thromboplastin and Hemolysis

Disseminated intravascular coagulation (DIC) of blood develops as a result of a sharp increase in the release of thromboplastic substances. The mechanism of disseminated thrombosis is switched in at the level of the microcirculatory bed with defibrination of the peripheral blood and subsequent hemorrhages and bleedings. The causes of DIC development may include complications of pregnancy and delivery, different kinds of shock including endotoxin shock, hemorrhage, hemolysis. Histomorphological findings in DIC are as follows: hemorrhagic syndrome, fibrin thrombi in capillaries, arterioles and venules of the skin, kidneys, adrenals, hypophysis, gastrointestinal tract, lungs and other organs followed by necroses and hemorrhages in these organs. Clinically, DIC is manifested by symptoms of insufficiency of the affected organs (acute renal insufficiency, Waterhouse-Fridericksen syndrome, etc).

Topics: Disseminated Intravascular Coagulation; Female; Hemolysis; Hemorrhage; Humans; Kidney; Male; Microcirculation; Necrosis; Obstetric Labor Complications; Pregnancy; Pregnancy Complications, Hematologic; Shock; Surgical Procedures, Operative; Thromboplastin; Thrombosis

Topics: Adenine Nucleotides; Aging; Anemia, Hemolytic; Animals; Blood Coagulation; Endotoxins; Erythrocytes, Abnormal; Fibrinogen; Hemolysis; Humans; Models, Biological; Platelet Adhesiveness; Purpura, Thrombotic Thrombocytopenic; Rabbits; Thrombin; Thrombocytopenia; Thromboplastin; Venoms

Topics: Adolescent; Adult; Blood Coagulation; Blood Coagulation Disorders; Child; Disseminated Intravascular Coagulation; Embolism, Fat; Endotoxins; Fatty Acids; Female; Fibrinolysis; Heart Defects, Congenital; Hemangioma; Hemolysis; Humans; Leukemia; Male; Middle Aged; Mononuclear Phagocyte System; Neoplasms; Pregnancy; Pregnancy Complications; Purpura, Thrombocytopenic; Skin Neoplasms; Thromboplastin

Topics: Adrenal Glands; Adrenocorticotropic Hormone; Animals; Antigen-Antibody Reactions; Blood Coagulation Disorders; Blood Coagulation Factors; Blood Coagulation Tests; Catecholamines; Dibenzylchlorethamine; Endotoxins; Female; Fibrinolysis; Haplorhini; Hemolysis; Hemorrhage; Humans; Kidney; Microscopy, Electron; Mononuclear Phagocyte System; Necrosis; Phagocytosis; Phenoxybenzamine; Pregnancy; Pregnancy, Animal; Shock, Septic; Shwartzman Phenomenon; Thromboplastin

Hemolytic diseases such as Sickle Cell Disease (SCD) are characterized by a natural propensity for both arterial and venous thrombosis. The ability of heme to induce tissue factor (TF) activation has been shown both in animal models of SCD, and in human endothelial cells and monocytes. Moreover, it was recently demonstrated that heme can induce coagulation activation in the whole blood of healthy volunteers in a TF-dependent fashion. Herein, we aim to further explore the cellular mechanisms by which heme induces TF-coagulation activation, using human mononuclear cells, which have been shown to be relevant to

Topics: Anemia, Sickle Cell; Animals; Endothelial Cells; Factor Xa; Heme; Hemolysis; Humans; Immunity, Innate; RNA, Messenger; Thromboplastin; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Topics: Acyclic Monoterpenes; Animals; Anti-Inflammatory Agents; Anticoagulants; Antioxidants; Chlorides; Diclofenac; Hemolysis; Humans; Inflammation; Interleukin-10; Interleukin-8; Lipopolysaccharides; Malondialdehyde; MAP Kinase Kinase Kinase 5; Molecular Docking Simulation; Nitric Oxide; Rats; Superoxide Dismutase; Thromboplastin; Tumor Necrosis Factor-alpha

Heme is a critical danger molecule liberated from hemeproteins in various conditions, including from hemoglobin in hemolytic diseases. Heme may cause thromboinflammatory damage by activating inflammatory and hemostatic pathways, such as complement, the TLRs, coagulation, and platelets. In this study, we explored the effect of single and dual inhibition of complement component C5 and TLR coreceptor CD14 on heme-induced thromboinflammation in an ex vivo human whole blood model. Heme induced a dose-dependent activation of complement via the alternative pathway. Single inhibition of C5 by eculizumab attenuated the release of IL-6, IL-8, TNF, MCP-1, MIP-1α, IFN-γ, LTB-4, MMP-8 and -9, and IL-1Ra with more than 60% (

Topics: Adult; Anemia, Sickle Cell; Animals; Blood Coagulation; Complement Activation; Complement C5; Cytokines; Granulocytes; Heme; Hemolysis; Humans; Inflammation; Lipopolysaccharide Receptors; Male; Monocytes; Swine; Thromboplastin

