Thrombin-catalyzed activation of human coagulation factor V

Abstract

Human coagulation factor V was purified from freshly frozen plasma by a method that gave high yields of single chain factor V. The purified protein has an Mr = 330,000 and consists of a single polypeptide chain with the following NH2-terminal sequence: Ala-Gln-Leu-Gly-Gln-Phe-Tyr-Val. Limited proteolysis of factor V by thrombin led to formation of factor Va which has a cofactor activity 25- to 30-fold that of factor V. Two intermediates and four end products were formed in the course of activation. From the NH2 terminus of factor V the end products are termed (apparent molecular weights in parentheses), fragment D (105,000), E (71,000), C1 (150,000), and F1F2 (71-74,000). The carboxyl-terminal fragment F1F2 is a doublet presumably resulting from cleavage of one of two approximately equally sensitive bonds. The end products but not the intermediates resemble those obtained on thrombin activation of bovine factor V, indicating a different order of peptide bond cleavages. The thrombin-catalyzed activation of purified factor V resembles the activation of factor V in plasma as judged from the activation pattern of a 125I-labeled factor V tracer in coagulating plasma. Fragments D and F1F2 are held together by noncovalent interactions and constitute the biologically active factor V molecule as judged from reconstitution experiments utilizing the isolated fragments. The two fragments (E and C1), derived from the interior of the molecule, are activation peptides.