Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

Abstract 4344

Localization of blood coagulation enzymes and cofactors to the surface of procoagulant anionic phospholipid membranes is a central paradigm in hemostasis and thrombosis. The prototype for these complexes is the prothrombinase complex that catalyzes the conversion of prothrombin to thrombin. This enzyme complex consists of the enzyme factor Xa, the cofactor factor Va and calcium bound to a phospholipid surface. Formation of the prothrombinase complex results in a greater than 10⁵-fold rate enhancement in the conversion of prothrombin to thrombin. Abnormal regulation of the prothrombinase complex has been demonstrated to be critical in the pathogenesis of thrombotic disorders.

Constituents of platelet membranes regulate the activity of the prothrombinase complex. We demonstrate that membranes containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) bind factor Va with high affinity (K_d∼10 nM) in the absence of phosphatidylserine (PS). These membranes support formation of a 60–70% functional prothrombinase complex at saturating factor Va concentrations. While reduced interfacial packing does contribute to factor Va binding in the absence of PS, it does not correlate with the enhanced activity of the Xa-Va complex assembled on PE-containing membranes. Instead, specific protein-PE interactions appear to contribute to the effects of PE. In support of this, soluble C6PE binds to recombinant factor Va₂ (K_d ∼6.5 mM) and to factor Xa (K_d ∼ 91 mM). C6PE and C6PS binding sites in factor Xa are specific, distinct and linked, as binding of one lipid enhances the binding and activity effects of the other. C6PE triggers assembly (K_d^app ∼40nM) of a partially active prothrombinase complex between factor Xa and factor Va2, compared to K_d^app for C6PS ∼2nM. We report here seven significant new observations:

1. PE effects k_cat more than K_M. A k_cat effect strongly implies an enzyme or cofactor conformational change, while a K_M effect could be due to conformational changes or to altered binding of enzyme-cofactor and substrate to the membrane.

2. PE promotes binding of factor Va to membranes.

3. PE promotes assembly of a prothrombinase complex due its effects on factors Va and Xa independent of the presence of a membrane and thus not due to its effects on membrane packing.

4. 30% PE does not produce a measurable effect on factor Xa membrane binding.

5. C6PE and C6PS bind synergistically to sites on factor Xa to alter its activity.

6. C6PE binds to factor Va with greater affinity than does C6PS, but binding of these lipids to sites on this protein is not synergistic.

7. Both PS and PE promote prothrombinase activity, but the magnitudes of these effects do not correlate with their effects on membrane interfacial packing.

These findings provide new insights into the possible synergistic roles of platelet PE and PS in regulating thrombin formation, particularly when exposed membrane PS may be limiting.