Cerium oxide nanoparticles (nanoceria [NC]) have attracted much attention in biomedicine due to their surface composition that confers interesting redox activities and regenerative properties. Studies have demonstrated that the application of NPs in biomedicine can influence components of hemostatic system, inducing blood clotting, alterations of blood cells, and endothelial cell functions. NC were tested in vitro to assess their hemocompatibility and anticoagulant, anti-inflammatory, and anti-senescence activity in human endothelial cells. Hemocompatibility has been evaluated in vitro looking at the impact of NC on coagulation times, fibrinogen, and platelet aggregation. The effect of NC on vascular endothelial cells were assayed by testing cell viability, antioxidant activity, anticoagulant (tissue factor [TF]-mRNA expression) and anti-inflammatory properties (VCAM-1 exposure, cytokine release), and senescence (telomere shortening). NC did not show significant effects on coagulation process, hemolysis, or platelet aggregation. In endothelial cells, NC did not affect cell viability, reduced oxidative stress, inhibited mRNA-TF expression, VCAM-1 expression, and cytokine release. Moreover, NC reduce telomere shortening, possibly counteracting premature senescence. The hemocompatibility combined with anticoagulant and anti-inflammatory phenotype and the ability of counteract the premature senescence in vascular cells make NC a promising therapeutic tool in oxidative stress-related conditions. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019.

Topics: Antioxidants; Blood Coagulation; Blood Platelets; Cell Survival; Cellular Senescence; Cerium; DNA; Fluorescence; Hemolysis; Hemostasis; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-6; Interleukin-8; Nanoparticles; Platelet Aggregation; Reactive Oxygen Species; RNA, Messenger; Thromboplastin; Vascular Cell Adhesion Molecule-1

The fluidity of cadaveric blood is an important characteristic in the post-mortem examination of cases of asphyxial death. Although it is empirically known that soft blood clots are present in cadaveric blood containing alcohol, the relationship between such clots and blood alcohol is unclear. We addressed this issue through in vitro studies using blood collected from healthy volunteers. Assessment of global hemostasis by rotational thromboelastometry revealed that ethanol treatment enhanced the procoagulant activity of whole blood. However, ethanol inhibited epinephrine-induced platelet aggregation, whereas plasma levels of von Willebrand factor and the activity of coagulation factors VIII and IX were unaffected. In contrast, tissue factor (TF) activity was higher in plasma obtained from ethanol-treated whole blood than that in plasma from untreated blood. Ethanol induced hemolysis of red blood cells, and the consequent hemoglobin (Hb) release promoted de novo synthesis of TF in isolated monocytes, as determined by real-time reverse transcription PCR, western blotting, and flow cytometry. However, ethanol itself did not induce TF expression in monocytes. Given that TF activates the extrinsic coagulation pathway and amplifies hemostatic reactions, Hb-induced TF expression in monocytes might contribute to soft blood clot formation.

Topics: Autopsy; Blood Coagulation; Cadaver; Ethanol; Flow Cytometry; Forensic Medicine; Hemolysis; Humans; Monocytes; Thromboplastin

Interaction between blood and bio-surfaces is important in many medical fields. With the aim of studying blood-mediated reactions to cellular transplants, we developed a whole-blood model for incubation of small volumes for up to 48 h.. Heparinized polyvinyl chloride tubing was cut in suitable lengths and sealed to create small bags. Multiple bags, with fresh venous blood, were incubated attached to a rotating wheel at 37°C. Physiological variables in blood were monitored: glucose, blood gases, mono- and divalent cations and chloride ions, osmolality, coagulation (platelet consumption, thrombin-antithrombin complexes (TAT)), and complement activation (C3a and SC5b-9), haemolysis, and leukocyte viability.. Basic glucose consumption was high. Glucose depletion resulted in successive elevation of extracellular potassium, while sodium and calcium ions decreased due to inhibition of energy-requiring ion pumps. Addition of glucose improved ion balance but led to metabolic acidosis. To maintain a balanced physiological environment beyond 6 h, glucose and sodium hydrogen carbonate were added regularly based on analyses of glucose, pH, ions, and osmotic pressure. With these additives haemolysis was prevented for up to 72 h and leukocyte viability better preserved. Despite using non-heparinized blood, coagulation and complement activation were lower during long-term incubations compared with addition of thromboplastin and collagen.. A novel whole-blood model for studies of blood-mediated responses to a cellular transplant is presented allowing extended observations for up to 48 h and highlights the importance of stringent evaluations and adjustment of physiological conditions.

Topics: Animals; Antithrombin III; Blood Gas Analysis; Blood Glucose; Carbon Dioxide; Cations; Cell Survival; Cell Transplantation; Chlorides; Collagen; Complement Activation; Glucose; Hemolysis; Humans; Hydrogen-Ion Concentration; Ions; Islets of Langerhans; Islets of Langerhans Transplantation; Leukocytes; Osmolar Concentration; Oxygen; Pancreas; Peptide Hydrolases; Rabbits; Thromboplastin; Time Factors

Hemoglobin SC disease is a very prevalent hemoglobinopathy; however, very little is known about this condition specifically. There appears to be an increased risk of thromboembolic events in hemoglobin SC disease, but studies evaluating the hemostatic alterations are lacking. We describe the findings of a cross-sectional observational study evaluating coagulation activation markers in adult patients with hemoglobin SC, comparing them with those in sickle cell anemia patients and healthy controls. A total of 56 hemoglobin SC and 39 sickle cell anemia patients were included in the study, all in steady state, and 27 healthy controls. None of the patients was taking hydroxyurea. Hemoglobin SC patients had a significantly up-regulated relative expression of tissue factor, as well as elevations in thrombin-antithrombin complex and D-dimer, in comparison to controls (P<0.01). Hemoglobin SC patients had lower tissue factor expression, and thrombin-antithrombin complex and D-dimer levels when compared to sickle cell anemia patients (P<0.05). Markers of endothelial activation (soluble thrombomodulin and soluble vascular cell adhesion molecule-1) and inflammation (tumor necrosis factor-alpha) were both significantly elevated in hemoglobin SC patients when compared to controls, being as high as the levels seen in patients with sickle cell anemia. Overall, in hemoglobin SC patients, higher hemolytic activity and inflammation were associated with a more intense activation of coagulation, and hemostatic activation was associated with two very prevalent chronic complications seen in hemoglobin SC disease: retinopathy and osteonecrosis. In summary, our results demonstrate that hemoglobin SC patients have a hypercoagulable state, although this manifestation was not as intense as that seen in sickle cell anemia.

