The activation of factor X and prothrombin by recombinant factor VIIa in vivo is mediated by tissue factor.

The human coagulation system continuously generates very small quantities of Factor Xa and thrombin. Current evidence suggests that basal level activation of the hemostatic mechanism occurs via Factor VIIa-dependent activation of Factor X, but direct proof has not been available for the participation of tissue factor in this pathway. To examine this issue, we infused relatively high concentrations of recombinant Factor VIIa (approximately 50 micrograms/kg body wt) into normal chimpanzees and observed significant increases in the plasma levels of Factor IX activation peptide, Factor X activation peptide, and prothrombin activation fragment F1+2. Metabolic turnover studies with radiolabeled Factor IX activation peptide, Factor X activation peptide, and F1+2 indicate that elevated levels of the activation peptides are due to accelerated conversion of the three coagulation system zymogens into serine proteases. The administration of a potent monoclonal antibody to tissue factor, which immediately neutralizes function of the Factor VIIa-tissue factor complex in vitro, abolishes the activation of Factor X and prothrombin mediated by the infused recombinant protein, and also suppresses basal level activation of Factor IX and Factor X. The above results suggest that recombinant Factor VIIa functions as a prohemostatic agent by interacting with endogenous tissue factor sites, but definitive proof will require studies in hemophilic animals using relevant hemostatic endpoints.

Factor IXa-factor VIIIa-cell surface complex does not contribute to the basal activation of the coagulation mechanism in vivo.

We have infused recombinant factor VIIa into patients with hereditary factor VII deficiency with marked reductions in plasma concentrations of factor IX activation peptide (FIXP), factor X activation peptide (FXP), and prothrombin activation fragment F1+2. These investigations show substantial elevations in these markers of coagulation activation and thereby demonstrate that the factor VII-tissue factor pathway is largely responsible for the activation of factor IX as well as factor X in the basal state (ie, the absence of thrombosis or provocative stimuli). We have administered a monoclonal antibody purified factor IX concentrate to individuals with hemophilia B. These studies show an increase in the plasma levels of FIXP that were initially greatly decreased, but no change in FXP or F1+2. We have also infused highly purified factor VIII concentrate into patients with hemophilia A. The data demonstrate no significant changes in the plasma concentrations of FXP and F1+2. The above observations indicate that factor IXa generated by the factor VII-tissue factor pathway is unable to activate factor X under basal conditions. Based upon the above findings, we outline a model of blood coagulation system function under basal conditions, and suggest a process by which the generation of factor Xa and thrombin might be accelerated during normal hemostasis and in the setting of thrombotic disorders.

Factor IX is activated in vivo by the tissue factor mechanism.

Despite significant progress in elucidating the biochemistry of the hemostatic mechanism, the process of blood coagulation in vivo remains poorly understood. Factor IX is a vitamin K-dependent glycoprotein that can be activated by factor XIa or the factor VII-tissue factor complex in vitro. To investigate the role of these two pathways in factor IX activation in humans, we have developed a sensitive procedure for quantifying the peptide that is liberated with the generation of factor IXa. The antibody population used for the immunoassay was raised in rabbits and chromatographed on a factor IX-agarose immunoadsorbent to obtain antibody populations with minimal intrinsic reactivity toward factor IX. We determined that the mean level of the factor IX activation peptide (FIXP) in normal individuals under the age of 40 years was 203 pmol/L and that levels increased significantly with advancing age. The mean concentration of FIXP was markedly reduced to 22.7 pmol/L in nine patients with hereditary factor VII deficiency (factor VII coagulant activity less than 7%) but was not significantly different from normal controls in nine subjects with factor XI deficiency (factor XI coagulant activity less than 8%). These data indicate that factor IXa generation in vivo results mainly from the activity of the tissue factor mechanism rather than the contact system (factor XII, prekallikrein, high molecular-weight kininogen, factor XI). Our results may also help to explain the absence of a bleeding diathesis in many patients with deficiencies of the contact factors of coagulation.

Detection of factor X activation in humans.

