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Thrombin Generation (thrombin + generation)
Terms modified by Thrombin Generation Selected AbstractsThrombin generation in haemophilia A patients with mutations causing factor VIII assay discrepancyHAEMOPHILIA, Issue 4 2010R. GILMORE Summary., Up to 40% of patients with mild haemophilia A have a discrepancy whereby factor VIII (FVIII) measurements by a two-stage chromogenic assay (FVIII:CCH) are disproportionately reduced compared with the FVIII one-stage clotting value (FVIII:C). Which assay best reflects the coagulation potential and clinical phenotype in this patient group is of clinical significance, yet remains unclear. We have assessed the global coagulant ability of haemophilia patients with FVIII assay discrepancy using calibrated automated thrombography (CAT). A total of 18 patients with mutations Arg531His/Cys or Arg698Trp causing FVIII discrepancy were investigated, together with 12 haemophilia patients with concordant FVIII values and 15 normal controls. Factor VIII levels in all patients and controls were measured using both one-stage clotting assay and two-stage chromogenic assay. Thrombin generation was assessed in platelet-poor plasma by CAT using a low tissue factor concentration (1 pm). FVIII:CCH values were below normal in all patients, and in the discrepant group were between 1.5- and 8-fold lower than FVIII:C values. CAT parameters were affected in all haemophilia patients. The endogenous thrombin potential (ETP) was reduced to 58,67% of the mean normal value (1301 nm min,1), whereas peak thrombin was further reduced to 27,30% of the mean normal value (178 nm) in both discrepant and concordant patient groups. Analysis of the discrepant patient group showed the most significant correlation between the one-stage FVIII:C assay and ETP (r2 = 0.44) and peak thrombin parameters (r2 = 0.27). [source] Influence of the surface on thrombin generationINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 4 2008T. W. STIEF Summary Thrombin generation depends on the surface of the blood vessel or container. With a new ultra-sensitive and -specific thrombin assay the surface-dependent thrombin generation was quantified. Citrated blood or plasma was preincubated for 1 h (37 °C). Citrated blood, plasma, or plasma with 0,10 g/l hemoglobin,erythrocyte microparticles (Hb,MP) were preincubated at 23 °C or at 37 °C. Plasma samples (50 ,l) were recalcified in polystyrol (PS) wells and incubated for different coagulation reaction times (CRT). Final supramolar arginine concentrations, 0.1% Triton X 100, and chromogenic thrombin substrate concentrations in the onefold km,range were added and the linear ,A/t was measured in the recalcified coagulation activity assay (RECA). Aprotinin or corn trypsin inhibitor were added. (i) Recalcification of plasma (in different monovettes) pre-incubated for 1 h (37 °C) generated the following thrombin activities after 7 min (37 °C): 0.74 IU/ml (polypropylene (PP)-citrate), 0.39 IU/ml (PP-EDTA), 0.06 IU/ml (PP-heparin), 1.38 IU/ml (PS), 0.63 IU/ml (1 ml volume PP), 0.13 IU/ml (15 ml volume PP), and 3.62 IU/ml (glass). (ii) Recalcification of preincubated whole blood generated up to about fivefold more thrombin. (iii) Thrombin generation is proportional to the plasmatic concentration of Hb,MP, 10 g/l Hb,MP generating about 4 IU/ml thrombin within 20 min CRT. (iv) The IC50 of aprotinin and corn typsin inhibitor on thrombin generation in RECA are about 2 KIU/ml and about 1 U/ml, respectively. The reaction wall, the preincubation temperature, and hemolysis influences thrombin generation. The RECA allows to diagnose the prothrombotic capacity of any material. [source] Chemotherapeutic agents doxorubicin and epirubicin induce a procoagulant phenotype on endothelial cells and blood monocytesJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 4 2009L. L. SWYSTUN Summary.,Background: Although chemotherapy is associated with an increased risk of thrombosis, the pathogenic mechanisms by which chemotherapeutic agents exert prothrombotic effects are unclear. Objectives: In this study we explored the possibility that chemotherapeutic agents doxorubicin, epirubicin, 5-fluorouracil and methotrexate induce a procoagulant phenotype on vascular endothelial cells and/or on blood monocytes. Methods: Thrombin generation was measured in defibrinated plasma exposed to chemotherapy-treated human umbilical vein endothelial cells (HUVECs). Tissue factor activity assays were performed on chemotherapy-treated HUVECs and blood monocytes. The effects of chemotherapy drugs on phosphatidylserine exposure and the protein C pathway were also measured. Results: Exposure of defibrinated plasma to either doxorubicin- or epirubicin-treated HUVECs resulted in an increase in plasma thrombin generation. The procoagulant activity of doxorubicin- and epirubicin-treated HUVECs reflects an increase in tissue factor activity and phosphatidylserine exposure. Doxorubicin-mediated increase in tissue factor activity is related to increased levels of phosphatidylserine rather than to protein disulfide isomerase activity, and is likely to involve reactive oxygen species generation. Unlike doxorubicin, epirubicin does not have an impact on the protein C anticoagulant pathway. Interestingly, neither methotrextate nor 5-fluorouracil altered endothelial or monocyte hemostatic properties. Conclusions: These studies suggest that doxorubicin and epirubicin have the greatest ,prothrombotic potential' by virtue of their ability to alter endothelial and monocyte hemostatic properties. [source] Thrombin generation in acute coronary syndrome and stable coronary artery