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Carboxypeptidase U (CPU, CPB2, TAFIa) is a basic carboxypeptidase that is able to attenuate fibrinolysis. The inactive precursor procarboxypeptidase U is converted to its active form by thrombin, the thrombin-thrombomodulin complex or plasmin. The aim of this study was to investigate and characterise the time course of CPU generation in healthy individuals. In plasma of 29 healthy volunteers, CPU generation was monitored during in vitro clot lysis. CPU activity was measured by means of an enzymatic assay that uses the specific substrate Bz-o-cyano-Phe-Arg. An algorithm was written to plot the CPU generation curve and calculate the parameters that define it. In all individuals, CPU generation was biphasic. Marked inter-individual differences were present and a reference range was determined. The endogenous CPU generation potential is the composite effect of multiple factors. With respect to the first CPU activity peak characteristics, we found correlations with baseline proCPU concentration, proCPU Thr325Ile polymorphism, time to clot initiation and the clot lysis time. The second CPU peak related with baseline proCPU levels and with the maximum turbidity of the clot lysis profile. In conclusion, our method offers a technique to determine the endogenous CPU generation potential of an individual. The parameters obtained by the method quantitatively describe the different mechanisms that influence CPU generation during the complex interplay between coagulation and fibrinolysis, which are in line with the threshold hypothesis.

Early onset preeclampsia (EOP) is a pregnancy-specific proinflammatory disorder that is characterised by competing thrombotic and bleeding risks. It was the aim of this study to characterise thrombin generation, a major determinant of thrombotic and bleeding risk, in order to better understand the haemostatic balance in patients with EOP. Patients with EOP were recruited at the Rotunda Hospital, Dublin. Twenty-six cases of EOP were recruited over a 21-month period, out of 15,299 deliveries at the Rotunda. Blood samples were collected into sodium citrate plus corn trypsin inhibitor anticoagulated vacutainers, platelet-poor plasma was prepared, and calibrated automated thrombography was used to assess thrombin generation. Results were compared to age and sex-matched non-pregnant controls (n=13) and age- and gestation-matched pregnant controls (n=20). The rate and extent of thrombin generation triggered by low-dose tissue factor (TF) was significantly reduced in patients with EOP compared to pregnant controls, most significantly in cases of severe EOP. EOP patients displayed a trend towards an increased response to endogenous activated protein C and thrombomodulin relative to pregnant controls. Plasma tissue factor pathway inhibitor (TFPI) activity was increased in EOP patients. Inhibition of TFPI abolished the attenuation of thrombin generation stimulated by low-dose TF. In conclusion, patients with EOP are characterised by an attenuated coagulation response characterised by reduced thrombin generation stimulated by low-dose TF and elevated plasma TFPI activity. These changes in coagulation may modulate thrombotic risk and bleeding risk in patients with EOP.

The main objective of the study is to compare the impacts of laparoscopic hysterectomy and total abdominal hysterectomy on the functions of coagulation and fibrinolysis system. Seventy-five patients who had undergone hysterectomy were randomly divided into laparoscopic hysterectomy group (n = 38) and total abdominal hysterectomy group (n = 37). The prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen, D-dimer, von Willebrand factor, α-granule membrane protein-140, thrombin-activated fibrinolysis inhibitor (TAFI) and platelet count were detected at preoperative 24 h (N0), postoperative 24 h (N1) and postoperative 48 h (N2). Compared with N0, values of PT, APTT and TT were significantly decreased at N1 in both groups, whereas von Willebrand factor, platelet count and α-granule membrane protein-140 levels at N1 were significantly increased (P < 0.05). There was no significant difference between N0 and N2 (P > 0.05). Compared with N0, fibrinogen, D-dimer and TAFI levels in both groups were significantly higher at N1 (P < 0.05), and there was no significant difference between N0 and N2 (P > 0.05). The intergroup comparison showed no significant difference of above indexes between two groups (P > 0.05). The univariate analysis showed that TAFI was negatively correlated with TT (r = -0.365, P < 0.01), APTT (r = -0.183, P < 0.05) and PT (r = -0.121, P < 0.05), whereas not correlated with other indicators. Laparoscopic hysterectomy may increase the risk of postoperative venous thrombosis.

