaluating the hemostatic correction achieved.  Six adults PwHA received a weekly dose of emicizumab of 3 mg/kg during weeks (W) 1 4 and 1.5 mg/kg from W5 onwards. Response to treatment was monitored weekly by emicizumab plasma concentration, thrombin generation (TG), and fibrin clot formation (FCF) and structure. TG and FCF results were compared to patient baseline, FVIII replacement, and healthy donors..  TG and FCF significantly increased in PwHA after the loading period, reaching a plateau that lasted until the end of monitoring. Similarly, fibrin clot network became denser with thinner fibrin fibers. However, TG contrary to FCF remained at the lower limits of reference values. Remarkably, despite having similar plateau concentrations of emicizumab some patients showed markedly different degrees of TG and FCF improvement..  Our study enriches the knowledge on the use of GCA to monitor non-factor replacement therapy, indicating that TG and FCF could act as direct markers of emicizumab biological activity. GCA allow to capture and visualize the individually variable response to emicizumab, leading a step forward to the personalization of patient treatment.

Topics: Adult; Antibodies, Bispecific; Factor VIII; Fibrin; Hemophilia A; Hemostatics; Humans; Thrombin

Platelet-targeted FVIII gene therapy can efficiently recover bleeding phenotype for hemophilia A (HA), yet characteristics of thrombus formation with this ectopic expression of factor VIII (FVIII) in platelets remain unclear. Here, we generated 2bF8

Topics: Animals; Blood Platelets; Factor VIII; Fibrin; Hemophilia A; Hemostatics; Humans; Mice; Mice, Knockout; Mice, Transgenic; Thrombosis

Factor VIII (FVIII) binding to endogenous von Willebrand factor (VWF) has constrained half-life extension of recombinant FVIII (rFVIII) products for hemophilia A. Efanesoctocog alfa (rFVIIIFc-VWF-XTEN; BIVV001) is a novel fusion protein designed to decouple FVIII from VWF in circulation and maximize half-life prolongation by XTEN. We compared functional clot formation and injury-induced platelet accumulation between efanesoctocog alfa and rFVIII.. The hemostatic potential of efanesoctocog alfa and rFVIII were assessed by measuring their dose-dependent effects on in vitro fibrin generation in hemophilic plasma and in vivo injury-induced platelet accumulation using intravital microscopy and repeat saphenous vein laser-induced injuries in hemophilia A mice.. Equal concentrations of efanesoctocog alfa or rFVIII (up to 1 IU/ml) added to plasma from patients with hemophilia A elicited similar kinetics for dose-dependent fibrin polymerization between factor products. In the presence of tissue plasminogen activator (tPA), clots formed had similar stability between products. Single intravenous doses (50, 100, or 150 IU/kg) of efanesoctocog alfa or rFVIII shortly before repeat saphenous vein laser-induced injuries increased platelet accumulation over time in a dose-dependent manner in hemophilia A mice. Platelet deposition kinetics were similar between products.. Equivalent doses of efanesoctocog alfa and rFVIII had similar efficacy in promoting fibrin clot formation and injury-induced platelet accumulation. The hemostatic potential of efanesoctocog alfa was indistinguishable from that of rFVIII.

Topics: Animals; Factor VIII; Fibrin; Hemophilia A; Hemostatics; Humans; Mice; Tissue Plasminogen Activator; von Willebrand Factor

Hemophilic arthropathy (HA) is characterized by joint damage following recurrent joint bleeds frequently observed in patients affected by the clotting disorder hemophilia. Joint bleeds or hemarthroses trigger inflammation in the synovial tissue, which promotes damage to the articular cartilage. The plasminogen activation system is integral to fibrinolysis, and the urokinase plasminogen activator, or uPA in particular, is strongly upregulated following hemarthroses. uPA is a serine protease that catalyzes the production of plasmin, a broad-spectrum protease that can degrade fibrin as well as proteins of the joint extracellular matrix and cartilage. Both uPA and plasmin are able to proteolytically generate active forms of matrix metalloproteinases (MMPs). The MMPs are a family of >20 proteases that are secreted as inactive proenzymes and are activated extracellularly. MMPs are involved in the degradation of all types of collagen and proteoglycans that constitute the extracellular matrix, which provides structural support to articular cartilage. The MMPs have an established role in joint destruction following rheumatoid arthritis (RA). They degrade cartilage and bone, indirectly promoting angiogenesis. MMPs are also implicated in the pathology of osteoarthritis (OA), characterized by degradation of the cartilage matrix that precipitates joint damage and deformity. HA shares a number of overlapping pathological characteristics with RA and OA. Here we discuss how the plasminogen activation system and MMPs might exacerbate joint damage in HA, lending insight into novel possible therapeutic targets to reduce the comorbidity of hemophilia.

Topics: Arthritis, Rheumatoid; Collagen; Enzyme Precursors; Fibrin; Fibrinolysin; Hemarthrosis; Hemophilia A; Humans; Matrix Metalloproteinases; Osteoarthritis; Peptide Hydrolases; Plasminogen; Proteoglycans; Urokinase-Type Plasminogen Activator

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

Macrophages make important contributions to inflammation and wound healing. We show here that macrophage polarization is deregulated in haemophilia in response to macrophage colony-stimulating factor (M-CSF) and partially in response to granulocyte-macrophage colony-stimulating factor (GM-CSF). As a result, haemophilia macrophages exhibit a specific impairment of M-CSF-mediated functions involved in wound healing such as clot invasion and phagocytosis. Haemophilia monocytes express reduced amounts of the receptors for M-CSF and GM-CSF, which correlates with a failure to express tumour necrosis factor α (TNFα) and CD163 in M-CSF-treated haemophilia macrophages and reduced expression of TNFα and CD206 after treatment with GM-CSF. Protein expression in response to M-CSF was regained with respect to CD163 and CD206 after embedding haemophilia monocytes in clotted plasma suggesting that a functioning coagulation system has positive effects on macrophage M2 polarization. Mimicking the functional deficits of haemophilia macrophages in normal macrophages was possible by adding leptin, which we found to be elevated in the blood of haemophilia patients, to a monocyte cell line. The increase of leptin occurred in conjunction with C-reactive protein in a body mass index-controlled cohort suggesting that haemophilia patients harbour chronic low-grade inflammation. Together, our data indicate that impaired clotting in haemophilia patients leads to increased inflammation and a deregulation in macrophage differentiation, which may explain the commonly observed deficits in wound healing and tissue regeneration.

Topics: Adult; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Blood Circulation; Blood Coagulation; Cell Differentiation; Cohort Studies; Enzyme-Linked Immunosorbent Assay; Erythrocytes; Fibrin; Flow Cytometry; Granulocyte-Macrophage Colony-Stimulating Factor; Hemophilia A; Humans; Lectins, C-Type; Leukocytes, Mononuclear; Macrophage Colony-Stimulating Factor; Macrophages; Male; Mannose Receptor; Mannose-Binding Lectins; Microscopy, Fluorescence; Monocytes; Phagocytosis; Receptors, Cell Surface; Tumor Necrosis Factor-alpha; Young Adult

Zeolite is a multifunctional material, which recently exhibited promising prospects in emerging biological and medical applications. This study reported a new bio-inorganic hybrid of zeolite-fibrin clot composite serving as haemostatic agent in haemophilia A. The zeolite-fibrin clot composite promoted haemocompatibility and helped to achieve short clotting time both in vitro (22 ± 3 s vs. >600 s) and in vivo (4.5 min vs. >60 min) compared to control in coagulation factor VIII deficiency bleeding model. Therefore, in situations of surgical operation or accidental injury, this effective and ready-to-use haemostatic agent may provide rapid haemostasis for haemophilia A patients.

Topics: Animals; Biocompatible Materials; Fibrin; Hemophilia A; Hemorrhage; Hemostasis; Hemostatics; Humans; Male; Mice; Zeolites

The activated partial thromboplastin time (APTT) waveform includes several parameters that are related to various underlying diseases. The APTT waveform was examined in various diseases. Regarding the pattern of APTT waveform, a biphasic pattern of the first or second derivative curve (DC) was observed in patients with hemophilia and patients positive for antiphospholipid (aPL) antibodies or coagulation factor VIII (FVIII) inhibitors. The time of the first and second DC and fibrin formation at 1/2 height were prolonged in patients with hemophilia, patients with inhibitors, patients positive for aPL, patients treated with anti-Xa agents, and patients with disseminated intravascular coagulation (DIC). These values all tended to decrease in pregnant women (at 28-36 weeks' gestation). The height of the second derivative peak 1 was significantly lower in patients with hemophilia, patients with FVIII inhibitors, patients positive for aPL, patients treated with anti-Xa agents, and patients with DIC; these values tended to be significantly higher in pregnant women. The height of the first DC was significantly lower in patients who were positive for FVIII inhibitors and was significantly higher in patients treated with anti-Xa agents and pregnant women. The height of the first and second DC was useful for the analysis of hemophilia, FVIII inhibitor, and aPL.

