Thromb Haemost 1982; 47(02): 145-149
DOI: 10.1055/s-0038-1657150
Original Article
Schattauer GmbH Stuttgart

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

Robert G Kopitsky
The Howard Hughes Medical Institute Laboratories, Department of Medicine and the Department of Biochemistry Duke University Medical Center, Durham NC, U.S.A.
,
Mary Ellen P Switzer
The Howard Hughes Medical Institute Laboratories, Department of Medicine and the Department of Biochemistry Duke University Medical Center, Durham NC, U.S.A.
,
Patrick A McKee
The Howard Hughes Medical Institute Laboratories, Department of Medicine and the Department of Biochemistry Duke University Medical Center, Durham NC, U.S.A.
› Author Affiliations
Further Information

Publication History

Received 16 December 1981

Accepted 19 February 1982

Publication Date:
13 July 2018 (online)

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.

 
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