Assembly of Prothrombinase on Endothelial Cells: Receptor-Mediated or Phospholipid-Driven?

Membrane binding by factors Xa and Va plays an essential role in facilitating their interaction to yield membrane-bound prothrombinase. This concept is backed by a large body of biochemical work using synthetic phospholipid vesicles typically composed of 25% phosphatidylserine (PS) and 75% phosphatidylcholine (PC). However, it remains uncertain whether kinetic and thermodynamic findings with these membranes can be extrapolated to explain the assembly of prothrombinase on cell surfaces relevant to coagulation in the vasculature. We examined the binding of fluorescent derivatives of Xa and Va to endothelial cells cultured in flow chambers using confocal imaging of fluorescence intensity. Cell-bound factor Xa was imaged using Alexa488 or Alexa532 covalently linked to the active site with a peptidyl chloromethyl ketone. Minimal fluorescence was observed on the endothelial surface without intentional activation of the cells by thrombin. Following thrombin activation, total fluorescence of bound Xa in the absence of added Va was marginally different from the unactivated cells but increased 16-fold when excess Va was present. Heterogeneity was evident in the distribution of fluorescence over the surface of the cells that supported Xa binding. Thus, the binding of Xa requires the presence of Va and is distributed in a heterogeneous fashion on the activated endothelial cell surface.  Factor Va bound to the endothelial cell was assessed using a derivative singly labeled at Cys⁵³⁹ with Alexa488 or Alexa532. Detection of bound Va also required prior activation by thrombin. The same robust signal for bound but heterogeneously distributed fluorescence was observed following activation both in the presence or absence of added Xa. Together the data indicate that Xa binding to the activated endothelial cell requires bound Va whereas Va binding is unaffected by the presence of Xa, implying a receptor-like role for Va. This is in marked contrast to the behavior on membranes containing 25% PS to which either Xa or Va can bind singly with good affinity. More definitive studies of prothrombinase assembly were conducted using donor fluorescence lifetime imaging on the cells using Xa_Alexa488 as donor and Va_Alexa532 as acceptor. Xa_Alexa488 bound in the presence of unlabeled Va exhibited an average lifetime of 3.1 ns which was decreased to 2.1 ns in the presence of Va_Alexa532. Equivalent lifetimes and energy transfer efficiency were measured using 25% PS containing membranes. Although Xa binding to the cells shows a near-absolute requirement for Va, the resulting cell-bound prothrombinase is comparable to that assembled on synthetic vesicles. An explanation could lie in the binding constraints associated with the exposure of limiting amounts of PS on the activated cell surface expected to disproportionately affect the binding of Xa and Va to membranes. These constraints were replicated using membrane bilayers supported on glass microspheres (lipospheres) containing either 0% or 5% PS balanced by PC and analysis of binding by flow cytometry. Background levels of binding were observed for all approaches in the absence of PS. Va binding was studied using a constant number of lipospheres and increasing concentrations of Va_Alexa488 in the presence of different concentrations of unlabeled Xa. Mean fluorescence per 5% PS containing liposphere increased saturably with increasing concentrations of Va_Alexa488 and was unaffected by Xa. The binding curves were consistent with a nM dissociation constant. In contrast, background levels of Xa were bound when monitored using varying concentrations of Xa_Alexa488 in the absence of Va.  A family of saturable curves was obtained at different fixed concentrations of unlabeled Va, signifying binding with nM affinity, with amplitude proportional to the concentration of Va. Thus, 5% PS containing lipospheres can replicate the paradoxical receptor-like role for Va in prothrombinase assembly on activated endothelial cells. The binding behavior on lipospheres could be fully accounted for by the markedly decreased affinity of Xa for 5% PS membranes and the major contribution of linked interactions in the stabilization of membrane-bound prothrombinase. Despite appearances to the contrary, a model developed from binding studies with pure phospholipids can be generalized to provide a thermodynamic accounting for the peculiarities of prothrombinase assembly on the endothelial cell.