disease: dependence on plasma factor compositionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2008K. BRUMMEL-ZIEDINS Summary.,Background:,Acute coronary syndrome (ACS) is associated with thrombin formation, triggered by ruptured or eroded coronary atheroma. We investigated whether thrombin generation based on circulating coagulation protein levels, could distinguish between acute and stable coronary artery disease (CAD). Methods and results:,Plasma coagulation factor (F) compositions from 28 patients with ACS were obtained after onset of chest pain. Similar data were obtained from 25 age- and sex-matched patients with stable CAD. All individuals took aspirin. Patients on anticoagulant therapy were excluded. The groups were similar in demographic characteristics, comorbidities and concomitant treatment. Using each individual's coagulation protein composition, tissue factor (TF) initiated thrombin generation was assessed both computationally and empirically. TF pathway inhibitor (TFPI), antithrombin (AT), factor II (FII) and FVIII differed significantly (P < 0.01) between the groups, with levels of FII, FVIII and TFPI higher and AT lower in ACS patients. When thrombin generation profiles from individuals in each group were compared, simulated maximum thrombin levels (P < 0.01) and rates (P < 0.01) were 50% higher with ACS while the initiation phases of thrombin generation were shorter. Empirical reconstructions of the populations reproduced the thrombin generation profiles generated by the computational model. The differences between the thrombin generation profiles for each population were primarily dependent upon the collective contribution of AT, FII and FVIII. Conclusion:,Simulations of thrombin formation based on plasma composition can discriminate between acute and stable CAD. [source] Thrombin generation during reperfusion after coronary artery bypass surgery associates with postoperative myocardial damageJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2006P. RAIVIO Summary.,Background: Cardiopulmonary bypass and coronary artery bypass grafting (CABG) result in significant thrombin generation and activation of fibrinolysis. Thrombin contributes to myocardial ischemia,reperfusion injury in animal studies, but the role of thrombin in myocardial damage after CABG is unknown. Objectives: We measured thrombin generation and fibrin turnover during reperfusion after CABG to evaluate their associations with postoperative hemodynamic changes and myocardial damage. Methods: One hundred patients undergoing primary, elective, on-pump CABG were prospectively enrolled. Plasma prothrombin fragment F1+2 and D-dimer were measured preoperatively and at seven time points thereafter. Mass of the Mb fraction of creatine kinase (Ck-Mbm) and troponin T (TnT) were measured on the first postoperative day. Results: Reperfusion induced an escalation of thrombin generation and fibrin turnover despite full heparinization. F1+2 during early reperfusion associated with postoperative pulmonary vascular resistance index. F1+2 at 6 h after protamine administration correlated with Ck-Mbm (r = 0.40, P < 0.001) and TnT (r = 0.44, P < 0.001) at 18 h postoperatively. Patients with evidence of myocardial damage (highest quintiles of plasma Ck-Mbm and TnT) had significantly higher F1+2 during reperfusion than others (P < 0.002). Logistic regression models identified F1+2 during reperfusion to independently associate with postoperative myocardial damage (odds ratios 2.5,4.4, 95% confidence intervals 1.04,15.7). Conclusions: Reperfusion caused a burst in thrombin generation and fibrin turnover despite generous heparinization. Thrombin generation during reperfusion after CABG associated with pulmonary vascular resistance and postoperative myocardial damage. [source] Thrombin generation in vascular tissueJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2006A. PATHAK Summary.,Background: Classically, it is thought that the vast majority of thrombin is generated on the surface of platelets, however, thrombotic events occur in patients despite treatment with potent antiplatelet agents. Methods and results: In freshly harvested left internal mammary artery (IMA) sections, addition of CaCl2 and platelet-poor plasma (PPP) were sufficient to stimulate a profound burst of thrombin and this effect was inhibited by antitissue factor antibodies. Ultracentrifugation of PPP to remove platelet microparticles had no effect on thrombin generation. Both the extrinsic and factor VIII-dependent pathways were necessary for IMA-supported thrombin generation as PPP derived from individuals deficient in factors V, VII, VIII or X did not support thrombin production. Small amounts of thrombin were generated utilizing factor IX (FIX)-deficient plasma, however, thrombin was not generated by aorta from FIX-deficient mice when FIX-deficient plasma was used. The addition of non-lipidated tissue factor (0.6 pm) and CaCl2 to actively proliferating cultured human aortic smooth muscle cells (SMC) resulted in a pronounced burst of thrombin generation occurring between 3 and 15 min after treatment. In the absence of tissue factor, thrombin was generated but at a slower rate and with a peak value 26% of that observed in the presence of tissue factor. Conclusion: Significant thrombin generation can occur on vascular tissue in the absence of platelets or platelet microparticles and on the surface of non-apoptotic SMC. [source] Thrombin generation for the control of heparin treatment, comparison with the activated partial thromboplastin timeJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2004R. Al Dieri Summary., Heparin can be quantified