Platelets are known to process human factor V during secretion and/or membrane binding. We studied the functional and structural changes produced in human factor V by purified human platelet calpain (calcium- activated thiol protease) and compared the alterations with those induced by thrombin. A maximum increase in coagulant activity of 2.5- fold was observed when factor V (1 U/mL, 33 nmol/L) was incubated with calpain (0.03 U/mL, 2.7 nmol/L) in comparison with a 8.8-fold increment for alpha-thrombin (0.7 U/mL, 8 nmol/L) at 25 degrees C. Thrombin additions to reactions initiated by calpain resulted in further activation comparable to that of thrombin alone, whereas the 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. Although thrombin activation eventually results in four final components designated C1 (150 kd), D (105 kd), E (71 kd), and F1F2 (71 to 74 kd), calpain yields initial components of 200 kd and 160 kd within one minute. Further digestion of the 200 kd species by calpain gives rise first to a polypeptide of 160 kd that is converted to a 140 kd and a 120 kd species by two minutes with an increase in coagulant activity. Immunoblotting of these fragments with the monoclonal antibody (MoAb) B10 directed to factor V and the thrombin-generated C1 fragment yields results demonstrating a common epitope in these calpain-generated components of 200, 160, 140 and 120 kd. The degradation of the initial 160 kd polypeptide gives rise to polypeptides of 100 and 65 kd, both undetectable on immunoblotting with MoAb B10. The 130, 87, 58, and 48 kd components are of less certain origin. Thus, platelet calpain generates a complex but reproducible cleavage pattern different from thrombin that may explain the partial activation observed. Nevertheless, calpain processing may play a role in early hemostatic reactions involving platelets before the appearance of the first thrombin molecule.