Abstract
Constituents of platelet membranes regulate the activity of the prothrombinase complex. We demonstrate that membranes containing phosphatidylcholine 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. Although 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 Va(2) (K(d) = ∼6.5 μm) and to factor Xa (K(d) = ∼91 μm). C6PE and C6PS binding sites of factor Xa are specific, distinct, and linked, because binding of one lipid enhances the binding and activity effects of the other. C6PE triggers assembly (K(d)(app) = ∼40 nm) of a partially active prothrombinase complex between factor Xa and factor Va(2), compared with K(d)(app) for C6PS ∼2 nm. 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.
Highlights
PS is an allosteric regulator lipid that appears on activated platelets along with PE
Prothrombinase Activity on PE Membranes—Previous studies reported that PE:PC membranes have either limited or no capacity to promote formation of an active prothrombinase complex [16]
We recorded the activity of the bovine prothrombinase complex on DOPE:DOPC membranes in the presence of varying factor Va concentrations, 0.2 nM factor Xa, 1.4 M prothrombin, 3 mM CaCl2, and 5 M phospholipid vesicles
Summary
PS is an allosteric regulator lipid that appears on activated platelets along with PE. We demonstrate that membranes containing phosphatidylcholine and phosphatidylethanolamine (PE) bind factor Va with high affinity (Kd ؍ϳ10 nM) in the absence of phosphatidylserine (PS) These membranes support formation of a 60 –70% functional prothrombinase complex at saturating factor Va concentrations. C6PE triggers assembly (Kdapp ؍ϳ40 nM) of a partially active prothrombinase complex between factor Xa and factor Va2, compared with Kdapp for C6PS ϳ2 nM These findings provide new insights into the possible synergistic roles of platelet PE and PS in regulating thrombin formation, when exposed membrane PS may be limiting. Localization of blood coagulation enzymes and cofactors to the surface of procoagulant anionic phospholipid membranes is a central paradigm in hemostasis and thrombosis [1, 2] The prototype for these complexes is the prothrombinase complex that catalyzes the conversion of prothrombin to thrombin. We test this hypothesis and ask whether there are additional effects of PE on membrane binding of factors Xa or Va or on the activity of factors Xa or Va that might contribute to the effects of PE on prothrombinase activity and prothrombinase assembly
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