Background: Increased thrombin formation and fibrin deposition on the valves were observed in patients with severe aortic valve stenosis (AS). Exercise enhances blood coagulation and fibrinolysis in healthy subjects, but its haemostatic effects in AS are unknown. We sought to investigate how stress echocardiography alters blood coagulation and fibrinolysis in AS patients free of significant atherosclerotic vascular disease., Methods: We studied 32 consecutive asymptomatic moderate-to-severe AS patients and 32 age- and sex-matched controls. We measured peak thrombin generated using calibrated automated thrombogram, clot lysis time (CLT), and fibrinolytic markers at four time points, i.e. at rest, at peak exercise, and 1h and 24h after a symptom-limited exercise test., Results: We observed that peak thrombin generated rose at peak exercise to 25% higher value in the patients than in controls (p<0.001) and reached its highest level 24h from exercise in AS patients while it decreased post-exercise in controls. Baseline CLT was 13% longer in AS patients (p=0.006) and associated with thrombin activatable fibrinolysis inhibitor (TAFI) activity (r=0.69, p<0.001), antiplasmin (r=0.47, p=0.007), and plasminogen (r=-0.55, p=0.001). In AS, CLT remained unaltered at peak exercise, while it decreased in controls, yielding the intergroup difference of 28% (p<0.001). Twenty-four hours after exercise CLT became 15% longer in AS patients (p<0.001). This hypofibrinolytic effect followed a similar pattern observed for TAFI activity., Conclusions: Asymptomatic moderate-to-severe AS patients respond to exercise with more pronounced and prolonged increase in thrombin generation, together with impaired fibrinolysis as compared to controls. Repeated episodes of exercise-induced prothrombotic state in AS might contribute to the progression of this disease., (Copyright © 2014 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Objective: Although the strong association between hyperprolactinaemia and platelet aggregation is well recognized, there are no studies on changes in coagulation and fibrinolytic status in patients with prolactinoma. To our knowledge, tissue plasminogen activator inhibitor-1 (PAI-1), plasma tissue factor pathway inhibitor (TFPI) and thrombin-activatable fibrinolysis inhibitor (TAFI) levels in these patients have not been investigated. Therefore, the main purpose of this study was to evaluate the markers of endogenous coagulation/fibrinolysis, including TFPI and TAFI, and to investigate the relationships between prolactin (PRL) and these haemostatic parameters and serum lipid profile in patients with prolactinoma., Research Methods and Procedures: Twenty-two patients with untreated, newly diagnosed prolactinoma and 20 age-matched healthy controls were included in the study. Platelet count, mean platelet volume, prothrombin time, activated partial thromboplastin time, fibrinogen, factors V, VII, VIII, IX and X activities, von Willebrand factor, antithrombin III (AT-III), protein C, protein S, tissue plasminogen activator (t-PA), PAI-1, TFPI and TAFI, as well as common lipid variables, were measured. The relationships between serum PRL and these haemostatic parameters were evaluated., Results: Compared with the control subjects, total cholesterol, low density lipoprotein cholesterol, apolipoprotein B, platelet count, fibrinogen, AT-III, PAI-1 and PAI-1/t-PA ratio were significantly increased in patients with prolactinoma (P < 0.0001, P < 0.001, P < 0.05, P < 0.05, P < 0.0001, P < 0.05, P < 0.0001 and P < 0.0001, respectively), whereas TFPI levels were significantly decreased (P < 0.01). Plasma TAFI Ag levels were not significantly different in patients with prolactinoma compared with the controls. In patients with prolactinoma, serum PRL was positively correlated with plasma FVII levels and apo B (r: 0.679, P < 0.05; r: 0.548, P < 0.05, respectively)., Conclusion: We found some important differences in the haemostatic parameters between the patients with prolactinoma and healthy controls. Increased platelet count, fibrinogen, PAI-1 and decreased TFPI in patients with prolactinoma may represent a potential hypercoagulable and hypofibrinolytic state, which might augment the risk for atherosclerotic and atherothrombotic complications. Thus, disturbances of the haemostatic system and dyslipidaemia may lead to the excess mortality in patients with prolactinoma., (© 2010 Blackwell Publishing Ltd.)