Topics: Antibodies, Antiphospholipid; Disseminated Intravascular Coagulation; Factor VIII; Factor Xa Inhibitors; Female; Fibrin; Hemophilia A; Humans; Male; Partial Thromboplastin Time; Pregnancy

Improved treatments are needed for hemophilia A and B, bleeding disorders affecting 400 000 people worldwide. We investigated whether targeting protein S could promote hemostasis in hemophilia by rebalancing coagulation. Protein S (PS) is an anticoagulant acting as cofactor for activated protein C and tissue factor pathway inhibitor (TFPI). This dual role makes PS a key regulator of thrombin generation. Here, we report that targeting PS rebalances coagulation in hemophilia. PS gene targeting in hemophilic mice protected them against bleeding, especially when intra-articular. Mechanistically, these mice displayed increased thrombin generation, resistance to activated protein C and TFPI, and improved fibrin network. Blocking PS in plasma of hemophilia patients normalized in vitro thrombin generation. Both PS and TFPIα were detected in hemophilic mice joints. PS and TFPI expression was stronger in the joints of hemophilia A patients than in those of hemophilia B patients when receiving on-demand therapy, for example, during a bleeding episode. In contrast, PS and TFPI expression was decreased in hemophilia A patients receiving prophylaxis with coagulation factor concentrates, comparable to osteoarthritis patients. These results establish PS inhibition as both controller of coagulation and potential therapeutic target in hemophilia. The murine PS silencing RNA approach that we successfully used in hemophilic mice might constitute a new therapeutic concept for hemophilic patients.

Topics: Animals; Blood Coagulation; Calcium-Binding Proteins; Carrier Proteins; Fibrin; Gene Silencing; Hemophilia A; Hemorrhage; Humans; Mice; Mice, Knockout; Thrombin

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

Essentials Platelet-Factor (F) VIII gene therapy is a promising treatment in hemophilia A. This study aims to evaluate if platelet-FVIII expression would increase the risk for thrombosis. Targeting FVIII expression to platelets does not induce or elevate thrombosis risk. Platelets expressing FVIII are neither hyper-activated nor hyper-responsive.. Background Targeting factor (F) VIII expression to platelets is a promising gene therapy approach for hemophilia A, and is successful even in the presence of inhibitors. It is well known that platelets play important roles not only in hemostasis, but also in thrombosis and inflammation. Objective To evaluate whether platelet-FVIII expression might increase thrombotic risk and thereby compromise the safety of this approach. Methods In this study, platelet-FVIII-expressing transgenic mice were examined either in steady-state conditions or under prothrombotic conditions induced by inflammation or the FV Leiden mutation. Native whole blood thrombin generation assay, rotational thromboelastometry analysis and ferric chloride-induced vessel injury were used to evaluate the hemostatic properties. Various parameters associated with thrombosis risk, including D-dimer, thrombin-antithrombin complexes, fibrinogen, tissue fibrin deposition, platelet activation status and activatability, and platelet-leukocyte aggregates, were assessed. Results We generated a new line of transgenic mice that expressed 30-fold higher levels of platelet-expressed FVIII than are therapeutically required to restore hemostasis in hemophilic mice. Under both steady-state conditions and prothrombotic conditions induced by lipopolysaccharide-mediated inflammation or the FV Leiden mutation, supratherapeutic levels of platelet-expressed FVIII did not appear to be thrombogenic. Furthermore, FVIII-expressing platelets were neither hyperactivated nor hyperactivatable upon agonist activation. Conclusion We conclude that, in mice, more than 30-fold higher levels of platelet-expressed FVIII than are required for therapeutic efficacy in hemophilia A are not associated with a thrombotic predilection.

Topics: Animals; Antithrombins; Blood Coagulation; Blood Platelets; Coagulants; Factor V; Factor VIII; Fibrin; Fibrin Fibrinogen Degradation Products; Fibrinogen; Genetic Therapy; Hemophilia A; Hemostasis; Humans; Leukocytes; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Phenotype; Platelet Activation; Thrombin; Thrombosis

Activated (super)Factor V ((super)FVa) is a novel engineered FV with excellent prohemostatic efficacy. (Super)FVa has three APC cleavage site mutations and an interdomain disulfide bond. Stability, pharmacokinetics, and immunogenic and thrombogenic potential are reported here.. Stability and circulating half-life were determined after incubation in buffer and human plasma, and after injection into FVIII-deficient mice. Immunogenicity potential was assessed by B- and T-cell specific epitope prediction and structural analysis using surface area and atomic depth computation. Thrombogenic potential was determined by quantification of lung fibrin deposition in wild-type mice after intravenous injection of (super)FVa (200 U/kg), recombinant human (rh) Tissue Factor (0.4-16 pmol/kg), rhFVIIa (3 mg/kg) or saline.. FVa retained full activity over 30 h in buffer, the functional half-life in human plasma was 4.9 h, and circulating half-life in FVIII-deficient mice was ~30 min. Predicted immunogenicity was not increased compared to human FV. While rh Tissue Factor, the positive control, resulted in pronounced lung fibrin depositions (mean 121 μg/mL), (super)FVa did not (6.7 μg/mL), and results were comparable to fibrin depositions with rhFVIIa (7.6 μg/mL) or saline (5.6 μg/mL).. FVa has an appropriate safety and stability profile for further preclinical development as a prohemostatic against severe bleeding.

Topics: Animals; Disease Models, Animal; Drug Stability; Factor Va; Factor VIII; Female; Fibrin; Half-Life; Hemophilia A; Hemostatics; Humans; Injections, Intravenous; Lung; Male; Mice, Inbred BALB C; Mice, Knockout; Mutagenesis, Site-Directed; Mutation; Protein Engineering; Protein Stability; Recombinant Proteins; Severity of Illness Index; Thrombin

Hemostasis occurs in two different topological scenarios: complete severing of a vessel or disruption of the vessel wall. Either to meet the daily rigors of active life or during an acute trauma, hemostasis involves the regulated and self-limiting production of thrombin to stop bleeding. In contrast, arterial and venous thrombosis typically involves the unregulated, intraluminal growth of a clot, in the absence of bleeding. For either hemostasis or thrombosis, the presence of flow and pressure gradients (delta-P, ΔP) dictates when and where thrombin and fibrin are located and in what quantity. For hemostatic clots, fibrin formation helped limit clot growth. We found that γ'-fibrinogen had a role in limiting clot growth via anti-thrombin activity at venous, but not arterial conditions. For hemophilic blood, severe factor deficiency (<1% healthy) led to a defect in both platelet and fibrin deposition under flow. However, moderate deficiency, which is associated with a less severe bleeding phenotype, had normalized platelet function but still lacked fibrin production. We conclude signaling levels of thrombin can be generated during moderate hemophilia to sufficiently activate platelets to achieve primary hemostasis, even if fibrin formation remains defective. Finally, as a clot grows, shear stresses can become sufficiently extreme in diseased arteries to drive von Willebrand Factor self-association into massive fibers, potentially the final burst of clot growth towards full thrombotic occlusion.

Topics: Blood Coagulation; Blood Platelets; Fibrin; Hemophilia A; Hemostasis; Humans; Platelet Activation; Stress, Mechanical; Thrombin; Thrombosis; von Willebrand Factor

Topics: Coagulants; Factor VIII; Fibrin; Hemophilia A; Hemostasis; Humans; Thrombin

In prior microfluidic studies with haemophilic blood perfused over collagen, we found that a severe deficiency (<1% factor level) reduced platelet and fibrin deposition, while a moderate deficiency (1-5%) only reduced fibrin deposition. We investigated: (i) the differential effect of rFVIIa (0.04-20 nm) on platelet and fibrin deposition, and (ii) the contribution of the contact pathway to rFVIIa-induced haemophilic blood clotting. Haemophilic or healthy blood with low and high corn trypsin inhibitor (CTI, 4 or 40 μg mL(-1) ) was perfused over collagen at an initial venous wall shear rate of 100 s(-1) . At 100 s(-1) wall shear rate, where FXIIa leads to thrombin production without added tissue factor, FXI-deficient blood (3%) or severely FVIII-deficient blood (<1%) produced no fibrin at either CTI level. Whereas rFVIIa potently enhanced platelet deposition, fibrin generation was not rescued. Distinct from the high CTI condition, engagement of the contact pathway (low CTI) in moderately FVIII-deficient (3%) or moderately FIX-deficient blood (5%) resulted in enhanced platelet and fibrin deposition following 4 nm rFVIIa supplementation. In mildly FVIII-deficient blood (15%) at <24 h since haemostatic therapy, rFVIIa enhanced both platelet and fibrin generation in either CTI condition although fibrin was produced more quickly and abundantly in low CTI. For tissue factor-free conditions of severe haemophilic blood clotting, we conclude that rFVIIa reliably generates low levels of 'signaling' thrombin sufficient to enhance platelet deposition on collagen, but is insufficient to drive fibrin polymerization unless potentiated by the contact pathway.

