FVIII/VWF complex displays a greater pro-haemostatic activity than FVIII preparations devoid of VWF: Study in plasma and cell-based models.

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

Novel or expanding current targets in fibrinolysis.

Globally the leading cause of long-term disability and mortality stems from cardiovascular diseases, which creates an enormous economic burden. Currently available treatments for intravascular thrombosis consist of a large repertoire of antithrombotic agents targeting coagulation and platelet function. However, the only agents available to enhance fibrinolysis are recombinant or modified forms of plasminogen activators. Their clinical use is limited by low efficacy, life-threatening side-effects (primarily caused by the high systemic dose required) and the inapplicability for prophylactic use. This review provides an update on the latest advances in targeting the antifibrinolytic proteins, plasminogen activator inhibitor-1 and thrombin-activatable fibrinolysis inhibitor, and will highlight novel therapeutic avenues to enhance fibrinolysis.

Thrombomodulin suppresses invasiveness of HT1080 tumor cells by reducing plasminogen activation on the cell surface through activation of thrombin-activatable fibrinolysis inhibitor.

Cell malignancy is negatively correlated with the expression of thrombomodulin (TM), a thrombin receptor expressed on the surface of various cells, including tumor cells. TM accelerates thrombin-activatable fibrinolysis inhibitor (TAFI) activation catalyzed by thrombin. The active form of TAFI (TAFIa) contributes to inhibition of plasmin formation through its carboxypeptidase B (CPB)-like activity. Here, we examined whether TM- and tumor cell-dependent TAFI activation participates in controlling pericellular fibrinolysis and cell invasion. Human fibrosarcoma HT1080 cells retained the ability to activate both prothrombin and plasminogen, but did not express TM. HT1080 cells mediated activation of TAFI in plasma in the presence of soluble-type TM (sTM) through cell-dependent prothrombin activation. HT1080 cells stably expressing TM (TM-HT1080) mediated plasma TAFI activation without added sTM, but HT1080 (wild-type) and Mock-transfected HT1080 cells (Mock) did not. Production of TAFIa suppressed cell-mediated plasminogen activation. Matrigel invasion by wild-type and Mock cells was enhanced two-fold by diluted plasma (10%), whereas the plasma-induced invasion of TM-HT1080 cells (65% of wild-type invasion) was lower than those of wild-type and Mock cells. Cell invasion by TM-HT1080 was partially enhanced by addition of a TAFIa/CPB inhibitor. These results suggest that TM suppresses pericellular fibrinolysis and plasma-induced tumor cell invasion, and that it is mediated, at least in part, by plasma TAFI activation.

Antithrombotic effects due to pharmacological modulation of thrombin-activatable fibrinolysis inhibitor in rats.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a basic carboxypeptidase zymogen that can be activated by thrombin. Activated TAFI (TAFIa) cleaves carboxyl-terminal lysine residues from partially degraded fibrin, rendering it resistant to fibrinolysis by endogenous tissue plasminogen activator (tPA). Carboxypeptidase inhibitor (CPI) isolated from potato inhibits TAFIa and reduces clot lysis time in rabbit and mouse plasma. In the present study, we report the effect of CPI on tPA-mediated clot lysis using rat plasma in vitro. CPI at 400, 600 and 800 ng/ml caused a dose-dependent enhancement of tPA-induced clot lysis. In vivo effect of CPI was also investigated using ferric chloride-induced arterial thrombosis model in rat. The results showed that i.v. administration of CPI significantly prolonged the 'time to occlusion' at the dose of 2 and 4 mg/kg. At 2 mg/kg i.v. dose in rat, CPI showed no effect on prothrombin time and activated partial thromboplastin time, indicating noninterference of CPI with other clotting factors in mediating its thrombolytic effect through TAFI inhibition. Furthermore, 2 mg/kg i.v. dose of CPI did not produce significant increase in bleeding time when tested in rat tail-transection bleeding model. These results provide evidence for a role of TAFI in arterial thrombosis in rats and suggest that TAFI inhibition could be explored as an attractive target for the development of new antithrombotic drugs.

Fenofibrate improves endothelial function and decreases thrombin-activatable fibrinolysis inhibitor concentration in metabolic syndrome.

