The role of platelets and recombinant factor VIIa on thrombin generation, platelet activation and clot formation.

In the present study we assessed the effect of platelet counts and rFVIIa on thrombin generation, platelet activation and clot formation after tissue factor pathway activation in human plasma aiming to investigate the mechanism by which rFVIIa induces haemostasis in patients with severe thrombocytopenia. Plasma samples with platelet counts from 5 x 10(9)/l to 150 x 10(9)/l were spiked with rFVIIa (1 micro g/ml) or buffer. Clotting was initiated in the presence of diluted thromboplastin. Thrombin generation was assessed using the Thrombogram-Thrombinoscope trade mark assay. The kinetics of platelet activation was assessed using flow cytometry to measure the expression the P-selectin on platelet membrane of washed platelets suspended in defibrinated homologous PPP. Thromboelastography was used to evaluate the effect of platelets and rFVIIa on the kinetics of clot formation and clot's firmness. In the presence of low platelet counts the endogenous thrombin potential (ETP) and the maximum concentration of generated thrombin (Cmax) were reduced by 60%-70%. The lag-time of thrombin generation and the time required to reach the Cmax (Tmax) were prolonged, the velocity of platelet activation was decreased and thrombus formation was delayed. Recombinant FVIIa accelerated thrombin generation and platelet activation but it did not significantly modify ETP or Cmax. Recombinant FVIIa enhanced platelet activation in a TF and thrombin dependent manner since its effect on the studied parameters was abolished when TF was omitted or when hirudin was added into the experimental system respectively. Recombinant FVIIa normalized the velocity of clot formation but it did not modify clot firmness, which depended mainly on platelets' count. In conclusion, in experimental conditions simulating severe thrombocytopenia rFVIIa in the presence of low amounts of TF, accelerates thrombin generation, without increasing the maximum amount of generated thrombin, thus leading in enhanced platelet activation and rapid clot formation.

On the mechanism of inhibition of tissue factor pathway by the synthetic pentasaccharide during coagulation of human plasma.

The tissue factor (TF) pathway is preponderant in the initiation of blood coagulation in normal haemostasis and in thrombotic states. In the present study we investigated the mechanisms by which the synthetic pentasaccharide may influence the regulation of the TF pathway during clotting of human platelet poor plasma (PPP). Clotting of normal PPP or plasmas immuno-depleted of a single clotting factor (factor VII, factor XII, factor XI, factor IX, factor VIII, factor X, factor V, factor II) was initiated by triggering the TF pathway in the presence of fondaparinux (0.5 microg/ml). Activated factor VII (FVIIa) levels were measured in serum obtained at several time intervals after re-calcification of PPP. A clotting assay highly specific for FVIIa was used. The synthetic pentasaccharide inhibited the generation of FVIIa by 66%. The inhibitory effect of fondaparinux on FVIIa was completely abolished when antithrombin activity of plasma was inhibited by a specific antibody. Following the activation of TF pathway in plasmas depleted of factor X or factor IX, the inhibitory effect of fondaparinux on FVIIa generation was completely abolished, whereas it was not significantly modified when the other clotting factor-depleted plasmas were clotted. When fondaparinux was added in the serum, after the maximal generation of FVIIa, it inhibited by 20-30% the activity of the FVIIa-TF complex. These data suggest that fondaparinux enhances the antithrombin-dependent downregulation of the TF pathway by decreasing the generation of FVIIa via the inhibition of the generation and the activity of activated factor IX and activated factor X, and by inhibiting the activity of the FVIIa-TF complex.

Inhibition of factor VIIa generation and prothrombin activation by treatment with enoxaparin in patients with unstable angina.

Factor VIIa (FVIIa) and thrombin generation occur in patients suffering an acute coronary event. We studied the effect of treatment with enoxaparin on FVIIa and prothrombin activation in patients with unstable angina. Anti-Xa activity, FVIIa, FVII coagulant activity (FVII:C) and FVII antigen (FVII:Ag), free tissue factor pathway inhibitor (TFPI), and prothrombin fragments 1 + 2 (F1+2) were measured in patients' plasma, over a 24-h treatment period with enoxaparin. All 14 patients recruited in the study (mean age 68 years) were treated with a subcutaneous injection of enoxaparin, 1 mg/kg twice daily. Blood was drawn just before, and at different time intervals after, the first injection. Before enoxaparin administration, the levels of FVIIa (4.02 +/- 0.8 ng/ml) and F1+2 (2.68 +/- 0.2 nmol/l) were significantly increased as compared with control subjects (2.3 +/- 0.3 ng/ml and 0.9 +/- 0.1 nmol/l respectively, P < 0.05). Free TFPI, FVII:C and FVII:Ag were within normal ranges. One hour after the first injection of enoxaparin, FVIIa and F1+2 levels decreased by 65% and 50%, respectively, and no significant fluctuations were noted throughout the observation period. The concentrations of FVII:C and FVII:Ag were not modified as compared with baseline values. After each injection, the peak concentrations of free TFPI and anti-Xa activity were observed at 2 and 4 h respectively. The kinetics of FVIIa and F1+2 inhibition did not follow those of anti-Xa activity and TFPI release.

On the mechanism of action of recombinant activated factor VII administered to patients with severe thrombocytopenia and life-threatening haemorrhage: focus on prothrombin activation.

We report the ex vivo effect of recombinant activated factor VII (rFVIIa) on prothrombin activation after whole blood clotting. Two patients with severe thrombocytopenia and life-threatening haemorrhage were successfully managed using a single dose of rFVIIa (90 microg/kg). Western blotting using antiprothrombin antibody showed that rFVIIa did not induce thrombin generation in citrated platelet-poor plasma. Patient sera showed significantly impaired prothrombin activation before and after rFVIIa administration. rFVIIa administration shortened the prothrombin time, activated partial thromboplastin time and Ivy bleeding time, and normalized the clot retraction. These data indicate that rFVIIa accelerated thrombin generation without significant increase of generated thrombin.