Contribution of the prothrombin fragment 2 domain to the function of factor Va in the prothrombinase complex.
The prothrombinase complex assembles through reversible interactions between factor Xa, factor Va and acidic phospholipid-containing membranes in the presence of calcium ions. This complex catalyses the conversion of prothrombin to thrombin through two proteolytic steps. We have used prethrombin 2 as a substrate analog for the first cleavage reaction of prothrombin activation (cleavage at Arg323-Ile324) catalyzed by the prothrombinase complex and have also relied on the known ability of prethrombin 2 to interact tightly but reversibly with fragment 2 or fragment 1.2. The kinetics of cleavage at Arg323-Ile324 have been assessed with these substrate analogs to investigate the contribution of cofactor-substrate interactions mediated by the fragment 2 domain to the ability of factor Va to enhance the catalytic efficiency of factor Xa within the prothrombinase complex. Initial velocity measurements indicated that the rate of prethrombin 2 cleavage by the factor Xa-PCPS binary complex was increased by a factor of approximately 1300 upon the addition of saturating concentrations of factor Va to assemble prothrombinase. Although the measured initial velocity was higher when either fragment 2 or fragment 1.2 was present, the factor Va-dependent enhancement in initial rate (2600- and 1500-fold) was comparable in each case. Steady state kinetic constants were obtained using prethrombin 2, prethrombin 2 plus fragment 2, and prethrombin 2 plus fragment 1.2 as substrates. For each substrate, the addition of saturating concentrations of factor Va to the Xa-PCPS binary complex led to increases in catalytic efficiency of between 1000 and 9000-fold. The kcat/Km for prethrombin 2 cleavage by prothrombinase was essentially identical to that obtained for prethrombin 2 saturated with fragment 2. Thus, comparable accelerating effects of factor Va are observed independent of the presence of the fragment 2 domain in the substrate. The results indicate that interactions between factor Va and the substrate mediated by the fragment 2 domain do not contribute in a dominant way to the ability of factor Va to enhance the catalytic efficiency of factor Xa within the prothrombinase complex.
Krishnaswamy
S
S
Department of Medicine, Emory University, Atlanta, Georgia 30322, USA. skris01@unix.cc.emory.edu
Walker
R K
RK
D000818:Animals; D001120:Arginine; D002417:Cattle; D005165:Factor V / Q000302:isolation & purification / Q000378:metabolism*; D015943:Factor Va / Q000302:isolation & purification / Q000378:metabolism*; D005170:Factor X / Q000302:isolation & purification / Q000378:metabolism*; D015951:Factor Xa / Q000302:isolation & purification / Q000378:metabolism; D007532:Isoleucine; D007700:Kinetics; D008081:Liposomes; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011516:Prothrombin / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism*
D008081:Liposomes; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; C022475:prothrombinase complex; D007532:Isoleucine; D015943:Factor Va; C044183:prothrombin fragment 2; D005165:Factor V; D011516:Prothrombin; D005170:Factor X; D001120:Arginine; D013917:Thrombin; D015951:Factor Xa