Harold R. Roberts

Platelet activity of high‐dose factor VIIa is independent of tissue factor

High‐dose recombinant factor VIIa has been successfully used as therapy for haemophiliacs with inhibitors. The mechanism by which high‐dose factor VIIa supports haemostasis is the subject of some controversy. Postulating a mechanism in which activity is dependent on tissue factor at the site of injury explains the localization of activity but not the requirement for high doses. Postulating a mechanism in which factor VIIa acts on available lipid independently of tissue factor explains the requirement for high doses but not the lack of systemic procoagulant activity. We report that factor VIIa bound weakly to activated platelets (Kd ∼ 90 nm). This factor VIIa was functionally active and could initiate thrombin generation in the presence of plasma concentrations of prothrombin, factor X, factor V, antithrombin III and tissue factor pathway inhibitor. The activity was not dependent on tissue factor. The concentration of factor VIIa required for detectable thrombin generation agreed well with the lowest concentration of factor VIIa required for efficacy in patients. High‐dose factor VIIa may function on the activated platelets that form the initial haemostatic plug in haemophilic patients. These observations are in agreement with clinical trials which have shown that high‐dose factor VIIa was haemostatically effective without causing systemic activation of coagulation.

Platelets and Thrombin Generation

This review examines the evidence that platelets play a major role in localizing and controlling the burst of thrombin generation leading to fibrin clot formation. From the first functional description of platelets, it has been recognized that platelets supply factors that support the activation of prothrombin. Studies have demonstrated that on activation, the amount of one specific lipid, phosphatidylserine, is significantly increased on the outer leaflet of platelet membranes. When it was found that phosphatidylserine containing lipid extracts could be substituted for platelets in clotting assays, this suggested the possibility that changes in platelet lipid composition were necessary and sufficient to account for platelet surface thrombin generation. Because a growing body of data suggest that platelet-binding proteins provide much of the specificity for platelet thrombin generation, we review in this report data suggesting that changes in lipid composition are necessary but not sufficient to account for platelet surface regulation of thrombin generation. Also, we review data suggesting that platelets from different individuals differ in their capacity to generate thrombin, whereas platelets from a single subject support thrombin generation in a reproducible manner. Individual differences in platelet thrombin generation might be accounted for by differences in platelet-binding proteins.

Pharmacokinetics and pharmacodynamics of recombinant factor VIIa

To evaluate the pharmacokinetics and pharmacodynamics of recombinant activated factor VII (rFVIIa).

Clinical experience with recombinant factor VIIa.

Recombinant factor VIIa (rFVIIa) represents a major therapeutic advance in the treatment of haemophilia patients with inhibitors. The efficacy and safety of rFVIIa has been extensively studied in over 1900 surgical and non-surgical bleeding episodes in over 400 patients with haemophilia A or B (with or without inhibitors) or acquired haemophilia. Of 103 evaluable surgical bleeding episodes, the response to treatment with rFVIIa was considered to be either excellent or effective in 81%, 86% and 92% of major, minor and dental bleeding episodes, respectively. Treatment has been evaluated in 518 serious bleeding episodes and the response was considered either excellent or effective in 62% of muscle, 80% of ear, nose and throat, 88% of central nervous system, 76% of joint, and 75% of internal or retroperitoneal bleeding episodes. An excellent safety profile has also been demonstrated: of 1957 treatments with rFVIIa, only 16 serious adverse events have been reported that were considered to be possibly, but not necessarily, related to treatment.

A randomized, double-blind comparison of two dosage levels of recombinant factor VIIa in the treatment of joint, muscle and mucocutaneous haemorrhages in persons with haemophilia A and B, with and without inhibitors

Recombinant factor VIIa (rFVIIa) was developed to provide an improved procoagulant component capable of ‘by‐passing’ inhibitor antibodies in the treatment of haemophilic patients. The primary objective of this study was to compare the efficacy of two dosage regimens of rFVIIa (given intravenously at periodic intervals) in the treatment of joint, muscle and mucocutaneous haemorrhages in persons with haemophilia A and B with and without inhibitors. The study was designed as a randomized, double‐blind, parellel group, international multicenter trial. Patients were randomly allocated to treatment A: 35 μ kg−1 or B:␣70 μ kg−1, in blocks of 2. Within each block, one patient was assigned to the 35 μ kg−1 dosing regimen and the other to 70 μ kg−1 dose. One hundred and fifty subjects from 20 sites were screened for this study and 116 had baseline assessments. Of these, 84 were treated on the protocol and 32 were not treated in the study, in most cases because they did not return to the clinic with an eligible bleeding episode. One hundred and seventy‐nine bleeding episodes were treated, of which 145 (81%) were acute haemarthroses. Both treatments were efficacious, with 71% having an excellent (59% and 60%) or effective (12% and 11%) response. Overall, the mean and median number of doses given per episode of joint bleeding were 3.1 and 2, respectively. The mean number of doses was 3.1 for the 70 μ kg−1 group and 2.7 for the 35 μ kg−1 group (P value = 0.142). The study concluded that rFVIIa in a dosage of 35 μ kg−1 or 70 μ kg−1 is both safe and reasonably effective in the treatment of joint or muscle haemorrhages in haemophilic patients with inhibitor antibodies to factor VIII or factor IX. It is concluded that the appropriate dose for the treatment of joint and peripheral muscle bleeding in haemophilic patients with inhibitors is 35–70 μ kg−1 given at 2–3 h intervals until haemostasis is achieved.

