The action of immobilized thrombin on factor VIII, fibrinogen and a synthetic tripeptide.
Bovine thrombin was insolubilized by attachment to cyanogen bromide-activated Sepharose (Sepharose-thrombin) or to activated (Affi-Gel 10) agarose containing a 10 A long arm (Affi-Gel-thrombin). Coupling in both instances approximated 7,000 units of thrombin per ml packed gel as determined by 125I-thrombin incorporation. The thrombin beads hydrolyzed the synthetic tripeptide Bz-Phe-Val-Arg-pNA (S-2160) at different rates, with the Sepharose-thrombin more active (220 esterase units per ml) than Affi-Gel thrombin (20.4 units per ml). The Km was significantly higher for the insolubilized thrombins (2 X 10(-3) M) than uncoupled thrombin (Km = 8 X 10(-5) M). The Sepharose-thrombin activated factor VIII significantly more rapidly than Affi-Gel-thrombin. Neither matrix-bound thrombin clotted a fibrinogen solution or liberated significant amounts of fibrinopeptides over 48 hr. This data indicates that a proteolysis of factor VIII, rather than a complex with thrombin, is the method of activation of factor VIII and that factor VIII is more accessible to the action of immobilized thrombin than is fibrinogen.
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D005169:Factor VIII / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D006629:Hirudins / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*
D006629:Hirudins; D009842:Oligopeptides; D005169:Factor VIII; D005340:Fibrinogen; D013917:Thrombin