The Effect of Factor Va on Prothrombinase Function

• Published in: Blood 2008-11, Vol.112 (11), p.1020-1020
• Main Authors: Kalafatis, Michael, Hirbawi, Jamila, Barhoover, Melissa A., Bukys, Michael A.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V112.11.1020.1020

Following injury to the vasculature, timely activation of prothrombin (Pro) to thrombin (IIa) induces fibrin formation and platelet activation. Activation of human Pro occurs because of two cleavages by membrane-bound factor Xa (fXa) (Arg271 followed by Arg320, pathway I, pre2 pathway). However, the catalytic efficiency of membrane-bound fXa is poor in the absence of the non-enzymatic cofactor, factor Va (fVa), and the overall reaction is incompatible with efficient IIa formation. Binding of fVa to fXa on a membrane surface in the presence of divalent metal ions results in the formation of prothrombinase (IIase). This enzymatic complex catalyzes the activation of Pro following the opposite pathway characterized by the formation of meizothrombin (Mz)(cleavage at Arg320 followed by Arg271, pathway II, meizo pathway). This pathway results in a dramatic increase in the catalytic efficiency of fXa. Thus, the activity of fXa within IIase is controlled by the presence of the non-enzymatic cofactor, fVa. Earlier data suggested that IIase activation of Pro may be best described as an ordered ping-pong reaction and, more recently, data using recombinant Pro established the existence of two forms of IIase. It has been also shown that IIase is a 1:1 stoichiometric association between fVa and fXa on a lipid surface. If IIase is one enzyme, theoretically, reversing the concentrations of reactants will have no effect on the pathway and catalytic efficiency of fXa in the presence of fVa for cleavage of Pro. We have performed such experiments and gel electrophoresis data showed that in the absence of fVa, membrane-bound fXa (10nM) activates Pro slowly through pathway I with production of prethrombin 2. Very little IIa is formed under these conditions (as indicated by the small amounts of B chain of IIa). In the presence of 10nM factor Xa and 100pM factor Va, there is a significant increase in the concentration of IIa as demonstrated by the increase in the appearance of B chain of IIa. However, IIa is still activated through pathway I as indicated by the presence of prethrombin 2 and the absence of fragment 1•2-A both of which are an indication of the activation of Pro via pathway I. In the presence of 100pM fXa with 10nM fVa, Pro is activated exclusively through the meizo pathway as demonstrated by the presence of fragment 1•2-A and the absence of prethrombin 2. These data demonstrate the existence of two enzymes: one form of IIase that exists at low, limiting concentratio