Topics: Adult; Biomarkers; Blood Coagulation; Cross-Sectional Studies; Endothelial Cells; Female; Gene Expression; Hemoglobin SC Disease; Hemolysis; Humans; Inflammation Mediators; Leukocytes; Male; Middle Aged; Thrombophilia; Thromboplastin

Low flow rate pumping of cell suspensions finds current applications in bioreactors for short-term dynamic cell culture and adhesion assays. The aim of this study was to develop an atraumatic pump and hemodynamically adapted test circuit to allow operating periods of at least several hours. A computer-controlled mini-pump (MP) was constructed based on non-occlusive local compression of an elastic tube with commercial bi-leaflet valves directing the pulsatile flow into a compliant circuit. Cell damage and activation in the system were tested with whole blood in comparison with a set with a conventional peristaltic pump (PP). Activation of circulating THP-1 monocytes was tested by measuring the expression of CD54 (ICAM-1). Additionally, monocyte-endothelial interactions were monitored using a parallel-plate flow chamber with an artificial stenosis. The system required a priming volume of only 20 mL, delivering a peak pulsatile flow of up to 35 mL/min. After 8 h, blood hemolysis was significantly lower for MP with 11 ± 3 mg/dL compared with PP with 100 ± 16 mg/dL. CD142 (tissue factor) expression on blood monocytes was 50% lower for MP. With MP, THP-1 cells could be pumped for extended periods (17 h), with no enhanced expression of CD54 permitting the long-term co-culture of THP-1 with endothelial cells and the analysis of flow pattern effects on cell adhesion. A low-damage assay setup was developed, which allows the pulsatile flow of THP-1 cells and investigation of their interaction with other cells or surfaces for extended periods of time.

Topics: Cell Adhesion; Cells, Cultured; Coculture Techniques; Endothelium, Vascular; Hemolysis; Humans; Hydrodynamics; In Vitro Techniques; Intercellular Adhesion Molecule-1; Leukocytes; Monocytes; Pulsatile Flow; T-Lymphocytes, Helper-Inducer; Thromboplastin

The mechanisms of crosstalk between haemolysis, coagulation and innate immunity are evolutionarily conserved from the invertebrate haemocyanin to the vertebrate haemoglobin (Hb). In vertebrates, extracellular Hb resulting from haemolytic infections binds bacterial lipopolysaccharide (LPS) to unleash the antimicrobial redox activity of Hb. Because bacterial invasion also upregulates tissue factor (TF), the vertebrate coagulation initiator, we asked whether there may be functional interplay between the redox activity of Hb and the procoagulant activity of TF. Using real-time PCR, TF-specific ELISA, flow cytometry and TF activity assay, we found that Hb upregulated the expression of functional TF in macrophages. ELISA, flow cytometry and immunofluorescence microscopy showed binding between Hb and TF, in isolation and in situ. Bioinformatic analysis of Hb and TF protein sequences showed co-evolution across species, suggesting that Hbβ binds TF. Empirically, TF suppressed the LPS-induced activation of Hb redox activity. Furthermore, Hb desensitised TF to the effects of antioxidants like glutathione or serum. This bi-directional regulation between Hb and TF constitutes a novel link between coagulation and innate immunity. In addition, induction of TF by Hb is a potentially central mechanism for haemolysis to trigger coagulation.

Topics: Antioxidants; Blood Coagulation; Cell Line; Cell Survival; Coagulants; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Hemoglobins; Hemolysis; Humans; Lipopolysaccharides; Luminescence; Macrophages; Monocytes; Mutation; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Thromboplastin; Up-Regulation

Increased thrombin generation (TG) was described in sickle cell disease (SCD) children. The aim of this study was to characterize TG at the individual level and assess its relationship with age, hemolysis, transcranial Doppler velocity (TCD), and hydroxyurea treatment.. TG was triggered in the platelet-poor plasma using tissue factor and phospholipids with addition of thrombomodulin in 97 SCD at steady state and 80 control children. Patients and controls were aged from 2 to 20 years, and they were distributed in four categories of age: [2-5], [6-10], [11-15], and [16-20] years. For each subject, ratio of endogenous thrombin potential (rETP) and peak height (rPeak) was calculated as subject's value divided by the mean value of controls of the same age range. rETP and rPeak of patients were considered abnormal when > mean + 2SD of controls. LDH, total hemoglobin, and reticulocyte count were measured as markers of hemolysis. Data on hydroxyurea treatment and TCD were collected from medical records.. Overall, 38.1% and 44.3% of patients showed elevated rETP and rPeak, respectively. rETP and rPeak decreased significantly with increasing age. In homozygous (SS) patients, TCD velocities and all markers of hemolysis correlated significantly with both rETp and rPeak. Negative correlations were observed between these ratios and the duration of hydroxyurea treatment.. Elevated TG in SCD children is mainly related to younger age and to the intensity of hemolysis. There probably a link between TG and cerebral vasculopathy in these patients. Hydroxyurea may have a beneficial effect, which could be related to the duration of treatment.