Procoagulant state, inflammation, and endothelial dysfunction have been documented in metabolic syndrome. Endothelial dysfunction is a strong predictor of cardiovascular events. Studies on the association of thrombin-activatable fibrinolysis inhibitor and thrombosis are still controversial, but substantial evidence suggests that increased thrombin-activatable fibrinolysis inhibitor or thrombin-activatable fibrinolysis inhibits or protects against arterial thrombosis. This study aimed to assess concomitantly the effects of fenofibrate therapy on thrombin-activatable fibrinolysis inhibitor concentrations and endothelial functions in patients with metabolic syndrome. Twenty-five patients (16 women; mean age 50.4 +/- 7.0) were enrolled in the study. Plasma thrombin-activatable fibrinolysis inhibitor, C-reactive protein, and fibrinogen levels were measured before fenofibrate administration and after 8 weeks of fenofibrate treatment. Endothelial function was assessed by endothelial-dependent flow-mediated dilatation from brachial artery. Pretreatment (baseline) thrombin-activatable fibrinolysis inhibitor level was 52.3 (1.2-119.7) decreasing to 7.7 (0.9-51.2; P < 0.001) after 8 weeks of fibrate treatment. Endothelial functions, which were measured with flow-mediated dilatation, were significantly improved after treatment (mean flow-mediated dilatation was 6.76 +/- 2.21 at baseline and 10.66 +/- 1.17% after 8 week of fenofibrate treatment, P < 0.001). Fenofibrate decreases thrombin-activatable fibrinolysis inhibitor levels and improves endothelial function in metabolic syndrome and, thus, suggests a potential for protection against cardiovascular effects. Further studies are warranted to confirm the effects of fibrates on thrombin-activatable fibrinolysis inhibitor and for conclusive evidence on the association between thrombin-activatable fibrinolysis inhibitor and thrombosis.

Decrements in the thrombin activatable fibrinolysis inhibitor (TAFI) levels in association with orlistat treatment in obesity.

Obesity and its associated metabolic complications can impair the physiologic regulation of fibrinolysis, leading to a hyper coagulable state. We aimed to assess circulating thrombin activatable fibrinolysis inhibitor (TAFI) levels in obese female patients and to test the effects of orlistat-induced weight loss on basal TAFI concentrations. Obese female outpatients age 18 and older, with a body mass index (BMI, calculated as weight in kilograms divided by the square of height in meters) of at least 30, were included into the study. Thirteen nonobese (median BMI, 22.60 kg/m(2)) age-matched females were taken as controls. Plasma TAFI levels were measured before orlistat administration and after 6 months of orlistat treatment in the obese group and only one measurement was done in the control group. Twenty-seven obese patients were recruited into the study. The median TAFI level of the control group was 124.00; this value was significantly lower than the basal TAFI level of the obese group (p < 0.001). TAFI levels after orlistat therapy were statistically significantly lower than basal TAFI levels (p < 0.001) in the obese group. Hemostatic abnormalities including TAFI alterations represent a link between obesity and vascular thrombosis. Effective interventions should be considered in improving the obesity-associated prothrombotic risk profile.

A comprehensive study on hemostasis in CAPD patients treated with erythropoietin.

OBJECTIVE: Bleeding diathesis and simultaneous thrombotic complications may be seen in dialyzed patients. Erythropoietin (EPO) may shift the precarious balance of the hemostatic system toward thrombosis. Platelets and tissue factor (TF) play a major role in plug formation. Tissue factor pathway inhibitor (TFPI) appears to play a primary role in regulating TF-induced coagulation. Thrombin activatable fibrinolysis inhibitor (TAFI) is a key protein linking coagulation and fibrinolysis. The aim of the study was to assess whether 6 months of EPO therapy affects platelet function, that is, platelet aggregation and P-selectin level; moieties of the extrinsic coagulation pathway: TF, TFPI, and TFPI/Xa complexes, and factors VII and X; markers of ongoing coagulation: thrombin-antithrombin complexes (TAT) and prothrombin fragments 1+2; a marker of ongoing fibrinolysis: plasmin-antiplasmin complexes (PAP); fibrinolytic activity: euglobulin clot lysis time (ECLT); and markers of endothelial cell injury: von Willebrand factor, thrombomodulin, E-selectin, and TAFI, in continuous ambulatory peritoneal dialysis (CAPD) patients. PATIENTS AND METHODS: 22 patients on CAPD were given EPO 6,000 U/week. 12 patients with chronic renal failure and 12 healthy volunteers served as control groups. All parameters were studied before, and after 1, 3, and 6 months of EPO therapy. SETTING: Department of Nephrology and Internal Medicine, Medical Academy of Bialystok, Poland. RESULTS: Platelet aggregation in whole blood did not change significantly during EPO treatment. A significant rise in arachidonic acid-induced platelet aggregation in platelet-rich plasma was observed after 3 and 6 months, and in collagen-induced platelet aggregation after 6 months of EPO therapy, compared to the baseline values. The TFPI concentration decreased significantly after 6 months of EPO therapy. The activity of factor VII increased transiently after 1 month of EPO therapy, compared to the baseline values. The TAFI concentration and activity in the CAPD group were significantly higher than in the control group. Erythropoietin therapy resulted in a significant decrease in TAFI concentration and activity after 6 months of EPO treatment. The ECLT was shortened significantly as early as after 1 month of EPO therapy. Thrombomodulin, von Willebrand factor concentration and activity, PAP, TAT, TFPI/Xa complexes, prothrombin fragments 1+2, factor X activity, P-selectin, E-selectin, and lipoprotein(a) did not change significantly during EPO treatment. CONCLUSION: Erythropoietin treatment has a minimal effect on hemostasis in CAPD patients. A tendency toward a decline in TAFI is of unknown clinical relevance so far, and awaits further research.