Topics: Blood Coagulation; Blood Coagulation Tests; Blood Platelets; Case-Control Studies; Collagen; Factor VIIa; Factor XI Deficiency; Fibrin; Hemophilia A; Hemophilia B; Humans; Microfluidic Analytical Techniques; Platelet Activation; Platelet Adhesiveness; Protein Binding; Recombinant Proteins

Patients with haemophilia A have seriously impaired thrombin generation due to an inherited deficiency of factor (F)VIII, making them form unstable fibrin clots that are unable to maintain haemostasis. Data on fibrin structure in haemophilia patients remain limited. Fibrin permeability, assessed by a flow measurement technique, was investigated in plasma from 20 patients with severe haemophilia A treated on demand, before and 30 minutes after FVIII injection. The results were correlated with concentrations of fibrinogen, FVIII and thrombin-activatable fibrinolysis inhibitor (TAFI), and global haemostatic markers: endogenous thrombin potential (ETP) and overall haemostatic potential (OHP). Fibrin structure was visualised using scanning electron microscopy (SEM). The permeability coefficient Ks decreased significantly after FVIII treatment. Ks correlated significantly with FVIII levels and dosage, and with ETP, OHP and levels of TAFI. SEM images revealed irregular, porous fibrin clots composed of thick and short fibers before FVIII treatment. The clots had recovered after FVIII replacement almost to levels in control samples, revealing compact fibrin with smaller intrinsic pores. To the best of our knowledge, this is the first description of fibrin porosity and structure before and after FVIII treatment of selected haemophilia patients. It seems that thrombin generation is the main determinant of fibrin structure in haemophilic plasma.

Topics: Adolescent; Adult; Aged; Blood Coagulation; Carboxypeptidase B2; Factor XII; Fibrin; Fibrinogen; Hemophilia A; Hemostasis; Humans; Microscopy, Electron, Scanning; Middle Aged; Molecular Structure; Porosity; Young Adult

Coagulation factor deficiencies create a range of bleeding phenotypes. Microfluidic devices offer controlled hemodynamics and defined procoagulant triggers for measurement of clotting under flow.. We tested a flow assay of contact pathway-triggered clotting to quantify platelet and fibrin deposition distal of dysfunctional thrombin production. Microfluidic metrics were then compared with PTT or % factor activity assays.. Whole blood (WB) treated with low level corn trypsin inhibitor (4 μg mL⁻¹) from nine healthy donors and 27 patients (deficient in factor [F] VIII, 19 patients; FIX, one patient; FXI, one patient; VWF, six patients) was perfused over fibrillar collagen at wall shear rate = 100 s⁻¹.. Using healthy WB, platelets deposited within 30 s, while fibrin appeared within 6 min. Compared with healthy controls, WB from patients displayed a 50% reduction in platelet deposition only at < 1% factor activity. In contrast, striking defects in fibrin deposition occurred for patients with < 13% factor activity (or PTT > 40 s). Full occlusion of the 60-μm high channel was completely absent over the 15-min test in patients with < 1% factor activity, while an intermediate defect was present in patients with > 1% factor.. Spontaneous bleeding in patients with < 1% factor activity may be linked to deficits in both platelet and fibrin deposition, a risk known to be mitigated when factor levels are raised to > 1% activity (PTT of ~40-60 s), a level that does not necessarily rescue fibrin formation under flow.

Topics: Blood Coagulation; Blood Platelets; Fibrin; Hemophilia A; Humans; Microfluidics

In recent years, a number of tissue factor pathway inhibitor (TFPI) antagonists have been developed to serve as bypassing agents to improve hemostasis in hemophilia A. Since TFPI antagonists and FVIII concentrates are procoagulants, their combined effect on spatial clot formation could be potentially pro-thrombotic.. To investigate the cooperative effect of TFPI inhibition and supplementation of FVIII in hemophilia A in a spatial, reaction-diffusion experiment in vitro.. Plasma was collected at different time points from hemophilia A patients undergoing prophylaxis and was supplemented in vitro with TFPI inhibitor BAX499 (formerly ARC19499) at concentrations from 0 up to 600nM. Clotting propagation in recalcified plasma activated by a surface with immobilized tissue factor (TF) was monitored by videomicroscopy.. Increasing concentration of BAX499 improved coagulation for all hemophilia A plasma samples activated with TF at 1.6pmole/m(2) by shortening lag time and increasing initial clot growth velocity and clot size. In contrast, plasma concentration of FVIII had little effect on lag time, but increased spatial clot growth velocity. There was a decrease in the BAX499 efficiency as FVIII concentration increased (lag time shortened by 50% if FVIII:C<5%, but the effect was only 25% if FVIII:C>30%).. The results indicate that BAX499 has an effect on clotting in hemophilia A plasma at low FVIII concentrations, however has little effect at high FVIII concentrations.

Topics: Aptamers, Nucleotide; Drug Interactions; Factor VIII; Fibrin; Hemophilia A; Humans; Lipoproteins

Clot waveform analysis extends the interpretation of aPTT measurement curves. The curve is mathematically processed to obtain information about fibrin formation kinetics including semiquantitative determination of thrombin, prothrombinase and tenase activity.. In this study the feasibility of clot waveform analysis for monitoring of haemophilia A was investigated using blood samples from healthy controls as well as haemophilia A patients under various clinical conditions.. Thrombin, prothrombinase and tenase activity show a high correlation to factor VIII levels. Tenase activity was found to exhibit a linear relationship to factor VIII levels over a very large concentration range and was able to discriminate patients with severe, moderate and mild haemophilia.. Clot waveform analysis is an easy, fast and cheap method to access disturbances in clot formation and can be done without any additional measurements beside an aPTT.

Topics: Blood Coagulation Tests; Data Interpretation, Statistical; Diagnosis, Computer-Assisted; Factor VIII; Feasibility Studies; Fibrin; Hemophilia A; Humans; Metabolic Clearance Rate; Pattern Recognition, Automated; Reproducibility of Results; Sensitivity and Specificity

Topics: Animals; Clinical Trials as Topic; Drug Discovery; Fibrin; Gene Expression Regulation; Hemophilia A; Humans; Recombinant Proteins; RNA Interference; Thrombophilia; Thrombosis

Coagulation factor VIII (FVIII) plays an essential role in haemostasis. To date, physiologic activity of FVIII circulating in the bloodstream (S-FVIII) is evaluated by classic coagulation assays. However, the functional relevance of FVIII (-von Willebrand factor complex) immobilised on thrombogenic surfaces (I-FVIII) remains unclear. We used an in vitro perfusion chamber system to evaluate the function of I-FVIII in the process of mural thrombus formation under whole blood flow conditions. In perfusion of either control or synthetic haemophilic blood, the intra-thrombus fibrin generation on platelet surfaces significantly increased as a function of I-FVIII, independent of S-FVIII, under high shear rate conditions. This I-FVIII effect was unvarying regardless of anti-FVIII inhibitor levels in synthetic haemophilic blood. Thus, our results illustrate coagulation potentials of immobilised clotting factors, distinct from those in the bloodstream, under physiologic flow conditions and may give a clue for novel therapeutic approaches for haemophilic patients with anti-FVIII inhibitors.

Topics: Blood Coagulation; Blood Platelets; Factor VIII; Fibrin; Hemophilia A; Hemorheology; Humans; Immobilized Proteins; Perfusion; Platelet Adhesiveness; Platelet Aggregation; Surface Properties; Thrombosis; von Willebrand Factor

The process of human blood clotting involves a complex interaction of continuous-time/continuous-state processes and discrete-event/discrete-state phenomena, where the former comprise the various chemical rate equations and the latter comprise both threshold-limited behaviors and binary states (presence/absence of a chemical). Whereas previous blood-clotting models used only continuous dynamics and perforce addressed only portions of the coagulation cascade, we capture both continuous and discrete aspects by modeling it as a hybrid dynamical system. The model was implemented as a hybrid Petri net, a graphical modeling language that extends ordinary Petri nets to cover continuous quantities and continuous-time flows. The primary focus is simulation: (1) fidelity to the clinical data in terms of clotting-factor concentrations and elapsed time; (2) reproduction of known clotting pathologies; and (3) fine-grained predictions which may be used to refine clinical understanding of blood clotting. Next we examine sensitivity to rate-constant perturbation. Finally, we propose a method for titrating between reliance on the model and on prior clinical knowledge. For simplicity, we confine these last two analyses to a critical purely-continuous subsystem of the model.