Topics: Adolescent; Age Factors; Anemia, Sickle Cell; Blood Platelets; Child; Child, Preschool; Female; Hemoglobins; Hemolysis; Homozygote; Humans; Hydroxyurea; Male; Phospholipids; Plasma; Thrombin; Thrombomodulin; Thromboplastin; Ultrasonography, Doppler, Transcranial; Young Adult

Tissue Factor (TF) initiates thrombin generation, and whole blood TF (WBTF) is elevated in sickle cell disease (SCD). We sought to identify the presence of TF-positive monocytes in SCD and their relationship with the other coagulation markers including WBTF, microparticle-associated TF, thrombin-antithrombin (TAT) complexes and D-dimer. Whether major SCD-related pathobiological processes, including haemolysis, inflammation and endothelial activation, contribute to the coagulation abnormalities was also studied. The cohort comprised children with SCD (18 HbSS, 12 HbSC, mean age 3·6 years). We demonstrated elevated levels of TF-positive monocytes in HbSS, which correlated with WBTF, TAT and D-dimer (P = 0·02 to P = 0·0003). While TF-positive monocytes, WBTF, TAT and D-dimer correlated with several biomarkers of haemolysis, inflammation and endothelial activation in univariate analyses, in multiple regression models the haemolytic markers (reticulocytes and lactate dehydrogenase) contributed exclusively to the association with all four coagulant markers evaluated. The demonstration that haemolysis is the predominant operative pathology in the associated perturbations of coagulation in HbSS at a young age provides additional evidence for the early use of therapeutic agents, such as hydroxycarbamide to reduce the haemolytic component of this disease.

Topics: Anemia, Sickle Cell; Antithrombin III; Biomarkers; Case-Control Studies; Child; Child, Preschool; Endothelium, Vascular; Fibrin Fibrinogen Degradation Products; Flow Cytometry; Hemolysis; Humans; Inflammation Mediators; Monocytes; Peptide Hydrolases; Thromboplastin

Sickle cell disease (SCD) is associated with a complex vascular pathophysiology that includes activation of coagulation and inflammation. However, the crosstalk between these 2 systems in SCD has not been investigated. Here, we examined the role of tissue factor (TF) in the activation of coagulation and inflammation in 2 different mouse models of SCD (BERK and Townes). Leukocytes isolated from BERK mice expressed TF protein and had increased TF activity compared with control mice. We found that an inhibitory anti-TF antibody abrogated the activation of coagulation but had no effect on hemolysis or anemia. Importantly, inhibition of TF also attenuated inflammation and endothelial cell injury as demonstrated by reduced plasma levels of IL-6, serum amyloid P, and soluble vascular cell adhesion molecule-1. In addition, we found decreased levels of the chemokines MCP-1 and KC, as well as myeloperoxidase in the lungs of sickle cell mice treated with the anti-TF antibody. Finally, we found that endothelial cell-specific deletion of TF had no effect on coagulation but selectively attenuated plasma levels of IL-6. Our data indicate that different cellular sources of TF contribute to activation of coagulation, vascular inflammation, and endothelial cell injury. Furthermore, it appears that TF contributes to these processes without affecting intravascular hemolysis.

Topics: Anemia, Sickle Cell; Animals; Blood Coagulation; Chemokine CCL2; Chemokine CXCL1; Disease Models, Animal; Endothelial Cells; Erythrocytes; Female; Hemolysis; Inflammation; Interleukin-6; Leukocytes; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Transgenic; Neutrophils; Serum Amyloid P-Component; Thromboplastin; Vascular Cell Adhesion Molecule-1

The coagulation and complement pathways simultaneously promote homeostasis in response to injury but cause tissue damage when unregulated. Mechanisms by which they cooperate are poorly understood. To delineate their interactions, we studied the effects of thrombin and C5 convertase on C5 in purified and plasma-based systems, measuring release of the anaphylatoxin C5a, and generation of C5b, the initial component of the lytic membrane attack complex. Thrombin cleaved C5 poorly at R751, yielding minimal C5a and C5b. However, thrombin efficiently cleaved C5 at a newly identified, highly conserved R947 site, generating previously undescribed intermediates C5(T) and C5b(T). Tissue factor-induced clotting of plasma led to proteolysis of C5 at a thrombin-sensitive site corresponding to R947 and not R751. Combined treatment of C5 with thrombin and C5 convertase yielded C5a and C5b(T), the latter forming a C5b(T)-9 membrane attack complex with significantly more lytic activity than with C5b-9. Our findings provide a new paradigm for complement activation, in which thrombin and C5 convertase are invariant partners, enhancing the terminal pathway via the generation of newly uncovered C5 intermediates. Delineating the molecular links between coagulation and complement will provide new therapeutic targets for diseases associated with excess fibrin deposition and complement activation.