A clot lysis time assay in which a tissue factor-induced fibrin clot is lysed by exogenously added tissue plasminogen activator has been recently reported. We evaluated the feasibility of clot lysis time in a routine hemostasis laboratory, and its correlation with thrombin activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 levels and changes with aging in 185 healthy participants. Clot lysis time was assessed by monitoring changes in turbidity during clot formation and subsequent lysis using a computerized kinetic spectrophotometric microtiter plate. After preliminary experiments, 100 and 160 ng/mL tissue plasminogen activator concentrations were chosen for the study. Clot lysis time was calculated by a new mathematical analysis of the lysis curve based on discrete derivative. Clot lysis time, thrombin activatable fibrinolysis inhibitor, and plasminogen activator inhibitor-1 plasma levels showed a normal distribution. For both concentrations of tissue plasminogen activator, clot lysis time progressively increased with increase in age (P < .0001) and was significantly correlated with thrombin activatable fibrinolysis inhibitor antigen, thrombin activatable fibrinolysis inhibitor activity, and plasminogen activator inhibitor-1 antigen (at least P < .01). During linear regression analysis, thrombin activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 antigen were found to significantly influence clot lysis time (at least P < .01). Clot lysis time determination has a good laboratory performance. Our new method of calculation is independent of the time of reading and allows a more accurate and consistent detection of both short and prolonged lysis times. Our data suggest the feasibility of the use of this test in the work of routine hemostasis laboratory.

Although the maintenance of precise balance between coagulation and fibrinolysis is of utmost importance for normal haemostasis, until recently these two systems were considered as completely separate mechanisms involved in the process of formation and dissolution of blood clot. Thrombin activatable fibrinolysis inhibitor (TAFI) is a recently described attenuator of the fibrinolytic rate and is considered to be the molecular link between coagulation and fibrinolysis. TAFI circulates in plasma as an inactive precursor and its conversion in active enzyme (TAFIa) occurs by the action of thrombin or plasmin, but most efficiently by thrombin in the presence of its cofactor thrombomodulin. Once generated, TAFI down-regulates fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin; thereby preventing the upregulation of plasminogen binding and activation. Because TAFI is activated by thrombin on one side, and acts as the attenuator of fibrinolysis on another side, it enables fine synchronization between these two systems. The antifibrinolytic function of TAFI mostly depends on TAFI concentration, the rate of its activation and the half-life of TAFIa in plasma. Changes in thrombin generation can have a profound effect on the rate of TAFI activation, and consequently on the rate of fibrinolysis. Therefore, it has been hypothesized that increased thrombin generation seen in thrombophilia patients may enhance TAFI activation, leading to a hypofibrinolytic state, which may further contribute to the thrombotic tendency. However, the results of several studies, in which relation between TAFI level and the occurrence of thromboembolic complications in carriers of hereditary thrombophilia have been investigated, were not consistent.

Objectives: To investigate the plasma levels of thrombin-activatable fibrinolysis inhibitor (TAFI) in women with polycystic ovary syndrome (PCOS) and its correlation with various metabolic, hormonal and hemostatic parameters., Methods: Forty-eight women with PCOS and 43 age- and BMI-matched ovulatory controls were recruited during a 20-month study period. Blood samples were drawn for all tests, which included plasma lipids and lipoproteins, reproductive hormones, glucose, insulin, TAFI antigen concentration, plasminogen activator inhibitor-1 (PAI-1) activity, fibrinogen concentration, thrombomodulin, thrombin-antithrombin (TAT) complexes, D-dimer, Protein C Antigen, Protein S Antigen, Antithrombin III (AT III) and activated protein C (APC) resistance., Results: Plasma TAFI levels of PCOS patients were found to be significantly higher than in healthy controls (93.8%+/-30.6%vs. 79.8% +/- 22.4%, p < 0.05). Plasma levels of D-dimer, AT III, PAI-1 and thrombomodulin were also significantly higher in women with PCOS compared with healthy controls. All the other hemostatic parameters (including TAT complexes; Protein C; APC; and Protein S) were comparable between the two study groups., Conclusion: This study showed that plasma levels of TAFI, PAI-1, D-dimer, AT III and thrombomodulin were significantly increased in women with PCOS compared with age- and BMI-matched controls.