Topics: Activated Protein C Resistance; Algorithms; Blood Coagulation; Blood Coagulation Factors; Computational Biology; Computer Simulation; Fibrin; Hemophilia A; Humans; Kinetics; Models, Biological; Models, Statistical; Protein Binding; Prothrombin Time; Systems Biology; Thrombin

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 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

It has been reported that thrombin generation test (TGT) may be a useful tool to monitor recombinant factor VIIa (rFVIIa). However, TGT does not reflect the stability of fibrin clot and its resistance to fibrinolysis which are crucial. Using whole-blood thromboelastography (TEG) and tissue plasminogen activator (tPA), we developed an in-vitro model to assess fibrin clot stability. Fibrin fibres were thicker in haemophiliacs compared with controls (P < 0.0001). After addition of rFVIIa 90 μg kg(-1), the diameter of fibrin fibres was dramatically decreased (P = 0.006). TEG-tPA assay showed a dose-dependent improvement of clot stability in the presence of rFVIIa. These data demonstrate a significant correlation between fibrin clot structure and its stability (P = 0.001). We also showed a correlation between thrombin generating capacity and clot resistance to fibrinolysis. Despite this overall correlation, a relatively large spreading around a general trend was observed, suggesting that the two assays bring complementary information on the haemostatic effect of rFVIIa.

Topics: Analysis of Variance; Blood Coagulation; Factor VIIa; Fibrin; Hemophilia A; Hemostasis; Hemostatics; Humans; Microscopy, Electron, Scanning; Models, Biological; Recombinant Proteins; Thrombelastography; Thrombin; Tissue Plasminogen Activator

The bypassing agent recombinant factor VIIa (rFVIIa) is efficacious in treating bleeding in hemophilia patients with inhibitors. Efforts have focused on the rational engineering of rFVIIa variants with increased hemostatic potential. One rFVIIa analog (V158D/E296V/M298Q-FVIIa, NN1731) improves thrombin generation and clotting in purified systems, whole blood from hemophilic patients and factor VIII-deficient mice.. We used calibrated automated thrombography and plasma clotting assays to compare effects of bypassing agents (rFVIIa, NN1731) on hemophilic clot formation, structure, and ability to resist fibrinolysis.. Both rFVIIa and NN1731 shortened the clotting onset and increased the maximum rate of fibrin formation and fibrin network density in hemophilic plasma clots. In the presence of tissue plasminogen activator, both rFVIIa and NN1731 shortened the time to peak turbidity (TTPeak(tPA)) and increased the area under the clot formation curve (AUC(tPA)). Phospholipids increased both rFVIIa and NN1731 activity in a lipid concentration-dependent manner. Estimated geometric mean concentrations of rFVIIa and NN1731 producing similar onset, rate, TTPeak(tPA), and AUC(tPA) as seen with 100% factors VIII and IX were: 24.5, 74.3, 29.7, and 37.1 nM rFVIIa, and 8.6, 31.2, 9.0, and 11.3 nM NN1731, respectively. In each case, the NN1731 concentration was significantly lower than rFVIIa.. These findings suggest that like rFVIIa, NN1731 improves the formation, structure, and stability of hemophilic clots. Higher lipid concentrations may facilitate assessment of both rFVIIa and NN1731 activity. NN1731 appears likely to support rapid clot formation in tissues with high endogenous fibrinolytic activity.

Topics: Animals; Blood Coagulation; Factor VII; Factor VIIa; Fibrin; Fibrinolysis; Hemophilia A; Humans; Mice; Microscopy, Confocal; Phospholipids; Plasma; Recombinant Proteins; Thrombin

Tissue factor pathway inhibitor (TFPI) is a major regulator of clotting initiation and a promising target for pro- and anticoagulation therapy. The aptamer BAX499 (formerly ARC19499) is a high-affinity specific TFPI antagonist designed to improve hemostasis. However, it is not clear how stimulation of coagulation onset by inactivating TFPI will affect spatial and temporal clot propagation.. To examine the BAX499 effect on clotting in a spatial, reaction-diffusion experimental system in comparison with that of recombinant activated factor VII (rVIIa).. Clotting in plasma activated by immobilized tissue factor (TF) was monitored by videomicroscopy.. BAX499 dose-dependently improved coagulation in normal and hemophilia A plasma activated with TF at 2 pmole m(-2) by shortening lag time and increasing clot size by up to ~2-fold. The effect was TFPI specific as confirmed by experiments in TFPI-depleted plasma with or without TFPI supplementation. Clotting improvement was half-maximal at 0.7 nm of BAX499 and reached a plateau at 10 nm, remaining there at concentrations up to 1000 nm. The BAX499 effect decreased with TF surface density increase. RVIIa improved clotting in hemophilia A plasma activated with TF at 2 or 20 pmole m(-2) , both by shortening lag time and increasing spatial velocity of clot propagation; its effects were strongly concentration dependent.. BAX499 significantly improves spatial coagulation by inhibiting TFPI in a spatially localized manner that is different to that observed with rVIIa.

Topics: Aptamers, Nucleotide; Blood Coagulation; Computer Simulation; Factor VIIa; Fibrin; Hemophilia A; Hemostasis; Humans; In Vitro Techniques; Lipoproteins; Male; Microscopy, Video; Models, Biological; Recombinant Proteins

The conversion of fibrinogen to fibrin and its crosslinking to form a stable clot are key events in providing effective hemostasis.. To evaluate the relationship of fibrinopeptide (FP) release and factor (F) XIII activation in whole blood from hemophiliacs.. We investigated FPA and FPB release, FXIII activation and fibrin mass in tissue factor-initiated coagulation in whole blood from individuals with hemophilia and healthy subjects.. In hemophiliacs, the rates of fibrin formation were delayed as compared to healthy individuals. FPA/FPB release and FXIII activation were decreased in hemophiliacs vs. healthy individuals: 5.4 +/- 0.7 microM min(-1) to 1.7 +/- 0.4 microM min(-1) (P = 0.003), 2.3 +/- 0.6 microM min(-1) to 0.5 +/- 0.1 microM min(-1) (P = 0.025), and 12.1 +/- 0.7 nM min(-1) to 3.1 +/- 0.7 nM min(-1) (P < 0.0005), respectively. More FPA was released in hemophiliacs (6.6 +/- 1.2 microM) prior to clot time (CT) than in healthy individuals (2.6 +/- 0.4 microM, P = 0.013), whereas FPB and activated FXIII levels remained comparable. FXIII activation, which normally coincides with FPA release, was delayed in hemophiliacs. At CT in normal blood, the FPA concentration was 2.6-fold higher than that of FPB (P = 0.003), whereas in hemophiliacs this ratio was increased to 6.6-fold (P = 0.001).. These data suggest that essential dynamic correlations exist between the presentations of fibrin I, fibrin II, and FXIIIa. The 'discordance' of fibrin formation in hemophiliacs results in clots that are more soluble than normal (43% lower mass; P = 0.02). The resulting poor physical clot strength probably plays a crucial role in the pathology of hemophilia.

Topics: Adult; Case-Control Studies; Factor XII; Fibrin; Hemophilia A; Humans; Male

Topics: Factor VIIa; Factor X; Fibrin; Gels; Hemophilia A; Hemostasis; Humans; In Vitro Techniques; Protein Binding; Recombinant Proteins; Thrombin; Tissue Scaffolds

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

Many patients with hemophilia, particularly those with inhibitory antibodies, utilize central venous access devices (CVADs) to facilitate frequent infusions. Infection of these devices is a common complication of factor replacement therapy. This communication reports our center's experience with CVAD infection in three patients with severe hemophilia A undergoing immune tolerance therapy (ITT) in whom intermittent infusions of recombinant tissue plasminogen activator (rTPA, Cathflo Activase) were utilized. In this small experience, patients experienced a decreased frequency of gram-positive infections when receiving routine rTPA treatments. Larger randomized trial should be performed in this patient population at high risk of CVAD infection.