Topics: Animals; Blood Coagulation; Chickens; Complement Activation; Complement C3-C5 Convertases; Complement C5; Erythrocytes; Hemolysis; Humans; Proteolysis; Signal Transduction; Thrombin; Thromboplastin

Sepsis is a life-threatening disorder resulting from systemic inflammatory and coagulatory responses to infection. High-mobility group box 1 protein (HMGB1), an abundant intranuclear protein, was recently identified as a potent lethal mediator of sepsis. However, the precise mechanisms by which HMGB1 exerts its lethal effects in sepsis have yet to be confirmed. We recently reported that plasma HMGB1 levels correlated with disseminated intravascular coagulation (DIC) score, indicating that HMGB1 might play an important role in the pathogenesis of DIC.. To investigate the mechanisms responsible for the lethal effects of HMGB1, and more specifically, to explore the effects of HMGB1 on the coagulation system.. Rats were exposed to thrombin with or without HMGB1, and a survival analysis, pathologic analyses and blood tests were conducted. The effects of HMGB1 on the coagulation cascade, anticoagulant pathways and surface expression of procoagulant or anticoagulant molecules were examined in vitro.. Compared to thrombin alone, combined administration of thrombin and HMGB1 resulted in excessive fibrin deposition in glomeruli, prolonged plasma clotting times, and increased mortality. In vitro, HMGB1 did not affect clotting times, but inhibited the anticoagulant protein C pathway mediated by the thrombin-thrombomodulin complex, and stimulated tissue factor expression on monocytes.. These findings demonstrate the procoagulant role of HMGB1 in vivo and in vitro. During sepsis, massive accumulation of HMGB1 in the systemic circulation would promote the development of DIC.

Topics: Animals; Blood Coagulation; Blood Coagulation Tests; Cells, Cultured; Coagulants; Cytokines; Disease Models, Animal; Disseminated Intravascular Coagulation; Enzyme Activation; Fibrin; Hemolysis; High Mobility Group Proteins; HMGB1 Protein; Humans; Inflammation; Kidney; Lung; Male; Monocytes; Protein C; Rats; Rats, Sprague-Dawley; Repressor Proteins; Thrombin; Thromboplastin; Thrombosis

The compatibility of a methacrylate-based bone cement (CMW 1, DePuy International Ltd, England) used for the fixation of joint prostheses was evaluated on plasma, an erythrocyte suspension and cultured human endothelial cells. The extract of the cement was tested, following 1 hour and 7 days of curing. After the contact in vitro of the extract with plasma, activated partial thromboplastin time, antithrombin III, thrombin-antithrombin complexes and fibrin degradation products were assayed. Hemolytic activity was tested by adding the cement extracts to a suspension of erythrocytes. After 4 hours of incubation at 37 degrees C, the hemoglobin concentration was determined on the supernatants by the colorimetric method. The effect of the cement on tissue factor and thrombomodulin production was evaluated on human umbilical vein endothelial cell cultures. Tissue factor was determined in cell lysates by enzyme immunoassay, following 4 hours' incubation of cultures with the cement extract. Thrombomodulin was assayed in cell lysates by enzyme immuno assay, after 24 hours' incubation with the cement extract. The response to all trans-retinoic acid (ATRA) was tested. The cement caused no significant modifications of the coagulation tests, had no hemolytic activity, did not determine tissue factor production and did not modify thrombomodulin, compared to the negative control. The response to stimulation with ATRA was similar to that of the negative control. We conclude that the cement extract does not affect the plasmatic phase of coagulation, has no effect on erythrocytes, does not induce the expression of procoagulant activity by endothelial cells and does not impair their antithrombotic property, within the limits of the tests performed.

Topics: Blood Coagulation; Blood Coagulation Tests; Bone Cements; Drug Evaluation, Preclinical; Endothelium; Erythrocytes; Hemoglobins; Hemolysis; Humans; Immunoenzyme Techniques; Materials Testing; Plasma; Polymethyl Methacrylate; Thrombomodulin; Thromboplastin; Time Factors

Temporin A (TA) and a cecropin A-temporin A hybrid peptide (CATA) were synthesized and assayed for their hemolytic, anticoagulant, and antifungal properties. CATA retains significant antifungal activity, is less hemolytic than TA, and inhibits blood coagulation. These results recommend further studies of the biological activities of CATA.

Topics: Amino Acid Sequence; Amino Acids; Anti-Infective Agents; Antifungal Agents; Antimicrobial Cationic Peptides; Blood Coagulation; Erythrocytes; Hemolysis; Humans; Molecular Sequence Data; Peptide Biosynthesis; Peptides; Protein Structure, Tertiary; Proteins; Prothrombin; Thromboplastin

We have previously reported that acidic phospholipids are exposed at the surface of human erythrocytes when the cells are subjected to electrical breakdown. It has now been shown that the prothrombinase assay, which was used previously for the determination of acidic phospholipids, is specific for phosphatidylserine under the conditions of our experiments. In the light of this finding, we have investigated and characterised factors that govern cell lysis, cell fusion, and the formation of giant cells induced by electrical breakdown with human erythrocytes in media of low ionic strength. Divalent cations (1.1 mM) protected the cells against haemolysis, in the order Mn2+ > Ca2+ > Ba2+ > Mg2+ >> Zn2+, whereas about 99% of the cells lysed immediately on breakdown in the presence of Na+ or K+ (2.1 mM), or Al3+ (0.95 mM). The lengths of pearl chains of fused erythrocytes formed was similarly greatest with Mn2+ and decreased progressively with Ba2+, Zn2+, Ca2+ and Mg2+. No cell fusion occurred with Na+, K+, or Al3+. It is suggested that interactions with phosphatidylserine, which is exposed at the cell surface by electrical breakdown, may enable Mn2+, Ba2+ and Ca2+ ions to inhibit cell lysis (via membrane resealing) and facilitate cell fusion. Following electrically-induced cell fusion, erythrocytes round-up into giant cells. It has previously been proposed that Ca2+ ions accelerate the rounding-up process. However, data are presented which show that, as with erythrocytes treated with Sendai virus, the formation of rounded, giant cells following cell fusion depends on the osmotic swelling properties of permeabilised erythrocytes. Osmotic swelling may also have induced any hemi-fused cells present to fuse completely. Zn2+ ions anomalously enabled erythrocytes to round-up very rapidly into giant cells following electrical breakdown. This phenomenon may result from an interaction of Zn2+ ions with cysteine groups in membrane proteins, which decreases the immediate loss of ions that occurs when erythrocytes are subjected to electrical breakdown in low-ionic-strength media.

