FACTOR V IS ACTIVATED AND CLEAVED BY PLATELET CALPAIN: COMPARISON WITH THROMBIN PROTEOLYSIS

Abstract

Platelets are known to process human and bovine factor V during secretion and/or membrane binding. We therefore studied the functional and structural changes produced in human factor V and Va by purified platelet calpain. A maximum increase in factor V coagulant activity of 2.5-fold over control incubations was observed for calpain (0.6 u/ml) at 25°C in comparison with a 10-fold increment for a thrombin (1 u/ml). Thrombin addition to reactions initiated by calpain resulted in further activation comparable to that of thrombin alone, while subsequent addition of calpain had no effect on the extent or pattern of the activation of factor V by thrombin. The cleavage pattern of factor V produced by these two enzymes are distinctly different. Calpain yields initial components of 210 kDa and 160 kDa within 1 min. Further digestion of the 210 kDa species give rise to polypeptides of 150, 140 and 120 kDa by 2 min with and increase in coagulant activity. The degradation of the 160 kDa polypeptide gives rise to smaller fragments of 130, 100, 90, and 87 kDa. Immunob lot ting of these fragments with the monoclonal antibody B10 directed to factor V and the thrombin generated Cl fragments yields results demonstrating an immunological relationship to the calpain generated components of 210, 160, 140 and 120 kDa. Thus platelet calpain generates a complex cleavage pattern different from thrombin which may explain the partial activation observed.