Background and Objectives: Cushing's syndrome (CS) is associated with an increased cardiovascular mortality and morbidity. Chronic endogenous and exogenous hypercortisolism frequently induce a hypercoagulable and thrombotic condition. Little is known about hemostatic features of patients with CS. To our knowledge, plasma tissue factor pathway inhibitor (TFPI) and thrombin-activatable fibrinolysis inhibitor (TAFI) levels in these patients have not been investigated. Therefore, the main purpose of this study was to evaluate the markers of endogenous coagulation/fibrinolysis, including TFPI and TAFI, and to investigate the relationships between cortisol and these hemostatic parameters and serum lipid profile in patients with CS., Design and Methods: Twenty-four patients with CS and 24 age-matched healthy controls were included in the study. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, factors V, VII, VIII, IX, and X activities, von Willebrand factor (vWF), antithrombin III (AT III), protein C, protein S, tissue plasminogen activator (t-PA), tissue plasminogen activator inhibitor-1 (PAI-1), TFPI and TAFI, as well as common lipid variables, were measured. The relationships between serum cortisol and these hemostatic parameters were examined., Results: Compared with the control subjects, platelet count, PT, fibrinogen, AT-III and PAI-1 were significantly increased in patients with CS (p<0.05, p<0.0001, p<0.01, p<0.05, and p<0.0001, respectively), whereas aPTT and TFPI levels were significantly decreased (p<0.0001 and p<0.01, respectively). Plasma TAFI Ag levels did not significantly change in patients with CS compared with the controls. In patients with CS, we showed a negative correlation between serum cortisol: 08:00 h and aPTT (r:-0.469, p<0.05). Serum cortisol: 24:00 h was positively correlated with PAI-1 Ag levels (r: 0.479, p<0.05)., Interpretation and Conclusions: In conclusion, we found some important differences in the hemostatic parameters between the patients with CS and healthy controls. Increased platelet count, fibrinogen, PAI-1, and decreased TFPI levels in these patients represent a potential hypercoagulable and hypofibrinolytic state, which might augment the risk for atherosclerotic and atherothrombotic complications. This condition may contribute to the excess of mortality due to cardiovascular disease seen in patients with CS.

Thrombin activatable fibrinolysis inhibitor (TAFI), when activated, forms a basic carboxypeptidase that can inhibit fibrinolysis. Potential physiologic activators include both thrombin and plasmin. In vitro, thrombomodulin and glycosaminoglycans increase the catalytic efficiency of TAFI activation by thrombin and plasmin, respectively. The most relevant (patho-) physiologic activator of TAFI has not been disclosed. Our purpose was to identify the physiologic activator of TAFI in vivo. Activation of protein C (a thrombin-thrombomodulin-dependent reaction), prothrombin, and plasminogen occurs during sepsis. Thus, a baboon model of Escherichia coli-induced sepsis, where multiple potential activators of TAFI are elaborated, was used to study TAFI activation. A monoclonal antibody (mAbTAFI/TM#16) specifically inhibiting thrombin-thrombomodulin-dependent activation of TAFI was used to assess the contribution of thrombin-thrombomodulin in TAFI activation in vivo. Coinfusion of mAbTAFI/TM#16 with a lethal dose of E coli prevented the complete consumption of TAFI observed without mAbTAFI/TM#16. The rate of fibrin degradation products formation is enhanced in septic baboons treated with the mAbTAFI/TM#16; therefore, TAFI activation appears to play a key role in the extent of fibrin(ogen) consumption during E coli challenge, and thrombin-thrombomodulin, in a baboon model of E coli-induced sepsis, appears to be the predominant activator of TAFI.