Topics: Anti-Bacterial Agents; Bacterial Infections; Catheterization, Central Venous; Child, Preschool; Desensitization, Immunologic; Disease Susceptibility; Drug Evaluation; Factor VIII; Fibrin; Fibrinolysis; Hemophilia A; Humans; Isoantibodies; Recombinant Proteins; Thrombophilia; Tissue Plasminogen Activator

Mural thrombus generation at sites of damaged vessel walls is essential for both physiological haemostasis and pathological intravascular thrombosis. While thrombi are established by the concerted action of platelet aggregation and blood coagulation, most previous in vitro coagulation assays have evaluated fibrin clot formation in a closed stirring situation that lacks blood cells including platelets. We describe here a modified flow chamber system, established originally for platelet functional studies, that enables real-time observation of intra-thrombus fibrin accumulation during platelet thrombogenesis under flow conditions. Analysis by confocal laser scanning microscopy during perfusion of whole blood anticoagulated to various extents revealed that the size and shape of mural thrombi can depend on the intra-thrombus fibrin development under high shear rate conditions. These observations were confirmed by perfusion of heparinized blood or blood from haemophilia patients with or without addition of activated factor VII. Thus, our experimental system provides visual evidence supporting the concept of "cell-based coagulation under whole blood flow", which might be the most physiologically relevant model of comprehensive thrombogenicity in vivo to date. This system promises to help formulate strategies for haemostatic management of congenital coagulation disorders as well as for antithrombotic therapy targeting fatal arterial thrombosis.

Topics: Adult; Anticoagulants; Arginine; Blood Coagulation; Blood Flow Velocity; Blood Platelets; Collagen; Dose-Response Relationship, Drug; Factor VIIa; Fibrin; Hemolysis; Hemophilia A; Hemorheology; Heparin; Humans; Male; Microscopy, Confocal; Microscopy, Electron, Scanning; Middle Aged; Perfusion; Pipecolic Acids; Sulfonamides; Surface Properties; Thrombosis

Recombinant activated factor VII (NovoSeven, rFVIIa) is used to abrogate bleeding in haemophiliacs with inhibitors and is hypothesised to work by increasing activated factor X generation on the platelet surface. We hypothesised that rFVIIa activity could be increased by the co-addition of platelet procoagulant surface. This study characterised the ability of a rehydrated, lyophilised (RL) platelet preparation to increase rFVIIa activity in haemophilic conditions. RL platelets supported thrombin generation in the presence of factors VIII and IX but, in the absence of factors VIII and IX, thrombin generation was significantly reduced. RL platelets supported rFVIIa-mediated thrombin generation in a rFVIIa-concentration dependent manner. In a cell-based in vitro model of haemophilia, the presence of RL platelets increased the rFVIIa-dependent thrombin generation rate 2.8-fold compared with rFVIIa alone. Similarly, the addition of RL platelets plus rFVIIa to the in vitro model of haemophilia and to haemophilic platelet-rich plasma shortened the onset of clot formation and increased clot stability in a fibrinolytic environment versus rFVIIa alone. These results suggest that RL platelets can support rFVIIa-mediated thrombin generation, and that co-administration of RL platelets with rFVIIa may increase the efficacy of rFVIIa in some patients.

Topics: Blood Coagulation; Blood Coagulation Factors; Cells, Cultured; Combined Modality Therapy; Dose-Response Relationship, Drug; Factor VII; Factor VIIa; Fibrin; Hemophilia A; Humans; Nephelometry and Turbidimetry; Platelet Activation; Platelet Adhesiveness; Recombinant Proteins; Thrombin; Tissue Preservation

Fibrin gel structure has been shown to be dependent on the thrombin concentration as well as the rate of thrombin generation. Accordingly, factor VIII (FVIII)- and FIX-deficient plasma (hemophilia A and B) form loose fibrin clots with high permeability constants. By adding rFVIIa in vitro to FVIII-deficient plasma containing platelets (frozen and thawed), the fibrin gel permeability constant normalized, indicating that extra rFVIIa (1.2 microg mL(-1) or higher) induced a tight fibrin structure. Thrombin generation is highly dependent on the number of platelets, and in this study it was demonstrated that the addition of rFVIIa (5 microg mL(-1)) normalizes the fibrin gel permeability in samples containing platelets (frozen-thawed) in numbers of at least down to 20 x 10(6) mL(-1). The effect of rFVIIa was not observed when unfrozen platelets instead of frozen-thawed platelets were added. Neither was any effect on the fibrin permeability seen, in the presence of annexin V, known to block the effect of phospholipids on the platelet surface. This indicates an important role of platelet phospholipids for the effect of rFVIIa. A similar effect on the fibrin permeability of rFVIIa was observed when added to platelet-rich plasma from a patient with Glanzmann thrombasthenia. Recombinant FVIIa has been found to induce hemostasis in patients with hemophilia and inhibitors against FVIII/FIX as well as in patients with Glanzmann thrombasthenia, indicating the importance of the formation of a tight fibrin gel structure, more resistant against premature proteolysis, for maintaining hemostasis. In conclusion, the addition of rFVIIa (5 microg mL(-1)) also substantially decreased the permeability constant of fibrin gels formed in FVIII-deficient plasma in the presence of low numbers of frozen-thawed platelets (down to 20 x 10(6) mL(-1)). A similar pattern was obtained in plasma from a Glanzmann patient. No effect was found in the presence of unfrozen instead of frozen-thawed platelets. Annexin V blocked any effect of rFVIIa. A normalization of the overall fibrinolysis potential (OFP) during the same condition supports the effect of rFVIIa on the fibrin permeability in the presence of a limited number of platelets.

Topics: Adult; Annexin A5; Blood Platelets; Cryopreservation; Factor VII; Factor VIIa; Female; Fibrin; Gels; Hemophilia A; Hemostasis; Humans; Microscopy, Confocal; Middle Aged; Permeability; Phospholipids; Recombinant Proteins; Thrombasthenia

Patients with hemophilia have an impaired thrombin generation and therefore form loose fibrin hemostatic plugs that are easily dissolved by fibrinolysis. This prevents maintained hemostasis in these patients, resulting in a severe bleeding disorder. Recombinant (F)VIIa has been shown to enhance thrombin generation on already thrombin-activated platelets in the absence of FVIII and FIX. An efficacy rate of 80-90% has been found in hemophilia patients with inhibitors against FVIII or FIX both in association with major surgery and in the treatment of serious bleedings. In a model measuring fibrin clot permeability in a platelet-containing system described by Blombäck et al. (1994) this was demonstrated to be dependent on the concentration of FVIII and FIX. The addition of rFVIIa in concentrations of 1.9, 4.8 and 9.6 microg mL(-1) normalized fibrin clot permeability. The concentration of 1.9 microg mL(-1) of rFVIIa normalized clot permeability in this system and the higher concentrations of rFVIIa added only slightly to the effect. No further decrease in clot permeability was found when rFVIIa in a concentration of 1.9 microg mL(-1) was added to a sample with a normal concentration (100%) of FVIII or FIX. Higher concentrations of rFVIIa added to the plasma containing 100% of FVIII or FIX induced only a slight further decrease of fibrin permeability constant, arguing against any unwanted effect of extra rFVIIa on clot permeability in the case of a normal hemostasis. Furthermore, the fibrin network was studied with 3D microscopy and the loose network found in the absence of FVIII or FIX increased in density with increasing FVIII or FIX concentrations. The addition of rFVIIa to FVIII- or FIX-deficient systems altered the network structure, making the fibers thinner and more tightly packed.

Topics: Blood Coagulation; Cells, Cultured; Dose-Response Relationship, Drug; Factor IX; Factor VII; Factor VIIa; Factor VIII; Fibrin; Hemophilia A; Hemophilia B; Humans; Models, Biological; Permeability; Porosity; Recombinant Proteins

Haemophilia is the most serious bleeding model that nature has provided us with, indicating the importance of factor FVIII and FIX in haemostasis. According to current knowledge, haemostasis is initiated by the formation of a complex between tissue factor (TF), exposed as a result of a vessel wall injury, and activated FVII (FVIIa) that is normally present in circulating blood. The TF-FVIIa complexes convert FX into FXa on the TF-bearing cell. FXa then activates prothrombin into thrombin. This limited amount of thrombin activates FVIII, FV, FXI and platelets. Thrombin-activated platelets change shape, resulting in exposure of negatively-charged phospholipids, which form the perfect template for full thrombin generation involving FVIII and FIX. In patients with haemophilia FVIII or FIX is missing. These individuals generate only initial limited amounts of thrombin as its generation is dependent on the presence of FVIII and FIX. Full thrombin generation is necessary for complete activation of FXIII and thrombin activatable fibrinolytic inhibitor to occur. Furthermore, full thrombin generation is important for the fibrin structure of the haemostatic plug. In the case of impaired thrombin generation, fibrin plugs will be loose and highly permeable. Such fibrin plugs are easily dissolved by normal fibrinolytic activity and thus prevent full and maintained haemostasis from occurring. The addition of rFVIIa to FVIII- or FIX-deficient plasma has been shown to increase thrombin generation in a cell-based in vitro model. Furthermore, extra rFVIIa was found to normalise fibrin clot permeability in vitro and to tighten the fibrin structure as studied by three-dimensional confocal microscopy. These findings indicate that administration of rFVIIa is capable of compensating for the lack of FVIII and FIX. Accordingly, the administration of exogenous rFVIIa has been found to stop bleedings in haemophilia patients and, provided it is given in doses high enough, to allow major surgery to be performed in severe haemophiliacs with inhibitors. As rFVIIa enhances thrombin generation on already activated platelets, it has been suggested that rFVIIa may also help to improve haemostasis in other situations involving impaired thrombin generation, such as platelet disorders (thrombocytopenia and functional platelet defects). Preliminary clinical data appear to support this. Patients with profuse bleeding due to extensive surgery or trauma often develop a complex coagulation pattern