with antifactor Xa and IIa tests (aXa, aIIa) but the anticoagulant power of heparin depends upon plasma properties as well as upon heparin concentrations and thus differs between subjects. Measuring the effect, as with the activated partial thromboplastin time (APTT) therefore is clinically more relevant. Here we investigate the use of the endogenous thrombin potential (ETP) for this purpose. In 12 volunteers 9000 IU of four heparins of different mol. wt distributions were injected. Samples were taken at 11 time points between 0 and 24 h. With the exception of the 0 and 24-h time points, heparin could be demonstrated by its aIIa and aXa activity in virtually all samples. The APTT showed the effect of this heparin in 34% of the samples; the ETP in 80%. This is partly due to the wide margins of the normal values, caused by large interindividual variation [coefficient of variation (CV) approximately 12% for the APTT, approximately 17% for the ETP]. The intraindividual variation is much smaller (CV approximately 4% for the APTT, approximately 5% for the ETP). Relative to the baseline value of the individual, the heparin effect was recognized by the APTT in 55% of the cases and by the ETP in 98%. There were no large differences between the different types of heparin. [source] Thrombin generation during cardiac bypass surgeryANAESTHESIA, Issue 11 2008E. Duff No abstract is available for this article. [source] Thrombin generation in whole bloodBRITISH JOURNAL OF HAEMATOLOGY, Issue 6 2008Raed Al Dieri No abstract is available for this article. [source] Thrombin generation in whole blood , response to Al Dieri & HemkerBRITISH JOURNAL OF HAEMATOLOGY, Issue 6 2008K. A. Tappenden No abstract is available for this article. [source] Activation of platelets in whole blood by recombinant factor VIIa by a thrombin-dependent mechanismBRITISH JOURNAL OF HAEMATOLOGY, Issue 4 2003Barry Wilbourn Summary. Using a diluted whole blood method of flow cytometric analysis, we have shown that platelets could be activated in vitro in the presence of high concentrations (100 nmol/l) of recombinant factor (F) VIIa (rFVIIa; NovoSeven®) and 2·5 mmol/l calcium chloride. This was demonstrated by a significant increase in the mean percentage of platelets expressing CD62P and their mean fluorescent intensity (MFI) after 30 min versus platelets incubated with calcium or rFVIIa alone or diluted blood alone. The presence of rFVIIa and calcium increased the exposure of the PAC-1 activation epitope of glycoprotein (Gp) IIb/IIIa. This effect was equally influenced by the presence of calcium alone but not by rFVIIa. The effect of rFVIIa was time and concentration dependent. Thrombin generation was also necessary, as the effect of rFVIIa was completely abrogated by the additional presence of hirudin. Furthermore, soy bean trypsin inhibitor (SBTI) but not corn trypsin inhibitor (CTI) abrogated CD62P exposure, suggesting that thrombin was derived via FX but not FXII activation. Exposure of CD62P demonstrated a significant lag phase, sometimes of the order of >,30 min, as well as large intersubject variation. Significant platelet activation was observed at a concentration as low as 25 nmol/l rFVIIa. Platelet,leucocyte aggregation was also increased in the presence of 25 nmol/l rFVIIa and calcium. No significant difference was observed between levels of CD62P in diluted whole blood and platelet-rich plasma adjusted to an identical platelet count after their exposure to rFVIIa and calcium for 30 min. [source] Development of an inhibitory antibody fragment to human tissue factor using phage display technologyDRUG DEVELOPMENT RESEARCH, Issue 3 2009S.M. Meiring Abstract Tissue factor is involved in the etiology of thrombotic diseases initiating the thrombosis associated with the inflammation that occurs during infection. The prevention of blood coagulation and inflammation is of primary importance in a number of pathological situations. A single-chain variable antibody fragment of molecular weight of 26,kD that inhibits the action of human tissue factor was selected by phage display technology, purified and tested for its tissue factor inhibitory effect, purified on a protein A column, and its purity evaluated on SDS-PAGE. The effects of the antibody fragment on prothrombin times, Factor Xa production, and thrombin generation were assessed with increasing fragment concentrations, using chromogenic and fluorometric substrates. The antibody fragment dose-dependently prolonged the prothrombin time (IC50=0.5,,M) and delayed the lag phase before the thrombin generation burst and the peak thrombin concentration in the thrombin generation assay. The effect on thrombin generation was more pronounced in thrombophilic plasma than in normal plasma. Antibody-based tissue factor inhibitors therefore may provide an effective treatment for thrombotic disease without serious bleeding complications. Drug Dev Res 2009. © 2009 Wiley-Liss, Inc. [source] From heparins to factor Xa inhibitors and beyondEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2005S. Alban Abstract Despite some disadvantages, unfractionated heparin (UFH) and oral anticoagulants have been the only anticoagulants for prophylaxis and therapy of thromboembolic disorders for several decades. Based on the increasing knowledge of the structure and pharmacology of heparin, low molecular weight heparins (LMWH) have been developed in the 1980s. Compared to UFH, their advantages are mainly based on their reduced nonspecific binding to proteins and cells resulting in improved pharmacokinetics. In 1991, LMWH were declared as the most efficient prophylaxis in high-risk patients. Although the use of