Role of Procoagulant Lipids in Human Prothrombin Activation. 2. Soluble Phosphatidylserine Upregulates and Directs Factor Xa to Appropriate Peptide Bonds in Prothrombin

• Published in: Biochemistry (Easton) 2002-01, Vol.41 (3), p.950-957
• Main Authors: Banerjee, Mou, Majumder, Rinku, Weinreb, Gabriel, Wang, Jianfang, Lentz, Barry R
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0116902

Activation of human prothrombin to thrombin (IIa) by factor Xa during blood coagulation requires proteolysis of two bonds and thus involves two possible activation pathways (parallel-sequential activation model). Hydrolysis of Arg322−Ile323 produces meizothrombin (MzIIa) as an intermediate, while hydrolysis of Arg273−Thr274 produces prethrombin 2−fragment 1.2 (Pre2−F1.2). A soluble lipid, dicaproylphosphatidylserine (C6PS), enhances activation by 60-fold [Koppaka et al. (1996) Biochemistry 35, 7482]. We report here that C6PS binding to factor Xa not only enhances the rate of activation but also alters the pathway. Activation was monitored using a chromogenic substrate (S-2238) to detect both IIa and MzIIa active site formation and SDS−PAGE to detect Pre2−F1.2 as well as IIa and MzIIa. Of the four kinetic constants needed to describe activation, two (MzIIa and Pre2−F1.2 consumption) were measured directly, and two (MzIIa and Pre2−F1.2 formation) were obtained by fitting the three time courses simultaneously to the parallel-sequential reaction model. The time courses of IIa, MzIIa, and Pre2−F1.2 formations were all well described below the C6PS critical micelle concentration (CMC) by this activation model. The rate of Arg322−Ile cleavage leading to MzIIa formation increased by 150-fold, while the rate of Arg273−Thr cleavage leading to Pre2−F1.2 formation was inhibited slightly. At concentrations of water-soluble C6PS above its CMC, all four proteolytic reactions increased in rate by 2−5-fold at the C6PS CMC. We conclude that soluble C6PS differentially affects the rate of individual bond cleavages during prothrombin activation in solution such that activation occurs almost exclusively via MzIIa formation. Finally, C6PS enhanced the rates of all proteolytic reactions to within a factor of 3 of the enhancement seen with PS-containing membranes. We conclude that PS-containing membranes regulate prothrombin activation by factor Xa mainly via interaction of individual PS molecules with factor Xa.