Topics: Factor VII; Factor VIIa; Fibrin; Hemophilia A; Hemostasis; Hemostatics; Humans; Recombinant Proteins; Thrombin

The action of recombinant factor VIIa (rFVIIa) in coagulation deficiencies with increased risk of bleeding was investigated using in vitro perfusion. Blood samples were drawn from healthy donors, a patient with hemophilia A and inhibitors, and six patients undergoing oral anticoagulant treatment. Fragmin 10 U/mL was used as anticoagulant. rFVIIa (10 microg/mL in plasma) was added to blood samples, incubated for 1 minute at 37 degrees C, and perfusion studies performed for 10 minutes at 600 x s(-1) through annular chambers containing damaged vascular segments. Subendothelial fibrin and platelets were expressed as a percentage of subendothelial surface screened. Under different conditions, rFVIIa consistently restored or improved fibrin formation on the damaged vascular subendothelium exposed to circulating blood. It restored fibrin deposition in blood from the hemophilia A patient; in patients undergoing acenocoumarol treatment, it reduced the international normalized ratio (INR) from 2.47 to 1.25 with a significant increase in fibrin deposition. Platelet deposition varied slightly between clinical conditions but was less evident in the hemophilia A patient. These data support the concept that rFVIIa facilitates fibrin formation in these clinical situations, promoting procoagulant activity at sites of vascular damage where tissue factor is exposed. This could improve hemostasis in patients with hemophilia A and inhibitors, and in patients treated with oral anticoagulants.

Topics: Adult; Anticoagulants; Blood Coagulation; Blood Platelets; Coagulants; Dalteparin; Factor VII; Factor VIIa; Fibrin; Hemophilia A; Humans; International Normalized Ratio; Male; Perfusion; Platelet Aggregation; Recombinant Proteins; Stress, Mechanical; Thrombocytopenia

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

After the formation of a platelet-plug, generation of fibrin is necessary for its stabilization. Both congenital and acquired deficiencies of clotting factors occur, leading to retarded formation of fibrin. In congenital disorders, preoperative correction is possible and necessary. In acquired deficiencies, the type and feasibility of correction depends on the cause of the deficiency.

Topics: Blood Coagulation Disorders; Fibrin; Hemophilia A; Hemophilia B; Hemostasis, Surgical; Humans; Vitamin K Deficiency; von Willebrand Diseases

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

To present current strategies for the treatment of hemophilia and von Willebrand's disease.. Prophylactic and corrective therapy with hemostatic and adjunctive agents: DDAVP (1-desamino-8-D-arginine vasopressin [desmopressin acetate]), recombinant coagulation products (human Factor VIII and human Factor VIIa) or virally inactivated plasma-derived products (high- or ultra-high-purity human Factor VIII or human Factor VIII concentrate containing von Willebrand factor activity, porcine Factor VIII, high-purity human Factor IX, human prothrombin-complex concentrate, human activated prothrombin-complex concentrate), adjunctive antifibrinolytic agents, topical thrombin and fibrin sealant. The induction of immune tolerance in patients in whom inhibitors develop should also be considered.. Morbidity and quality of life associated with bleeding and treatment.. Relevant clinical studies and reports published from 1974 to 1994 were examined. A search was conducted of our reprint files, MEDLINE, citations in the articles reviewed and references provided by colleagues. In the MEDLINE search the following terms were used singly or in combination: "hemophilia," "von Willebrand's disease," "Factor VIII," "Factor IX," "von Willebrand factor," "diagnosis," "management," "home care," "comprehensive care," "inhibitor," "AIDS," "hepatitis," "life expectancy," "complications," "practice guidelines," "consensus statement" and "controlled trial." The in-depth review included only articles written in English from North America and Europe that were relevant to human disease and pertinent to a predetermined outline. The availability of treatment products in Canada was also considered.. Minimizing morbidity and maximizing functional status and quality of life were given a high value.. Proper prophylactic or early treatment with appropriate hemostatic agents minimizes morbidity and functional disability and improves quality of life. Economic gains are realized through the reduction of mortality and morbidity and their associated costs. The patient has a better opportunity to contribute to society through gainful employment and the fulfillment of social roles. Potential harms include HIV infection, hepatitis B, hepatitis C and the development of inhibitor antibodies to clotting-factor concentrates. The risk of viral transmission has been minimized through the development of procedures for the viral inactivation of plasma-derived clotting-factor concentrates and through the use of recombinant coagulation-factor concentrates and other non-plasma-derived hemostatic agents.. DDAVP is the drug of choice for patients with mild hemophilia or type 1 or 2 (except 2B) von Willebrand's disease whose response to DDAVP in previous testing has been found to be adequate. Therapeutic blood components of choice include recombinant products and virally inactivated plasma-derived products. In Canada the recommended products are recombinant Factor VIII for hemophilia A, high-purity plasma-derived Factor IX for hemophilia B and plasma-derived Factor VIII concentrates containing adequate von Willebrand factor (e.g., Haemate P) for von Willebrand's disease. Dosages vary according to specific indications. Adjunctive antifibrinolytic agents, topical thrombin and fibrin sealant are useful for the treatment of oral or dental bleeds and localized bleeds in accessible sites. In patients with inhibitor antibodies, high-dose human or porcine Factor VIII is usually effective when the inhibitor titre is less than 5 Bethesda units/mL. In nonresponsive patients, or in those whose inhibitor titre is higher, "bypassing" agents (e.g., activated prothrombin-complex concentrate and recombinant Factor VIIa) are useful. Long-term management may include immune-tolerance induction.. These recommendations were reviewed and approved by the Association of Hemophilia Clinic Directors of Canada (AHCDC) and the Medical and Scientific Advisory Committee of the Canadian Hemophilia Society. No similar consensus statements or practice guidelines are available for comparison.. These recommendations were developed at the request of the Canadian Blood Agency, which funds the provision of all coagulation-factor concentrates for people with congenital bleeding disorders, and were developed and endorsed by the AHCDC and the Medical and Scientific Advisory Committee of the Canadian Hemophilia Society.

Topics: Antifibrinolytic Agents; Canada; Deamino Arginine Vasopressin; Factor IX; Factor VIII; Fibrin; Health Care Costs; Hemophilia A; Humans; Recombinant Proteins; Thrombin; von Willebrand Diseases

Thrombus formation on collagen fibrils was quantified at venous (100/s) and arterial (650/s and 2,600/s) wall shear rates in blood from patients with various subtypes of von Willebrand disease (vWD) and with hemophilia A (HA). Nonanticoagulated blood was drawn directly from an antecubital vein over purified type III collagen fibrils exposed in parallel-plate perfusion chambers. Blood-collagen interactions were differentiated and quantified by morphometry as platelet adhesion, thrombus height, thrombus volume, and deposition of fibrin strands. Sixteen patients with vWD, including four type III, six type I, four type IIA, and two type IIB, were compared with 26 normal subjects and nine patients with HA, including six severe HA and three mild HA. Platelet adhesion and thrombus formation at 2,600/s were significantly decreased in blood from patients with vWD type III, IIA, and IIB, but not in blood from patients with type I and in HA. The abnormal thrombus formation was apparently not related to the decreased levels of factor VIII (F.VIII), because thrombus height and volume were normal in severe and mild HA. Thrombus formation at 650/s was also significantly decreased in patients with vWD type III, IIA, and IIB and slightly reduced in type I. Significant reduction in thrombus volume and height was also observed in blood from patients with severe HA, but not in mild HA. Thrombus dimensions were not affected at 100/s in the vWD subtypes. However, significantly decreased thrombus height and virtually absent fibrin deposition were observed in blood from patients with severe HA. Apparently, F.VIII supports thrombus formation at low and intermediate shear conditions, presumably through the generation of thrombin. In contrast, von Willebrand factor (vWF) mediates not only platelet adhesion, but also thrombus formation at intermediate and high shear rates. Thus, the relative contribution of coagulation (F.VIII) and platelet function (vWF) in thrombus formation appears to be shear rate dependent, but having optimal effects at different shear conditions.