Topics: Cadmium; Cations, Divalent; Cell Fusion; Cell Size; Cobalt; Electric Stimulation; Erythrocytes; Giant Cells; Hemolysis; Humans; Osmolar Concentration; Phosphatidylserines; Phospholipids; Thromboplastin; Zinc

The procoagulant activity of human erythrocytes, which provides a measure of the translocation of acidic phospholipids from the inner to the outer monolayer of the plasma membrane, has been compared with the percentage cell fusion in experiments on the effects of electrical breakdown pulses under differing experimental conditions. After treatment with breakdown pulses of 20 microseconds or longer (5 kV cm-1), the plasma membranes of erythrocytes in 250 mM sucrose exhibited an almost complete loss of asymmetry with respect to acidic phospholipids. As the breakdown voltage was increased from 2 to 5 kV cm-1 (with breakdown pulses of 99 microseconds), the surface exposure of acidic phospholipids and cell fusion increased approximately in parallel. Furthermore, with 99 microseconds pulses and a voltage of 3 kV cm-1, a decrease in the osmolarity from 250 to 150 mM of the sucrose medium was accompanied by an increase in both the surface exposure of acidic phospholipids and the extent of cell fusion. Breakdown pulses of 2-5 microseconds were sufficient to cause a marked loss of asymmetry, but no cell fusion was observed unless the pulse length was at least 20 microseconds. Kinetic experiments indicated that exposure of the acidic phospholipids at the cell surface was more likely to be due to a direct effect of the electric field pulses on plasma membrane structure than to secondary effects, such as the action of endogenous proteinases on the membrane skeleton. It seems possible that a localised, surface exposure of acidic phospholipids may contribute to the 'long-lived fusogenic state' (Sowers, A.E. (1986) J. Cell Biol. 102, 1358-1362) and the 'transient permeant structures' (Teissié, J. and Rols, M.P. (1986) Biochem. Biophys. Res. Commun. 140, 258-266) that enable cell fusion to occur when contact between cells is established after they have been subjected to field pulses. Our observations also provide circumstantial support for the concept that changes in the phospholipid asymmetry of membranes may be important in physiologically-occurring instances of biomembrane fusion.

Topics: Animals; Cattle; Cell Fusion; Electric Stimulation; Erythrocytes; Hemolysis; Humans; Hydrogen-Ion Concentration; Hypotonic Solutions; Kinetics; Osmosis; Phospholipids; Sucrose; Surface Properties; Thromboplastin

Functionally inhibitory antibody to the plasma membrane complement inhibitor CD59 has been used to investigate control of the terminal complement proteins at the endothelial cell surface. Antibodies against purified human erythrocyte CD59 (polyclonal anti-CD59 and monoclonal antibodies [MoAbs] 1F1 and 1F5) were found to bind specifically to monolayers of cultured human umbilical vein endothelial cells, and by Western blotting to recognize an 18- to 21-Kd endothelial protein. When bound to the endothelial monolayer, anti-CD59 (immunoglobulin G or Fab fragment) potentiated membrane pore formation induced upon C9 binding to C5b-8, and augmented the C5b-9-induced cellular responses, including stimulated secretion of von Willebrand factor and expression of catalytic surface for the prothrombinase enzyme complex. Although potentiating endothelial responses to the terminal complement proteins, anti-CD59 had no effect on the response of these cells to stimulation by histamine. Taken together, these data suggest that human endothelial cells express the CD59 cell surface inhibitor of the terminal complement proteins, which serves to protect these cells from pore-forming and cell-stimulatory effects of the C5b-9 complex. These data also suggest that the inactivation or deletion of this cell surface regulatory molecule would increase the likelihood for procoagulant changes in endothelium exposed to complement activation in plasma.

