Thrombin Potentiation of Factor VIII Procoagulant Activity: Assessment by the Two-Stage Assay

Summary Factor VIII (FVIII) procoagulant activity is the function of a plasma glycoprotein that is missing or inactive in patients with classic hemophilia. Numerous studies have shown that trace thrombin causes rapid enhancement followed by gradual inactivation of FVIII procoagulant activity. Recent evidence suggests that thrombin activation of the FVIII/von Willebrand factor (vWF) protein is required for inactivation to occur. All of these studies have used the one-stage partial thromboplastin time to assay FVIII activity. Other investigators have used the two-stage assay of FVIII activity and have been unable to demonstrate thrombin-induced enhancement of FVIII activity, although inactivation has consistently occurred. We performed experiments designed to help resolve this disagreement, using the two-stage assay specifically modified to detect thrombin potentiation of FVIII activity. The length of the first-stage incubation time was found to be critical in demonstrating the initial effect of thrombin on FVIII activity. Taking advantage of this finding we were able to show a 4.1 ± 0.5-fold enhancement of FVIII activity upon incubating purified FVin/vWF with 0.04 NIH unit thrombin per ml. The apparent enhancement of FVEQ activity declined with increasing thrombin concentration. Incubation with 0.08, 0.16, and 0.32 NIH unit thrombin per ml resulted in only 3.2 ± 0.5, 2.6 ± 0.5 and 1.5 ± 0.3-fold enhancement, respectively, of FVIII activity. As with results from the one-stage assay, activation was followed by slow inactivation of FVIH/vWF. Using the two-stage assay we also showed 100% inactivation and 100% inhibition of FVIII activity by plasmin and human anti-FVUI IgG, respectively. Plasmin inactivation of FVIII activity showed a dose-response effect. Thrombin was unable to activate plasmin-degraded FVin/vWF. Our results show that thrombin potentiation of FVni activity is easily demonstrable in the two-stage assay. These findings support the contention that activation of FVm activity by thrombin is prerequisite for inactivation and underscore the importance of thrombin activation of FVHI/vWF in the intrinsic clotting system.

[1]  H. Hemker,et al.  The role of phospholipid and factor VIIIa in the activation of bovine factor X. , 1981, The Journal of biological chemistry.

[2]  P. McKee,et al.  Reactions of thrombin with human factor VIII/von Willebrande factor protein. , 1980, The Journal of biological chemistry.

[3]  E. Davie,et al.  Preparation and properties of bovine factor VIII (antihemophilic factor). , 1980, Biochemistry.

[4]  S. Pizzo,et al.  Is there a precursive, relatively procoagulant-inactive form of normal antihemophilic factor (factor VIII)? , 1979, Blood.

[5]  Y. Nemerson,et al.  Activation of factor X by factors IXa and VIII; a specific assay for factor IXa in the presence of thrombin-activated factor VIII. , 1978, Blood.

[6]  T. Kirkwood,et al.  Discrepancy between One‐stage and Two‐stage Assay of Factor VIII:C , 1978, British journal of haematology.

[7]  E. Koller,et al.  The Influence of Thrombin on the Clotting Activity of Factor VIII. A Study with Insolubilized Thrombin , 1978, Thrombosis and Haemostasis.

[8]  J. Andersen,et al.  MOLECULAR STRUCTURAL STUDIES OF HUMAN FACTOR VIII * , 1975, Annals of the New York Academy of Sciences.

[9]  M. Karpatkin,et al.  Methods for the Production of Clinically Effective Intermediate and High‐Purity Factor‐VIII Concentrates , 1971, British journal of haematology.

[10]  J. Niemetz,et al.  Activated Coagulation Factors: In‐Vivo and in‐Vitro Studies , 1969, British journal of haematology.

[11]  R. G. Macfarlane,et al.  The Interaction of Factors VIII and IX , 1964 .

[12]  S. Rapaport,et al.  The importance of activation of antihemophilic globulin and proaccelerin by traces of thrombin in the generation of intrinsic prothrombinase activity. , 1963, Blood.

[13]  K. Brinkhous,et al.  Effect of antihemophilic factor on one-stage clotting tests; a presumptive test for hemophilia and a simple one-stage antihemophilic factor assy procedure. , 1953, The Journal of laboratory and clinical medicine.

[14]  L. Hoyer,et al.  The effect of thrombin on human factor VIII. Cleavage of the factor VIII procoagulant protein during activation. , 1981, The Journal of laboratory and clinical medicine.

[15]  H. Lee,et al.  Rapid isolation and purification of antibody to factor VIII by protein A. , 1979, Thrombosis research.

[16]  S. Chavin,et al.  The role of factors VIII and IX in the activation of bovine blood coagulation factor X. , 1979, Thrombosis research.

[17]  H. Hemker,et al.  Human Blood Coagulation , 1969, Boerhaave Series for Postgraduate Medical Education.

[18]  T. Newcomb,et al.  EFFECTS OF THROMBIN ON FACTOR V. , 1963, Annals of biochemistry and experimental medicine.