LMWH is increasing and they are today also applied for therapy and in other indications like acute coronary syndrome, they are considered not optimal concerning efficacy and safety. With the approval of fondaparinux for the prevention of venous thromboembolic disease in high-risk orthopedic patients, there might be a paradigm shift in the field of anticoagulants. Fondaparinux, a synthetic, chemically defined pentasaccharide, is the first selective inhibitor of factor Xa. By its highly specific binding to antithrombin, it selectively inhibits factor Xa and consequently prevents thrombin generation. In contrast to UFH and LMWH, it does not bind to any other cells and other proteins than antithrombin. This leads to a favourable linear pharmacokinetic profile, allowing once-daily subcutaneous application of a fixed dose without monitoring in thromboembolism prophylaxis. In addition to the evaluation of fondaparinux for further indications, chemical modifications of this pentasaccharide such as the long-acting idraparinux are currently under investigation. [source] Mouse recombinant protein C variants with enhanced membrane affinity and hyper-anticoagulant activity in mouse plasmaFEBS JOURNAL, Issue 22 2009Michael J. Krisinger Mouse anticoagulant protein C (461 residues) shares 69% sequence identity with its human ortholog. Interspecies experiments suggest that there is an incompatibility between mouse and human protein C, such that human protein C does not function efficiently in mouse plasma, nor does mouse protein C function efficiently in human plasma. Previously, we described a series of human activated protein C (APC) Gla domain mutants (e.g. QGNSEDY-APC), with enhanced membrane affinity that also served as superior anticoagulants. To characterize these Gla mutants further in mouse models of diseases, the analogous mutations were now made in mouse protein C. In total, seven mutants (mutated at one or more of positions P10S12D23Q32N33) and wild-type protein C were expressed and purified to homogeneity. In a surface plasmon resonance-based membrane-binding assay, several high affinity protein C mutants were identified. In Ca2+ titration experiments, the high affinity variants had a significantly reduced (four-fold) Ca2+ requirement for half-maximum binding. In a tissue factor-initiated thrombin generation assay using mouse plasma, all mouse APC variants, including wild-type, could completely inhibit thrombin generation; however, one of the variants denoted mutant III (P10Q/S12N/D23S/Q32E/N33D) was found to be a 30- to 50-fold better anticoagulant compared to the wild-type protein. This mouse APC variant will be attractive to use in mouse models aiming to elucidate the in vivo effects of APC variants with enhanced anticoagulant activity. [source] Effect of two oral doses of 17,-estradiol associated with dydrogesterone on thrombin generation in healthy menopausal women: a randomized double-blind placebo-controlled studyFUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 2 2010Alexandra Rousseau Abstract Oral hormone therapy is associated with an increased risk of venous thrombosis. Drug agencies recommend the use of the lowest efficient dose to treat menopausal symptoms for a better risk/ratio profile, although this profile has not been totally investigated yet. The aim of the study was to compare the effect of the standard dose of 17,-estradiol to a lower one on thrombin generation (TG). In a 2-month study, healthy menopausal women were randomized to receive daily 1mg or 2 mg of 17,-estradiol (E1, n = 24 and E2, n = 26; respectively) with 10 mg dydrogesterone or placebo (PL, n = 22). Plasma levels factors VII, X, VIII and II were assessed before and after treatment as well as Tissue factor triggered TG, which allows the investigation of the different phases of coagulation process. The peak of thrombin was higher in hormone therapy groups (E1: 42.39 ± 50.23 nm, E2: 31.08 ± 85.86 nm vs. 10.52 ± 40.63 nm in PL, P = 0.002 and P = 0.01). Time to reach the peak was also shortened (PL: 0.26 ± 0.69 min vs. E1: ,0.26 ± 0.80 min, E2: ,0.55 ± 0.79 min, P <10,3 for both comparisons) and mean rate index of the propagation phase of TG was significantly increased. Among the studied clotting factors, only the levels of FVII were significantly increased after treatment administration. The two doses of 17,-estradiol induced in a similar degree an acceleration of the initiation and propagation phase of tissue factor triggered thrombin generation and a significant increase of FVII coagulant activity. [source] Effect of recombinant factor VIIa variant (NN1731) on platelet function, clot structure and force onset time in whole blood from healthy volunteers and haemophilia patientsHAEMOPHILIA, Issue 5 2007D. F. BROPHY Summary., NN1731 is a novel variant of recombinant factor VIIa (rFVIIa) that binds to activated platelets, but has greater enzymatic activity than rFVIIa in generating FXa and thrombin. The effect of NN1731 on clot structure and platelet function was characterized ex vivo in whole blood from healthy volunteers and haemophilic patients. Blood samples from six healthy volunteers, nine haemophilia A patients with and without inhibitors and one acquired haemophilia A patient, were spiked with increasing concentrations (0.32, 0.64 and 1.28 ,g mL,1) of rFVIIa and NN1731. Platelet contractile force (PCF) or platelet function, clot elastic modulus (CEM) or clot structure, and force onset time (FOT) or the thrombin generation time (TGT) were determined using the Hemodyne Hemostasis Analysis System (HASÔ). Baseline PCF, CEM and FOT values in patients were abnormal compared to healthy volunteers' baseline