Topics: Biomechanical Phenomena; Blood Coagulation; Collagen; Fibrin; Hemophilia A; Humans; Platelet Adhesiveness; von Willebrand Diseases

The bolus intravenous infusion of factor Xa in combination with phosphatidylcholine/phosphatidylserine (PCPS) vesicles, at a dose of 2.6 x 10(-11) moles and 4.0 x 10(-8) moles/kg body weight respectively, has previously been shown to correct the bleeding diathesis of haemophilic (factor VIII deficient) dogs (Br J Haematol 1988; 69: 491-7). The cuticle bleeding time (CBT) was used as the test to evaluate objectively this response. Transmission electron microscopy was performed to document the evolution of haemostatic plugs forming in the vascular bed of the injured cuticles of both normal and factor VIII deficient dogs with and without treatment with factor Xa/PCPS. The dosage previously shown to normalize the CBT in haemophilic and significantly shorten it in normal animals was used. Subjective and objective observations, using morphometric techniques, were made over a period of 25 min following injury induction. The administration of factor Xa/PCPS was associated with complete and sustained normalization of haemostatic plug development in the haemophiliacs including platelet recruitment, activation and compaction and subsequently fibrinous transformation. In the case of the normals, platelet activation, etc, was exaggerated and fibrinous transformation appeared to be accelerated. The haemostatic plugs forming in the treated haemophiliacs were indistinguishable from those forming in the normals and significantly different, with regard to all parameters measured, from the morphological appearances noted in the untreated haemophiliacs. These data suggest that the correction of the haemostatic defect previously observed results from the normalization of haemostatic plug formation and that this is sustained despite the promotion of systemic fibrinolysis that is also known to occur.

Topics: Animals; Bleeding Time; Blood Platelets; Dogs; Drug Therapy, Combination; Factor Xa; Fibrin; Hemophilia A; Hemostasis; Liposomes; Microscopy, Electron; Phosphatidylcholines; Phosphatidylserines

Topics: Acquired Immunodeficiency Syndrome; Drug Contamination; Factor VIII; Fibrin; Hemophilia A; Hot Temperature; Humans; von Willebrand Factor

A standardized injury of the nail cuticle of normal, factor VII and factor VIII deficient dogs was used to study the evolution of the morphological changes occurring within the forming and formed haemostatic plug at the site of vascular injury. The morphological changes occurring were documented by light and transmission electron microscopy (TEM). Randomized measurements were made of the distances between adjacent platelets as a function of platelet interdigitation or compaction and the degree of dilatation of the open canalicular system (OCS) was used as an indicator of the degree of platelet activation. Fibrin deposition was noted both in terms of its location and the point in time at which it first appeared. TEM demonstrated major differences between the factor VIII deficient and the normal and factor VII deficient groups. In the normal animals the intermembrane distance showed noticeable changes with the platelets becoming tightly interdigitated at the time bleeding stopped. During the same period the OCS became dilated. These changes, which were not seen in the factor VIII deficient animals, continued until many platelets lost their intracellular content and became balloon cells or ghosts and fibrinous transformation became prominent. Although those events did occur in the factor VII deficient state, each was delayed and resulted in significant differences between the factor VII and normal animals suggesting that the extrinsic pathway may play an important role in initiating the changes noted. The results suggested that the generation of thrombin and/or factor Xa is essential to promote the initial stabilization of the platelet plug as well as initiating its subsequent consolidation by fibrinous transformation.

Topics: Animals; Blood Platelets; Dogs; Factor VII Deficiency; Fibrin; Hematoma; Hemophilia A; Hemostasis; Humans; Microscopy, Electron; Time Factors; Toes

Thrombin activity during separation and cryoprecipitation of CPD-blood was monitored by fibrinopeptide A (FPA) determinations. After pooling and lyophilization of cryoprecipitate, the total amount of contaminating fibrin was estimated by N-terminal amino acid analyses. In addition, retention of fibrin in standard transfusion filters (170 micron) was examined by gamma counting of 125I des-AA fibrin monomer enriched cryoprecipitate prior and subsequent to filtration. Prior to pooling of cryoprecipitate, thrombin activity, as estimated by FPA levels, was most pronounced during collection of blood from the blood donors and during cryoprecipitation and thawing of the plasma bags. Comparison of these FPA concentrations to the total amount of fibrin in pooled, freeze dried cryoprecipitate, as estimated by N-terminal analyses, revealed a considerable generation of fibrin during the process of lyophilization. In freeze dried cryoprecipitate, 5.3 per cent (range 3.0-7.5 per cent) of the fibrinogen had been converted to fibrin, implying a fibrin content of 20.3-60.3 mg per bottle of 500 U factor VIII. The amount of fibrin in two bottles of a commercially available factor VIII concentrate, also containing 500 U of factor VIII, was 14.1 and 19.8 mg, respectively. Sham transfusions of 125I des-AA fibrin monomer enriched cryoprecipitate revealed that only 1.0 per cent (range 0-2.5 per cent) of the fibrin was retained in the standard transfusion filters. Thus, substantial amounts of fibrin may be transfused to patients upon treatment with freeze dried cryoprecipitate.

Topics: Drug Contamination; Factor VIII; Fibrin; Fibrinopeptide A; Freeze Drying; Hemophilia A; Humans

Topics: Blood Coagulation Factors; Factor VIII; Fibrin; Hemophilia A; Humans; Wound Healing

The purpose of these studies was to establish the validity of 125I fibrin autoradiography--SDS gel techniques for monitoring degradation products from whole plasma or blood clots. These methods can be used to study fibrin degradation not only in patients with congenital factor XIII deficiency, but also in patients with disseminated intravascular coagulation or deep vein thrombosis during the course of thrombolytic therapy. Such an assay might complement existing immunologic techniques to characterize fibrin degradation in vivo by providing an in vitro analysis of the rate and pattern of fibrin degradation in whole blood or plasma. Fibrin degradation was traced by Coomassie blue staining for protein and by autoradiography on SDS-PAGE of degradation products released from a 125I-labeled fibrin tracer. The degradation of non-crosslinked clots from purified fibrin supplemented with plasmin showed a typical release of X, Y, D, and E fibrin fragments. Subsequently, all X and Y fragments were digested to D and E fragments. The degradation of non-crosslinked washed clots prepared from plasma supplemented with plasmin reflected the same pattern. The degradation of non-crosslinked washed clots prepared from EDTA anticoagulated plasma without added plasmin also showed release of X, Y, D, and E fragments. However, in contrast to the non-crosslinked washed clots supplemented with plasmin, there was no additional degradation of the X and Y fragments. These studies established that the pattern of degradation of the 125I-radiolabeled fibrin tracer was similar to that of the total protein released from the fibrin clot as observed by protein staining.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Blood Coagulation; Calcium; Cross-Linking Reagents; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Fibrin; Fibrin Fibrinogen Degradation Products; Fibrinolysin; Fibrinolysis; Glycoproteins; Hemophilia A; Humans; Protein C

Haemostatic plug formation in four patients with severe haemophilia A (VIII:C less than 1%) was studied in skin biopsies taken at 3, 10 and 30 min and 2 h after a template bleeding time wound had been made. The primary haemostatic plug showed relatively minor changes, consisting of a delay in platelet degranulation and interdigitation. Some platelet aggregates not attached to vessels were encountered in the wound. Subsequently the primary haemostatic plug changed into a firm stable degranulated mass of interdigitated platelets. The major abnormality occurred during the fibrinous transformation. At 2 h many haemostatic plugs consisted of a thin peripheral layer of fibrin and platelet remnants around a central area containing red and white blood cells with a varying amount of plasma and only relatively few fibrin fibres. These observations suggest that fibrin formation in the periphery of the plug is less dependent of factor VIII than in central areas. The lack of fibrin formation in the centre of the plug compensating for the platelet lysis at 2 h may have caused the central erosion of the plug.

