Departments of Medicine and Biochemistry, University of Vermont, Burlington, Vermont 05405 Received July 25, 1989; Revised Manuscript Received September 22, 1989 abstract: The activation of human factor V by factor Xa and thrombin was studied by functional assessment of cofactor activity and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by either autoradiography of 125I-labeled factor V activation products or Western blot analyses of unlabeled factor V activationproducts. Cofactoractivity was measured by the ability of the factor V/Va peptides to support the activation of prothrombin. The factor Xa catalyzed cleavage of factor V was observed to be time, phospholipid, and calcium ion dependent, yielding a cofactor with activity equal to that of thrombin-activated factor V (factor Va). Thecleavage patterndiffered markedly from the one observed in the bovine system. The factor Xa activated factor V subunits expressing cofactor activity were isolated and found to consist of peptides of MT 220000 and 105000. Although thrombin cleaved the Mx 220000 peptide to yield peptides previously shown to be products of thrombin activation, cofactor activity did not increase. N-Terminal sequence analysisconfirmed that both factor Xa and thrombin cleave factor V at the same bond to generate the Mt 220000 peptide. The factor Xa dependent functional assessment of 125I-labeled factor V coupled with densitometric analyses of the cleavage products indicated that the cofactor activity of factor Xa activated factor V closely paralleled theappearance of the MT 220 000 peptide. This observation facilitated the study of the kinetics of factor V activation by allowing the activation of factor V to be monitored by the appearance of the Mt 220000 peptide (factor Xa activation) or the Mr 105 000 peptide (thrombin activation). Factor Xa catalyzed activation of factor V obeyed Michaelis-Menten kinetics and was characterized by a Km of

10.4 nM, a kal of 2.6 min-1, and a catalytic efficiency (kat/Km) of 4.14 X 106 M" 1 s" 1. The thrombin-catalyzed activation of factor V was characterized by a Km of 71.7 nM, a kat of 14.0 min" 1, and a catalytic efficiency of 3.26 X 106 M" 1 s" 1. This indicates that factor Xa is as efficient an enzyme towardfactor V as thrombin.