Both increased and decreased fibrinolytic activity have been reported in patients with hyperthyroidism. Elevated levels of plasma plasminogen activator inhibitor-1 (PAI-1) antigen have been found in hyperthyroid patients. Thrombin activatable fibrinolysis inhibitor (TAFI) is a novel plasma protein, which inhibits fibrinolysis through removal of C-terminal lysines from partially degraded fibrin. Previously, we showed that plasma TAFI antigen levels were increased in patients with overt and subclinical hypothyroidism. The aim of this study is to investigate plasma levels of TAFI and PAI-1 antigens in hyperthyroid patients. PAI-1 and TAFI antigen levels were measured in the plasma of 29 patients with hyperthyroidism (14 overt hyperthyroid and 15 subclinical hyperthyroid), and 26 healthy individuals. Although there were increased levels of PAI-1 antigen in hyperthyroid patients, plasma TAFI antigen levels were significantly lower compared to controls (80.79 ng/ml vs. 32.42 ng/ml, p = 0.000 for PAI-1; 10.42 microg/ml vs. 12.24 microg/ml, p = 0.009 for TAFI). Elevated PAI-1 antigen levels were positively correlated with free thyroid hormones, although TAFI antigen levels were in negative correlation with free thyroxine. Furthermore, an inverse correlation between PAI-1 and TAFI antigen levels was found. Our study demonstrated that TAFI antigen levels were decreased in patients with hyperthyroidism. Inverse correlation with PAI-1 suggests that the decrease in TAFI antigen levels may be due to activation of TAFI pathway. Further studies evaluating the underlying mechanisms of low TAFI antigen levels in hyperthyroidism should be undertaken.

Dengue virus causes a febrile illness: Dengue fever (DF), and less frequently a life-threatening illness: Dengue hemorrhagic fever (DHF). Although severe bleeding remains a major cause of death in DHF, the pathogenesis of bleeding is poorly understood. This prospective cohort study was designed to determine the extent of activation of endothelial cells and the hemostatic system in correlation with clinical severity, and also to detect the best prognostic factor(s) for DHF. Endothelial cell activation, coagulation, anticoagulant and fibrinolysis parameters were measured in 42 children with Dengue infections (20 with DF and 22 with DHF) during three phases of illness. In DHF patients, during the febrile phase, von Willebrand factor antigen (vWF:Ag), tissue factor (TF) and plasminogen activator inhibitor (PAI-1) were significantly elevated, while platelet counts and ADAMTS 13 (a disintegrin and metalloprotease with thrombospondin repeats) were significantly low compared to DF patients. During the toxic phase, soluble thrombomodulin (sTM), tissue plasminogen activator (t-PA) and PAI-1 were also significantly increased, while ADAMTS 13 and thrombin activatable fibrinolysis inhibitor (TAFIa) were significantly low compared to DF patients. Abnormal vWF multimers were seen only in DHF patients. For endothelial cell injury and release of procoagulant components, activation of the coagulation cascade with thrombin generation, increased antifibrinolytic factors and consumption of natural anticoagulants, each appeared to play an important role in the development of hemorrhage in Dengue patients. Using logistic regression analysis, we found plasma VWF:Ag to be the best indicator of progression to DHF.

To determine the role of CD14 in lipopolysaccharide (LPS)-induced effects on coagulation and fibrinolysis in humans, 16 healthy subjects received an intravenous injection of LPS preceded by intravenous IC14, a recombinant chimeric monoclonal antibody against human CD14, or placebo. LPS-induced coagulation activation (tissue-factor mRNA in whole blood cells and plasma concentrations of F1+2) was not influenced by IC14, whereas the antibody reduced the increase in thrombin-antithrombin complexes and soluble fibrin. LPS injection also was associated with an early activation of fibrinolysis (plasma concentrations of tissue-type plasminogen activator and plasmin-alpha(2)-antiplasmin complexes), followed by an inhibitory response (plasminogen activator inhibitor type 1), which were attenuated by IC14. Furthermore, LPS reduced thrombin-activatable fibrinolysis-inhibitor antigen levels and increased soluble thrombomodulin levels, which were not influenced by IC14. These results suggest that different hemostatic responses during endotoxemia may proceed via CD14-dependent and -independent pathways.