Topics: Adult; Bleeding Time; Blood Platelets; Fibrin; Hemophilia A; Hemostasis; Humans; Microscopy, Electron; Middle Aged; Skin; Time Factors; Wounds, Penetrating

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

The purpose of this study was to determine the effects of the dilution of plasma (1/3 in saline) on the kinetics of fibrin generation in the activated partial thromboplastin time (APTT) assay. The diluted APTT is considered to increase the sensitivity of the APTT test however, studies in our laboratory using an electro-optical fibrin detection system failed to show significant differences in APTT values obtained with diluted and undiluted canine plasma. Seventeen plasmas, including plasmas moderately and markedly deficient in intrinsic factor activity were assayed in the undiluted and diluted APTT assay using two methods for fibrin endpoint detection; a visual "tilt-tube" technique and an electro-optical detection system. In the former technique the endpoint was the formation of a visible fibrin web or clot; in the latter procedure the end point was the first detection of a change in optical density of the plasma. Optical density changes during fibrin formation were also recorded ( thrombokinetograms ). The results indicated that the electro-optical fibrin detection system failed to identify a prolongation of the APTT as a result of 1/3 plasma dilution; a prolongation that was consistently observed with the visual fibrin detection technique. Plasma dilution however, did significantly reduce the rate of fibrin production as indicated by the thrombokinetogram profile. It was concluded that the dilution of plasma with saline, as has been used to increase the sensitivity of the APTT assay procedure, has little effect on the time of onset of fibrin formation in a given plasma. The major effect appears to be on the way in which fibrin forms in that the polymerization/crosslinkage events associated with macroscopic fibrin production are delayed.

Topics: Animals; Blood Coagulation Tests; Dog Diseases; Dogs; Fibrin; Hemophilia A; Intrinsic Factor; Partial Thromboplastin Time; Plasma; Sodium Chloride

Systemic hemostatic agents are reviewed. Among the agents discussed are vitamin K preparations (phytonadione, menadione, menadione sodium bisulfite, menadiol sodium diphosphate); and blood products (whole blood, plasma, cryoprecipitate, factor VIII concentrates, factor IX concentrates and fibrinogen concentrates). Normal and abnormal hemostasis and fibrinolysis are discussed, as is the general management of systemic hemostatic defects. Specific disorders covered are clotting factor deficiencies, hemophilia A, factor VIII inhibitors, von Willebrand disease, hemophilia B (Christmas disease), other congenital coagulation disorders, acquired deficiency of factors II, VII, IX and X, and defibrination syndrome.

Topics: Antibodies; Blood Coagulation Disorders; Factor VIII; Fibrin; Fibrinolysis; Hemophilia A; Hemophilia B; Hemostasis; Hemostatics; Humans; Transfusion Reaction; von Willebrand Diseases

Topics: Fibrin; Hemophilia A; Hemostatic Techniques; Humans; Occlusive Dressings; Oral Hemorrhage; Tampons, Surgical; Tooth Extraction

Topics: Antigens; Blood Coagulation; Factor VIII; Fibrin; Hemophilia A; Humans; Immunoelectrophoresis; Immunoelectrophoresis, Two-Dimensional; Precipitins; Time Factors

Topics: Arteriosclerosis; Blood Coagulation; Blood Coagulation Factors; Fibrin; Fibrinolysis; Hemophilia A; Hemostasis; Humans; Thromboembolism; Thrombosis

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; Fibrin; Fibrinogen; Fibrinolysis; Hemophilia A; Heparin; Hot Temperature; Humans; Thrombin

Evidence has been presented that fibrin formation may be detected directly in agarose gels containing citrated plasma when they are treated with thrombin or calcium chloride. A new assay is described for haemophilic factor-VIII antibody based on inhibition of fibrin formation within an agarose gel containing normal plasma.

Topics: Antibodies; Antibody Formation; Blood Coagulation Tests; Calcium Chloride; Factor VIII; Fibrin; Gels; Hemophilia A; Humans; Polysaccharides; Sepharose; Time Factors

Topics: Absorption; Acetoacetates; Amides; Antibodies; Antigen-Antibody Reactions; Factor VIII; Fibrin; Hematoporphyrins; Hemophilia A; Hot Temperature; Humans; Iodoacetates; Light

Topics: Animals; Antibodies; Blood Coagulation Tests; Blood Platelets; Blood Volume; Cortisone; Disease Models, Animal; Disseminated Intravascular Coagulation; Endotoxins; Factor VIII; Fibrin; Fibrinogen; Hematocrit; Hemophilia A; Iodine Radioisotopes; Kidney; Kidney Cortex Necrosis; Rabbits

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

A patient with classical hemophilia (factor VIII deficiency) was found to have a new abnormal fibrinogen (fibrinogen St. Louis). Other family members exhibited either defect alone. Fibrinogen St. Louis was inherited as an autosomal dominant and was not associated with clinical bleeding. When compared with normal fibrinogen, fibrinogen St. Louis was found to have defective fibrin polymerization and possibly a slower release of fibrinopeptides. The prolonged thrombin times were partially corrected by calcium chloride and protamine sulfate. Ultracentrifugal sedimentation, electrophoretic mobility, DEAE chromatographic pattern, carbohydrate content, N-terminal amino acids, immunodiffusion, and immunoelectrophoretic patterns and electrophoresis of reduced and alkylated fragments were all normal. In contrast to fibrinogen St. Louis, the most similar other fibrinogen variant (fibrinogen Zurich) was found to be heterogeneous by several criteria and to have reduced hexose content.

Topics: Adult; Amino Acids; Blood Coagulation; Blood Coagulation Factors; Blood Coagulation Tests; Blood Protein Electrophoresis; Calcium Chloride; Carbohydrates; Chromatography, DEAE-Cellulose; Clot Retraction; Factor VIII; Female; Fibrin; Fibrinogen; Hemophilia A; Humans; Hydrogen-Ion Concentration; Immunodiffusion; Immunoelectrophoresis; Male; Middle Aged; Pedigree; Peptides; Platelet Adhesiveness; Protamines; Prothrombin Time; Ultracentrifugation

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

Topics: Afibrinogenemia; Blood Coagulation Disorders; Blood Coagulation Factors; Factor IX; Factor VII; Factor VIII; Factor X; Factor XIII; Fibrin; Fibrinogen; Hemophilia A; Hemophilia B; Humans; Prothrombin; Thrombin; Vitamin K

Topics: Adult; Animals; Blood Coagulation; Blood Coagulation Tests; Clot Retraction; Cryoglobulins; Fibrin; Fibrinogen; Fibrinolysis; Hemophilia A; Humans; Middle Aged; Peptide Hydrolases; Snakes; Venoms

Topics: Blood Coagulation; Blood Viscosity; Fibrin; Hemophilia A; Hemophilia B; Humans; Kinetics; Multiple Sclerosis; Parkinson Disease; Polymers; Thrombelastography

Topics: Adenine Nucleotides; Adolescent; Antibodies; Blood Coagulation Tests; Blood Platelet Disorders; Blood Platelets; Blood Transfusion; Clot Retraction; Factor VIII; Fibrin; Hemophilia A; Humans; Male; Precipitin Tests

Topics: Animals; Blood Coagulation; Blood Platelets; Cell Membrane Permeability; Cytoplasm; Dogs; Fibrin; Hemophilia A; Hemophilia B; Microscopy, Electron; Organoids; Time Factors

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: Adaptation, Biological; Afibrinogenemia; Blood Coagulation Factors; Blood Platelet Disorders; Blood Platelets; Capillaries; Fibrin; Hemophilia A; Hemorrhage; Humans; Physiology; Thrombocytopenia; Thrombocytosis

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

Topics: Biochemical Phenomena; Biochemistry; Coagulants; Fibrin; Fibrinogen; Glycoproteins; Hemophilia A; Peptides

Topics: Adolescent; Adult; Animals; Anticoagulants; Blood Coagulation Disorders; Bone Development; Child; Child, Preschool; Female; Fibrin; Fractures, Bone; Fractures, Ununited; Hematoma; Hemophilia A; Heparin; Humans; Infant; Male; Periosteum; Pseudarthrosis; Rabbits; Radiography; Radius Fractures; Warfarin; Wound Healing

Topics: Blood Coagulation Disorders; Blood Coagulation Factors; Blood Platelets; Coagulants; Fibrin; Fibrinogen; Fibrinolysis; Hemophilia A; Heparin; History; Humans; Phosphotransferases; Thrombin

Topics: Anemia; Anemia, Hypochromic; Biomedical Research; Blood Chemical Analysis; Collagen Diseases; Enzyme Inhibitors; Fibrin; Hemophilia A; Hepatitis; Hepatitis A; Jaundice; Jaundice, Obstructive; Leukemia; Liver Cirrhosis; Multiple Myeloma; Nephritis; Nephrotic Syndrome; Physiology; Purpura; Thrombin; Uremia

Topics: Blood Coagulation; Cerebrospinal Fluid; Fibrin; Hemophilia A; Medicine; Tuberculosis; Tuberculosis, Meningeal

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

Topics: Blood Transfusion; Fibrin; Hemophilia A; Hemorrhagic Disorders; Humans; Hypoprothrombinemias; Prothrombin; Syndrome; Thrombocytopenia

Topics: Fibrin; Hemophilia A; Humans; Hypoprothrombinemias; Medicine; Prothrombin; Sex Chromosome Disorders