Thrombin Activates Factor XI on Activated Platelets in the Absence of Factor XII

Thrombin can activate factor XI in the presence of dextran sulfate or sulfatides. However, a physiological cofactor for thrombin activation of factor XI has not been identified. We examined this question in a cell-based, tissue factor-initiated model system. In the absence of factor XII, factor XI enhanced thrombin generation in this model. The effect on thrombin generation was reproduced by 2 to 5 pmol/L factor XIa. A specific inhibitor of factor XIIa did not diminish the effect of factor XI. Thus, factor XI can be activated in a model system that does not contain factor XIIa or nonphysiological cofactors. Preincubation of factor XI with activated platelets and thrombin or factor Xa enhanced subsequent thrombin generation in the model system. Preincubation of factor XI with thrombin or factor Xa, but without platelets, did not enhance thrombin generation, suggesting that these proteases might activate factor XI on platelet surfaces. Thrombin and factor Xa were then directly tested for their ability to activate factor XI. In the presence of dextran sulfate, thrombin or factor Xa activated factor XI. Thrombin, but not factor Xa, also cleaved detectable amounts of factor XI in the presence of activated platelets. Thus, thrombin activates enough factor XI to enhance subsequent thrombin generation in a model system. Platelet surfaces might provide the site for thrombin activation of functionally significant amounts of factor XI in vivo.

Thrombolytic Therapy in Thrombosis: A National Institutes of Health Consensus Development Conference

Excerpt For over three decades, the primary method of therapy used by almost all physicians for the management of acute deep-vein thrombosis and pulmonary embolism has been anticoagulation. This fo...

Impact of procoagulant concentration on rate, peak and total thrombin generation in a model system

Summary.  Using a cell‐based model system of coagulation, we performed a systematic examination of the effect of varying individual procoagulant proteins (over the range of 0–200% of pooled plasma levels) on the characteristics of thrombin generation. The results revealed a number of features unique to the different coagulation factors, as well as common features allowing them to be grouped according to the patterns observed. Variation of those factors contributing to formation of the tenase complex, factor (F)VIII, factor (F)IX and factor (F)XI, primarily affected the rate and peak of thrombin production, but had little to no effect on total thrombin production. The effect of decreased FXI was milder than seen with decreased FVIII or FIX, and more variable between platelet donors. In contrast, varying the concentration of factors that contribute to formation of the prothrombinase complex, prothrombin or factor (F)V (with FV‐deficient platelets), significantly affected all three measures of thrombin production: rate, peak and total. Additionally, while no thrombin generation was observed with no factor X, only very small amounts (between 1% and < 10% of normal plasma levels) were required to normalize the measured parameters. Finally, our results with this cell‐based system highlight differences in thrombin generation on cell surfaces (platelets) compared with phospholipids, and suggest that platelets contribute more than simply a surface for the generation of thrombin.

High-dose factor VIIa increases initial thrombin generation and mediates faster platelet activation in thrombocytopenia-like conditions in a cell-based model system

Clinical experience has shown that high doses of recombinant factor VIIa (rFVIIa) may ensure haemostasis in thrombocytopenic patients. We have used a cell‐based model system to mimic thrombocytopenia and analyse the effect of rFVIIa. Lowering the platelet density from 200 × 109/l (reflecting normal conditions) to 100, 50, 20 and 10 × 109/l revealed a platelet density‐dependent decrease in the maximal rate of thrombin generation, a prolongation in the time to maximal thrombin activity and a lower maximal level of thrombin formed. The platelet activation, measured as the time to half‐maximal P‐selectin (CD62) exposure, was not significantly dependent on the platelet density in the range of 200 × 109/l to 10 × 109/l, although there was a tendency for slower platelet activation at 20 × 109 and 10 × 109 platelets/l than at the higher platelet densities. Addition of 50–500 nmol/l rFVIIa to samples with 20 × 109 or 10 × 109 platelets/l shortened the lag phase of thrombin generation as well as the time to half‐maximal platelet activation. Our data indicate that high doses of rFVIIa may help to provide haemostasis in thrombocytopenic patients by increasing the initial thrombin generation, resulting in faster platelet activation and thereby compensating for the lower number of platelets present.

Thrombogenic Materials in Prothrombin Complex Concentrates

Abstract Prothrombin complex concentrates are now available for use for treatment of bleeding complications associated with deficiencies of factors II, VII, IX, or X. The purpose of this report is ...