Topics: Antibodies, Monoclonal; Antigens, Differentiation; CD59 Antigens; Cell Membrane; Complement Activation; Complement C5; Complement C5b; Complement C9; Complement System Proteins; Endothelium, Vascular; Epitopes; Hemolysis; Histamine; Humans; Membrane Glycoproteins; Membrane Proteins; Platelet Activation; Thromboplastin; von Willebrand Factor

Topics: Animals; Blood Coagulation; Blood Coagulation Factors; Blood Platelets; Blood Vessels; Disseminated Intravascular Coagulation; Endotoxins; Factor VII; Hemolysis; Humans; Leukocytes; Thromboplastin

Topics: Animals; Erythrocytes; Hemolysis; Humans; Male; Methods; Prothrombin Time; Rats; Thromboplastin

Topics: Animals; Anticoagulants; Bee Venoms; Hemolysis; In Vitro Techniques; Rabbits; Thromboplastin; Time Factors; Viper Venoms

A series of novel aromatic diamidines was synthesized and evaluated for antiproteolytic activity. The compounds were distignuished by inclusion of an aromatic ring structure--either benzene or bisbenzene or naphthalene--in the link between two amidinobenzene moieties. A highly potent inhibitor of bovine thrombin was discovered in alph, alph'-bis(4-amidino-2-iodophenoxy)-p-xylene with a Ki value of 1.1 X 10(-7) M (pH 8.1, 37 degrees), while alpha, alpha'-bis(4-amidino-2-iodophenoxy)-m-xylene was found to be an outstanding inhibitor of porcine pancreatic kallikrein (Ki = 3.1 X 10(-8) M). Several of the compounds investigated also demonstrated a considerable blocking effect on typsin and on the complement-dependent immune lysis of red cells.

Topics: Amidines; Amidohydrolases; Animals; Benzamidines; Blood Coagulation; Blood Coagulation Tests; Complement Inactivator Proteins; Depression, Chemical; Hemolysis; Humans; In Vitro Techniques; Kallikreins; Pancreas; Rats; Swine; Thrombin; Thromboplastin; Trypsin Inhibitors

Evidence of disseminated intravascular coagulation (DIC) was dought in normal baboons infused with autologous hemolyzed whole blood, preceded or followed by infusion of dextran (molecular weight, 70,000). Mean peak plasma hemoglobin following a rapid single injection was 370 mg/100 ml in 2 animals and 1,236 mg/100 ml in 1 animal, while levels during continuous 5 hour infusion in 2 animals averaged 326 and 474 mg/100 ml, respectively. Dextran infusion immediately preceded hemoglobin injection in 2 baboons and followed hemoglobin injection by 1 1/2 and 2 1/2 hours, respectively, in 2 baboons. Coagulation studies showed a moderate although significant fall in platelet count with prolongation of the partial thromboplastin time following hemoglobin infusion, and shortening of the thrombin time after dextran. Fibrin degradation products developed in four of five experiments after hemolysate injection. The induction of acute experimental hemoglobinemia results, therefore, in the development of coagulation changes consistent with milk DIC. Preliminary infusion of dextran (molecular weight, 70,000) may facilitate this response by either initiating the development or impeding the clearance of fibrin degradation products.

Topics: Acute Disease; Animals; Blood Cell Count; Blood Coagulation; Blood Platelets; Dextrans; Disseminated Intravascular Coagulation; Fibrin; Fibrinogen; Haplorhini; Hemoglobins; Hemolysis; Molecular Weight; Papio; Prothrombin Time; Thrombin; Thromboplastin; Time Factors

Total hemolytic complement activity and the third component of complement were found to be significantly depressed in vivo in rabbits following the induction of disseminated intravascular coagulation by both thrombin and thromboplastin. Production of severe thrombocytopenia by the administration of platelet antiserum prior to the infusion of thrombin or thromboplastin partially prevented complement activation. The data show that, when clotting is triggered, complement activation takes place and that platelets are required to some extent for this reaction.

Topics: Animals; Antigen-Antibody Complex; Blood Platelets; Cattle; Complement System Proteins; Disseminated Intravascular Coagulation; Endotoxins; Escherichia coli; Female; Goats; Hemolysis; Immune Sera; Infusions, Parenteral; Male; Rabbits; Sodium Chloride; Thrombin; Thromboplastin

A new centrifugal blood pump system has been developed for left ventricular bypass by the addition of non-thrombogenic blood surface materials and an ultrathin-walled cannula for the retrograde cannulation of the left ventricle. Partial LV bypass at 3 to 6 L/min was undertaken in 55 calves without thoracotomy. In 20 it was continued for 24 hours, with 13 survivors who were eventually sacrificed. Eleven of the last 14 experiments were completed without mishap. Heparin was employed only during pump insertion. Hematologic changes were limited to moderate platelet depression, and tolerable hemolysis (average serum level 21 mg% in the last 13 experiments). Normal clotting parameters and the absence of significant fibrin split product formation correlated with the absence of gross thrombosis and few minor renal emboli observed at autopsy. This pump system appears to have several advantages over previously described equipment for LV bypass.

Topics: Animals; Assisted Circulation; Blood Cell Count; Blood Platelets; Blood Pressure; Cattle; Fibrinogen; Hematocrit; Hemoglobins; Hemolysis; Leukocyte Count; Oxyhemoglobins; Prothrombin Time; Thromboembolism; Thromboplastin

Recovery of intrathoracic and intraperitoneal blood and reinfusion by autotransfusion has been demonstrated to be safe and practical in selected trauma patients. Autotransfusion is ideally applicable to the trauma patient in whom replacement of six or fewer units of blood is required. In addition, autotransfusion provides readily available blood for patients with unusual blood types and for those in whom multiple transfusions may rapidly deplete available stores. The properties of an ideal autotransfusion device include rapid assembly, relatively low cost, ease of operation, in-line filtration, minimized air blood interface, simplified anticoagulation, and safety from air embolism and coagulopathies.