values. Overall, haemophilia blood samples with or without inhibitors spiked with NN1731 had significantly greater PCF, CEM and shorter FOT values relative to samples spiked with corresponding doses of rFVIIa. The variability in response to treatment between patients was greater with rFVIIa compared to NN1731. At 1.28 ,g mL,1 (90 ,g kg,1), NN1731 normalized PCF, CEM and FOT in nine of 10 patients, while rFVIIa normalized these parameters in four of 10 patients. Increasing in vitro concentrations of NN1731 normalized platelet function, clot structure and thrombin generation consistently in haemophilia blood with or without inhibitors. NN1731 may be a promising haemostatic agent for patients with bleeding disorders. These results should be confirmed in an in vivo study. [source] FEIBA®: mode of actionHAEMOPHILIA, Issue 2004P. L. Turecek Summary., FEIBA®(factor eight inhibitor bypassing activity) has a history of more than 30 years of successful use in controlling bleeding in haemophilic patients who have developed inhibitory antibodies against factor (F)VIII or FIX. Recently it was shown that FEIBA® contains the proenzymes of the prothrombin complex factors, prothrombin, FVII, FIX and FX, but only very small amounts of their activation products, with the exception of FVIIa, which is contained in FEIBA® in greater amounts. FEIBA® controls bleeding by induction and facilitation of thrombin generation, a process for which FV is crucial. A number of biochemical in vitro and in vivo studies have shown that FXa and prothrombin play a critical role in the activity of FEIBA®. Consequently, they are considered to be key components of this product. The prothrombinase complex has been found to be a major target site for FEIBA®. Apart from prothrombin and FXa, FEIBA® contains other proteins of the prothrombin complex, which could also facilitate haemostasis in haemophilia patients with inhibitors. [source] Cholestasis enhances liver ischemia/reperfusion-induced coagulation activation in ratsHEPATOLOGY RESEARCH, Issue 2 2010Jaap J. Kloek Aim:, Cholestasis is associated with increased morbidity and mortality in patients undergoing major liver surgery. An additional risk is induced when vascular inflow occlusion is applied giving rise to liver ischemia/reperfusion (I/R) injury. The role of the coagulation system in this type of injury is elusive. The aim of the current study was to assess activation of coagulation following hepatic I/R injury in cholestatic rats. Methods:, Male Wistar rats were randomized into two groups and subjected to bile duct ligation (BDL) or sham laparotomy. After 7 days, both groups underwent 30 min partial liver ischemia. Animals were sacrificed before ischemia or after 6 h, 24 h, and 48 h reperfusion. Results:, Plasma AST and ALT levels were higher after I/R in cholestatic rats (P < 0.05). Hepatic necrosis, liver wet/dry ratio and neutrophil influx were increased in the BDL group up to 48 h reperfusion (P < 0.05). Liver synthetic function was decreased in the BDL group as reflected by prolonged prothrombin time after 6 h and 24 h reperfusion (P < 0.05). I/R in cholestatic rats resulted in a 12-fold vs. 7-fold (P < 0.01) increase in markers for thrombin generation and a 6-fold vs. 2-fold (P < 0.01) increase in fibrin degradation products (BDL vs. control, respectively). In addition, the cholestatic rats exhibited significantly decreased levels of antithrombin (AT) III and increased levels of the fibrinolytic inhibitor plasminogen activator inhibitor (PAI-1) during reperfusion. Conclusions:, Cholestasis significantly enhances I/R-induced hepatic damage and inflammation that concurs with an increased activation of coagulation and fibrinolysis. [source] Influence of the surface on thrombin generationINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 4 2008T. W. STIEF Summary Thrombin generation depends on the surface of the blood vessel or container. With a new ultra-sensitive and -specific thrombin assay the surface-dependent thrombin generation was quantified. Citrated blood or plasma was preincubated for 1 h (37 °C). Citrated blood, plasma, or plasma with 0,10 g/l hemoglobin,erythrocyte microparticles (Hb,MP) were preincubated at 23 °C or at 37 °C. Plasma samples (50 ,l) were recalcified in polystyrol (PS) wells and incubated for different coagulation reaction times (CRT). Final supramolar arginine concentrations, 0.1% Triton X 100, and chromogenic thrombin substrate concentrations in the onefold km,range were added and the linear ,A/t was measured in the recalcified coagulation activity assay (RECA). Aprotinin or corn trypsin inhibitor were added. (i) Recalcification of plasma (in different monovettes) pre-incubated for 1 h (37 °C) generated the following thrombin activities after 7 min (37 °C): 0.74 IU/ml (polypropylene (PP)-citrate), 0.39 IU/ml (PP-EDTA), 0.06 IU/ml (PP-heparin), 1.38 IU/ml (PS), 0.63 IU/ml (1 ml volume PP), 0.13 IU/ml (15 ml volume PP), and 3.62 IU/ml (glass). (ii) Recalcification of preincubated whole blood generated up to about fivefold more thrombin. (iii) Thrombin generation is proportional to the plasmatic concentration of Hb,MP, 10 g/l Hb,MP generating about 4 IU/ml thrombin within 20 min CRT. (iv) The IC50 of aprotinin and corn typsin inhibitor on thrombin generation in RECA are about 2 KIU/ml and about 1 U/ml, respectively. The reaction wall, the preincubation temperature, and hemolysis influences thrombin generation. The RECA allows to diagnose the prothrombotic capacity of any material. [source] Severe hemophilia with mild bleeding phenotype: molecular characterization and global coagulation profileJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 