A sensitive radioimmunoassay (RIA) for the fragment that is liberated from factor X when this zymogen is activated by factor VII/VIIa-tissue factor or factor IXa was developed. Antisera were raised in rabbits to a synthetic 15 amino acid peptide containing the COOH-terminal sequence of the activation fragment coupled to bovine serum albumin with glutaraldehyde. The reactivity of the antibody population obtained toward the factor X zymogen was negligible (less than 1/36,000 that of the activation peptide on a molar basis). However, because other plasma constituents contributed to a nonspecific basal signal in the RIA, a procedure by which the peptide could be reproducibly extracted from plasma was developed. The mean level of this species in normal individuals younger than the age of 40 was 66.4 pmol/L, and elevations up to 550 pmol/L were observed in patients with evidence of disseminated intravascular coagulation. The validity of these measurements of factor X activation is supported by the fact that the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native peptide and can be quantitatively recovered. The mean concentration of the activation fragment was markedly decreased to 25.7 pmol/L in patients with hereditary factor VII deficiency (P = .0001 v normal controls), whereas the mean level in subjects with factor VIII deficiency was 61.1 pmol/L (P greater than .1 v normal controls). These data indicate that the basal (ie, in the absence of thrombosis or provocative stimuli) levels of FXP under in vivo conditions result mainly from the activity of the extrinsic pathway.

Elevated factor Xa activity in the blood of asymptomatic patients with congenital antithrombin deficiency.

The presence of congenital antithrombin deficiency has been consistently shown to predispose patients to venous thrombosis. We have utilized the prothrombin fragment F1+2 radioimmunoassay to quantitate factor Xa activity in the blood of 22 asymptomatic individuals with this clinical disorder not receiving antithrombotic therapy. The mean level of F1+2 was significantly elevated in these patients as compared to normal controls (3.91 vs. 1.97 nM, P less than 0.001). The metabolic behavior of 131 I-F1+2 was found to be similar in antithrombin-deficient subjects and normal individuals. The hemostatic system hyperactivity as measured by the F1+2 assay could be specifically corrected by raising the plasma antithrombin levels of the above asymptomatic individuals into the normal range. This study provides the first demonstration that the prethrombotic state can be biochemically defined as an imbalance between the production and inhibition of factor Xa enzymatic activity within the human circulation. It is known that antithrombin and alpha 1-proteinase inhibitor (PI) are the major inhibitors of factor Xa in human plasma in the absence of heparin. To further evaluate the mechanism by which antithrombin functions as an inhibitor of factor Xa in humans, we studied five patients who exhibited severe congenital deficiencies of alpha 1-PI. Our results indicated that the plasma of these subjects showed virtually identical decreases in plasma antifactor Xa activity in the absence of heparin when compared to antithrombin-deficient individuals, but the plasma F1+2 levels in the alpha 1-PI deficient population were not significantly different than normal. This data suggests that alpha 1-PI does not function as a major inhibitor of factor Xa in vivo, and that a tonically active heparin-dependent mechanism exists in humans for accelerating the neutralization of this enzyme by antithrombin.

Detection of protein C activation in humans.

We have developed a radioimmunoassay (RIA) for the dodecapeptide that is liberated from protein C when this zymogen is activated by thrombin bound to thrombomodulin present on the vascular endothelium. The protein C activation peptide (PCP) was synthesized using the solid-phase method of Merrifield. Antisera were raised in rabbits to the synthetic analogue coupled to bovine serum albumin with glutaraldehyde. The antibody population obtained was used together with a 125I-labeled tyrosinated ligand and various concentrations of unlabeled PCP to construct a double antibody RIA capable of measuring as little as 10 pM of this component. We have established that the synthetic dodecapeptide has the same immunoreactivity as the native peptide and that the reactivity of protein C is less than 1/2,000 that of PCP on a molar basis. The extremely low levels of peptide in normal individuals as well as the nonspecific contributions of plasma constituents to the immunoreactive signal, necessitated the development of a procedure by which the PCP could be reproducibly extracted from plasma and concentrated approximately 20-fold. This methodology permitted us to demonstrate that the plasma PCP levels in 17 normal donors averaged 6.47 pM, and that elevations up to 180 pM were observed in individuals with evidence of disseminated intravascular coagulation. The validity of these measurements of protein C activation is supported by the fact that, in both of these situations, the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native dodecapeptide. We have also noted that the mean PCP concentration in seven patients fully anticoagulated with warfarin averaged 2.61 pM. Our studies also show that PCP is cleared from the plasma of primates with a t1/2 of approximately 5 min. Given that the t1/2 of activated protein C is estimated to be 10-15 min, the latter enzyme appears to exert its effects on the activated cofactors of the coagulation system at concentrations considerably less than 1.0 nM.