Topics: Bilirubin; Blood Banks; Blood Cell Count; Blood Coagulation Tests; Blood Platelets; Blood Transfusion, Autologous; Fibrinogen; Hematocrit; Hemoglobins; Hemolysis; Hemorrhage; Humans; Leukocyte Count; Liver; Liver Function Tests; Postoperative Complications; Prothrombin Time; Thoracic Surgery; Thorax; Thromboplastin; Wounds and Injuries

Topics: Adenosine Diphosphate; Albumins; Animals; Blood Coagulation Tests; Blood Platelets; Cells, Cultured; Chromium Radioisotopes; Citrates; Collagen; Culture Media; Cytoplasm; Glucose; Hemolysis; Humans; L-Lactate Dehydrogenase; Phosphoric Monoester Hydrolases; Rabbits; Swine; Thromboplastin

Topics: Animals; Blood Cell Count; Blood Coagulation Tests; Blood Platelets; Depression, Chemical; Disseminated Intravascular Coagulation; Ethanol; Factor V; Female; Fibrinogen; Hemoglobins; Hemolysis; Indicators and Reagents; Male; Methods; Rabbits; Stimulation, Chemical; Thromboplastin; Time Factors

Topics: Animals; Blood Cell Count; Blood Coagulation Tests; Blood Platelets; Disseminated Intravascular Coagulation; Factor V; Female; Fibrinogen; Hematocrit; Hemoglobins; Hemolysis; Heparin; Infusions, Parenteral; Male; Prothrombin Time; Rabbits; Thromboplastin; Water

Topics: Blood Coagulation; Blood Platelets; Calcium; Factor XII; Hemoglobins; Hemolysis; Humans; Methemoglobin; Prothrombin Time; Thrombin; Thromboplastin

Topics: Blood Coagulation; Blood Coagulation Tests; Blood Platelets; Erythrocytes; Glass; Hemolysis; Humans; Indicators and Reagents; Phospholipids; Platelet Adhesiveness; Prothrombin; Prothrombin Time; Silicones; Surface Properties; Thromboplastin

Topics: Animals; Blood Coagulation; Cell Membrane; Cell Survival; Chromium Isotopes; Erythrocyte Aging; Hemolysis; Immune Sera; Injections, Intravenous; Rabbits; Silicon Dioxide; Thromboplastin

Topics: Adult; Aminocaproates; Blood Coagulation; Fibrinogen; Freezing; Hemolysis; Humans; Middle Aged; Prothrombin Time; Thrombelastography; Thrombin; Thromboplastin; Time Factors; Trypsin Inhibitors

Topics: Erythrocytes; Fibrinogen; Hemolysis; Humans; Prothrombin Time; Thrombelastography; Thrombin; Thromboplastin

Topics: Acidosis; Adrenocorticotropic Hormone; Animals; Antibodies; Anticoagulants; Antigens; Blood Coagulation Disorders; Blood Coagulation Factors; Blood Volume; Colloids; Cortisone; Endotoxins; Female; Fibrinolysis; Fluorescent Antibody Technique; Hemolysis; Humans; Hyperlipidemias; Kidney Cortex Necrosis; Leukocytes; Mononuclear Phagocyte System; Norepinephrine; Peptide Hydrolases; Phagocytosis; Phenoxybenzamine; Pregnancy; Rabbits; Shwartzman Phenomenon; Sympatholytics; Thromboplastin

Topics: Adult; Alanine Transaminase; Aminocaproates; Angioedema; Animals; Aspartate Aminotransferases; Bilirubin; Blood Cell Count; Blood Coagulation Disorders; Blood Coagulation Tests; Blood Platelets; Creatine Kinase; Creatinine; Dogs; Factor V; Factor VIII; Fibrinogen; Haplorhini; Hemolysis; Humans; Hyperbilirubinemia, Hereditary; Male; Methyltestosterone; Muscular Diseases; Myoglobinuria; Necrosis; Norepinephrine; Plasminogen; Rats; Spectrophotometry; Thromboembolism; Thromboplastin

Topics: Blood Coagulation Disorders; Hemolysis; Humans; Mononuclear Phagocyte System; Thromboplastin; Uremia

Topics: Abruptio Placentae; Adult; Animals; Anuria; Bilirubin; Blood Coagulation Disorders; Dogs; Erythrocytes; Female; Fibrin; Fibrinolysis; Hematologic Diseases; Hemolysis; Humans; Mice; Pregnancy; Rabbits; Rats; Shock, Hemorrhagic; Thrombin; Thromboplastin

Topics: Adenine Nucleotides; Animals; Blood Coagulation; Densitometry; Electron Transport; Erythrocytes; Guinea Pigs; Hemolysis; Hemostasis; Thromboplastin

Topics: Blood Coagulation; Cell Death; Erythrocytes; Fibrin; Fibrinolysis; Hemolysis; Humans; Thrombolytic Therapy; Thromboplastin

Topics: Animals; Blood Coagulation Disorders; Blood Coagulation Tests; Blood Platelets; Dogs; Fibrinogen; Fibrinolysin; Hemolysis; Heparin; Prothrombin Time; Research; Shock; Shock, Hemorrhagic; Silicones; Thromboplastin

Topics: Anemia; Blood Coagulation; Blood Coagulation Factors; Blood Coagulation Tests; Blood Platelets; Calcium; Factor IX; Factor V; Factor VII; Factor VIII; Factor X; Factor XI; Factor XII; Fibrinogen; Hemolysis; Humans; Kidney Diseases; Liver Diseases; Prothrombin; Thromboplastin

Topics: Cell Death; Hemolysis; Thromboplastin