4 2010E. SANTAGOSTINO Summary.,Background: Patients with severe hemophilia may show very varied bleeding tendencies, and the reasons for this heterogeneous clinical expression are unclear. The factor VIII/FIX genotype is the main determinant of the residual factor activity; however, different bleeding phenotypes have also been reported in patients sharing the same mutation. Such global coagulation tests as thrombin generation assays are tools with which to investigate different coagulation profiles among severe hemophiliacs. Objectives, patients and methods: This case,control study was aimed at comprehensively evaluating the role of genotype and endogenous thrombin potential (ETP) as predictors of the clinical phenotype in severe hemophiliacs with an extremely mild bleeding tendency (cases, n = 22), in comparison with those showing a typical bleeding tendency (controls, n = 50). Results: Cases were more frequently affected by hemophilia B than by hemophilia A, and showed a lower incidence of severe FVIII/FIX gene defects (referred to as null mutations), higher FVIII and FIX antigen levels and higher ETP values in platelet-rich plasma than controls (P < 0.05). By multivariate logistic regression, only non-null mutations were confirmed as an independent predictor of a mild clinical phenotype. Conclusions: These results indicate that non-null mutations represent the main determinant of the bleeding tendency, and that ETP measurement in platelet-rich plasma is able to identify severe hemophiliacs with a mild clinical phenotype. [source] Platelet functions beyond hemostasisJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 11 2009S. S. SMYTH Summary., Although their central role is in the prevention of bleeding, platelets probably contribute to diverse processes that extend beyond hemostasis and thrombosis. For example, platelets can recruit leukocytes and progenitor cells to sites of vascular injury and inflammation; they release proinflammatory and anti-inflammatory and angiogenic factors and microparticles into the circulation; and they spur thrombin generation. Data from animal models suggest that these functions may contribute to atherosclerosis, sepsis, hepatitis, vascular restenosis, acute lung injury, and transplant rejection. This article represents an integrated summary of presentations given at the Fourth Annual Platelet Colloquium in January 2009. The process of and factors mediating platelet,platelet and platelet,leukocyte interactions in inflammatory and immune responses are discussed, with the roles of P-selectin, chemokines and Src family kinases being highlighted. Also discussed are specific disorders characterized by local or systemic platelet activation, including coronary artery restenosis after percutaneous intervention, alloantibody-mediated transplant rejection, wound healing, and heparin-induced thrombocytopenia. [source] Factor IX mutants with enhanced catalytic activityJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2009R. HARTMANN Summary.,Background:,Activated coagulation factor IX (FIXa) has low catalytic activity towards its physiologic substrate FX when activated FVIII (FVIIIa) is absent. One reason for this is that the FIX surface loop 99 stabilizes FIXa in a conformation that limits access of FX to the active site. Objectives:,To investigate the effect of mutations in loop 99 and in the active site on FIXa activity with and without FVIIIa. Methods:,Five full-length FIX mutants with amino acid exchanges in the catalytic domain of FIX were constructed and characterized by measuring their activity in FX activation in model systems and in plasma. Results and Conclusions:,The mutants showed no or marginally improved catalytic properties in FX activation by the intrinsic tenase complex (FIXa,FVIIIa,Ca2+,phospholipid). The combination of mutations Y94F and K98T hardly affected FX activation in the presence of FVIIIa, but yielded a FIX molecule that, in FIX-depleted plasma, had , 2.5-fold higher clotting activity and , 3.5-fold higher activity in a thrombin generation assay than plasma-derived FIX (pdFIX). Two FIXa mutants had considerably increased activities towards FX in the absence of FVIIIa. FIXa-Y94F/K98T/Y177F/I213V/E219G (FIXa-L) and FIXa-Y94F/A95aK/K98T/Y177F/I213V/E219G (FIXa-M) activated FX with catalytic efficiencies (kcat/Km) that, as compared with activated pdFIX, were increased 17-fold and six-fold, respectively. However, in plasma, their zymogen forms performed similarly to pdFIX. This indicates that the introduced mutations not only affected the activity of FIXa but may have also influenced the lifetime of the activated mutant molecules in plasma by modifying their activation and/or inhibition rates. [source] Description of the chemical and pharmacological characteristics of a new hemisynthetic ultra-low-molecular-weight heparin, AVE5026JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2009C. VISKOV Summary.,Background and objectives: AVE5026 is a novel, hemisynthetic, ultra-low-molecular-weight heparin (ULMWH), which is in clinical development for prevention of venous thromboembolism. Its unique structural features result from the highly selective depolymerization of heparin by the phosphazene base that protects the antithrombin (AT)-binding site from destruction. In the present paper, we describe the chemical and biological characteristics of AVE5026, as well as its effects on experimental thrombosis as compared to those of the low-molecular-weight heparin (LMWH) enoxaparin after a single subcutaneous (s.c.) administration in certain animal models. Method and results: AVE5026 has a higher anti-factor Xa (anti-FXa) activity (,160 U mg,1) along with a catalytic anti-thrombin (anti-FIIa) activity (,2 U mg,1) as a result of its structure being strongly enriched in specific AT-binding oligosaccharides. In human plasma, potent inhibition of thrombin generation by AVE5026 was closely related to its anti-FXa activity. In a rat venous thrombosis model, AVE5026 showed a dose-dependent antithrombotic activity comparable to that of enoxaparin (ED50-AVE5026 = 1.6 mg kg,1, ED50-enoxaparin = 2.8 mg kg,1). Interestingly, non-occlusive venous thrombosis in rabbits was inhibited by an ED50 of 0.1 mg kg,1 AVE5026, whereas 0.316 mg kg,1 enoxaparin was not active. In a canine model, similarly to enoxaparin (ED50 = 1.3 mg kg,1), AVE5026 dose-dependently inhibited arterial thrombosis (ED50 = 2.0 mg kg,1). At equipotent doses, AVE5026 did not affect bleeding parameters, whereas enoxaparin showed increased hemorrhage in rats, rabbits and dogs. Conclusion: These unique structural attributes distinguish AVE5026 from the LMWH class. Based on these data in well-established arterial and venous thrombosis models, AVE5026 could represent a valuable alternative in thrombosis prevention with an improved benefit-risk profile as compared to that of enoxaparin. [source] Critical roles for thrombin in acute and chronic inflammationJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2009D. CHEN Summary., Thrombin can amplify inflammation induced by other stimuli, either through ischemia (consequent upon thrombosis), indirectly through generation of downstream mediators such as activated protein C, or directly via signals through protease activated receptors (PAR). This paper will summarize recent data from our laboratory indicating that thrombin is required to initiate CCR2-dependent leukocyte recruitment and that it is the principal determinant of the outcome after vascular injury, via PAR-1 activation of a distinct subset of smooth muscle cell progenitors. In both, tissue factor (TF) initiates thrombin generation and the thrombin acts locally, exemplifying that the initiation phase can generate autocrine or paracrine signalling molecules. Thrombin is an important constituent of innate immunity, able to amplify and modify responses to invading pathogens or tissue damage. With novel anti-thrombin therapeutics and agents to target PAR, a new understanding of the importance of thrombin may allow the development of innovative anti-inflammatory strategies. [source] Regulation of TFPI function by protein SJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2009T. M. HACKENG Summary., Protein S is an anticoagulant cofactor of full-length tissue factor pathway inhibitor (TFPI) that facilitates optimal factor Xa-inhibition and efficient down-regulation of thrombin generation in plasma. Protein S and TFPI are constitutively active in plasma and therefore provide an effective anticoagulant barrier against unwanted procoagulant activity in the circulation. In this review, we describe the current status on how TFPI-activity depends on protein S, and show that TFPI and protein S are major regulators of thrombin generation both in the absence and presence of activated protein C (APC). As there is covariation of plasma TFPI and protein S levels both in health and in disease, these findings suggest that the risk of venous thrombosis associated with protein S deficiency states might be in part explained by the accompanying low plasma TFPI levels. [source] Chemotherapeutic agents doxorubicin and epirubicin induce a procoagulant phenotype on endothelial cells and blood monocytesJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 4 2009L. L. SWYSTUN Summary.,Background: Although chemotherapy is associated with an increased risk of thrombosis, the pathogenic mechanisms by which chemotherapeutic agents exert prothrombotic effects are unclear. Objectives: In this study we explored the possibility that chemotherapeutic agents doxorubicin, epirubicin, 5-fluorouracil and methotrexate induce a procoagulant phenotype on vascular endothelial cells and/or on blood monocytes. Methods: Thrombin generation was measured in defibrinated plasma exposed to chemotherapy-treated human umbilical vein endothelial cells (HUVECs). Tissue factor activity assays were performed on chemotherapy-treated HUVECs and blood monocytes. The effects of chemotherapy drugs on phosphatidylserine exposure and the protein C pathway were also measured. Results: Exposure of defibrinated plasma to either doxorubicin- or epirubicin-treated HUVECs resulted in an increase in plasma thrombin generation. The procoagulant activity of doxorubicin- and epirubicin-treated HUVECs reflects an increase in tissue factor activity and phosphatidylserine exposure. Doxorubicin-mediated increase in tissue factor activity is related to increased levels of phosphatidylserine rather than to protein disulfide isomerase activity, and is likely to involve reactive oxygen species generation. Unlike doxorubicin, epirubicin does not have an impact on the protein C anticoagulant pathway. Interestingly, neither methotrextate nor 5-fluorouracil altered endothelial or monocyte hemostatic properties. Conclusions: These studies suggest that doxorubicin and epirubicin have the greatest ,prothrombotic potential' by virtue of their ability to alter endothelial and monocyte hemostatic properties. [source] Complementary roles of platelets and coagulation in thrombus formation on plaques acutely ruptured by targeted ultrasound treatment: a novel intravital modelJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2009M. J. E. KUIJPERS Summary.,Background:,Atherothrombosis is a major cause of cardiovascular events. However, animal models to study this process are scarce. Objectives:,We describe the first murine model of acute thrombus formation upon plaque rupture to study atherothrombosis by intravital fluorescence microscopy. Methods:,Localized rupture of an atherosclerotic plaque in a carotid artery from Apoe,/, mice was induced in vivo using ultrasound. Rupture of the plaque and formation of localized thrombi were verified by two-photon laser scanning microscopy (TPLSM) in isolated arteries, and by immunohistochemistry. The thrombotic reaction was quantified by intravital fluorescence microscopy. Results:,Inspection of the ultrasound-treated plaques by histochemistry and TPLSM demonstrated local damage, collagen exposure, luminal thrombus formation as well as intra-plaque intrusion of erythrocytes and fibrin. Ultrasound treatment of healthy carotid arteries resulted in endothelial damage and limited platelet adhesion. Real-time intravital fluorescence microscopy demonstrated rapid platelet deposition on plaques and formation of a single thrombus that remained subocclusive. The thrombotic process was antagonized by thrombin inhibition, or by blocking of collagen or adenosine diphosphate receptor pathways. Multiple thrombi were formed in 70% of mice lacking CD40L. Conclusions:,Targeted rupture of murine plaques results in collagen exposure and non-occlusive thrombus formation. The thrombotic process relies on platelet activation as well as on thrombin generation and coagulation, and is sensitive to established and novel antithrombotic medication. This model provides new possibilities to study atherothrombosis in vivo. [source] Platelet hyperprocoagulant activity in Type 2 diabetes mellitus: attenuation by glycoprotein IIb/IIIa inhibitionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2008M. RAZMARA Summary.,Background:,Platelets are hyperactive in Type 2 diabetes mellitus (T2DM), and antiplatelet treatment with glycoprotein (GP) IIb/IIIa inhibitors provides better thrombotic protection in DM than in non-diabetic subjects. Objective:,We hypothesized that diabetic platelets are hyperprocoagulant, and that this hyperactivity can be inhibited by GPIIb/IIIa blockade. Methods:,Patients with T2DM and gender/age/body mass index-matched non-diabetic controls were recruited (n = 12 for both) to study the effect of GPIIb/IIIa blockade on platelet procoagulant activity. Platelet phosphotidylserine (PS), factor (F) Va expression, and platelet-derived microparticle (PDMP) generation were measured by whole blood flow cytometry. Platelet-dependent thrombin generation and plasma clotting time were monitored in recalcified platelet-rich plasma. Results:,Compared to controls, basal platelet activation was similar, while thrombin receptor activating peptide stimulated activation was enhanced in patients with T2DM. Diabetic platelets also displayed more profound elevations of platelet PS exposure, FVa binding, and PDMP generation upon stimulation. These alterations resulted in a hyperprocoagulant state, as evidenced by a marked increase in the platelet procoagulant index, enhanced thrombin generation, and a shortened plasma clotting time. GPIIb/IIIa blockade by c7E3 or SR121566 decreased platelet PS exposure and FVa binding, and diminished platelet procoagulant activity in patients with T2DM. Conclusions:,Platelets have increased procoagulant activity in patients with T2DM. The hyperprocoagulant activity is counteracted by GPIIb/IIIa blockade. [source] Ex vivo inhibition of thrombus formation by an anti-glycoprotein VI Fab fragment in non-human primates without modification of glycoprotein VI expressionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2008P. OHLMANN Summary.,Objectives:,Glycoprotein (GP)VI is an attractive target for the development of new antithrombotic drugs. Its deficiency protects animals in several models of thrombosis, arterial stenosis and ischemia-,reperfusion while inducing no major bleeding tendency. The Fab fragment of one anti-GPVI monoclonal antibody (9O12.2) inhibits all GPVI functions in vitro. The aim of this study was to determine the ex vivo effects of 9O12.2 Fab on hemostasis, coagulation and thrombosis in non-human primates. Methods and results:,Blood samples were collected from cynomolgus monkeys before and after (30, 90 and 150 min, 1 and 7 days) a bolus injection of 9O12.2 Fab (4 mg kg,1) or vehicle. Platelet counts and coagulation tests (prothrombin time, activated partial thromboplastin time) were not modified following Fab injection. The PFA-100 closure time increased during the first hours and returned to initial values on day + 1. Platelet-bound Fab was detected from 30 min to 24 h after Fab injection without GPVI depletion at any time. Collagen-induced platelet aggregation was selectively and fully inhibited at 30 min. Thrombus formation on collagen in flowing whole blood (1500 s,1) was delayed and decreased, and collagen-induced or tissue factor-induced thrombin generation in platelet-rich plasma was profoundly inhibited. Conclusion:,The anti-GPVI 9O12.2 Fab inhibits thrombus formation ex vivo in non-human primates with a composite effect on platelet activation and thrombin generation in the absence of GPVI depletion. [source] Recollections on thrombin generationJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2 2008H. C. HEMKER Summary., Against an autobiographic background, a historical sketch is given of the development of the technique of thrombin generation, from subsampling to duly calibrated continuous measurement with fluorogenic substrates. Its application to various problems in the pathophysiology of hemostasis and thrombosis is discussed. [source] | ||||||||||||||||||||||