The roles of factor Va and protein S in formation of the activated protein C/protein S/factor Va inactivation complex

Abstract

BackgroundActivated protein C (APC)‐mediated inactivation of factor (F)Va is greatly enhanced by protein S. For inactivation to occur, a trimolecular complex among FVa, APC, and protein S must form on the phospholipid membrane. However, direct demonstration of complex formation has proven elusive.ObjectivesTo elucidate the nature of the phospholipid‐dependent interactions among APC, protein S, and FVa.MethodsWe evaluated binding of active site blocked APC to phospholipid‐coated magnetic beads in the presence and absence of protein S and/or FVa. The importance of protein S and FV residues were evaluated functionally.ResultsActivated protein C alone bound weakly to phospholipids. Protein S mildly enhanced APC binding to phospholipid surfaces, whereas FVa did not. However, FVa together with protein S enhanced APC binding (>14‐fold), demonstrating formation of an APC/protein S/FVa complex. C4b binding protein‐bound protein S failed to enhance APC binding, agreeing with its reduced APC cofactor function. Protein S variants (E36A and D95A) with reduced APC cofactor function exhibited essentially normal augmentation of APC binding to phospholipids, but diminished APC/protein S/FVa complex formation, suggesting involvement in interactions dependent upon FVa. Similarly, FVaNara (W1920R), an APC‐resistant FV variant, also did not efficiently incorporate into the trimolecular complex as efficiently as wild‐type FVa. FVa inactivation assays suggested that the mutation impairs its affinity for phospholipid membranes and with protein S within the complex.ConclusionsFVa plays a central role in the formation of its inactivation complex. Furthermore, membrane proximal interactions among FVa, APC, and protein S are essential for its cofactor function.

Anticoagulant protein S—New insights on interactions and functions

“…16,52,53,99 Protein S Gla36 and Asp95 (dark green) are both needed for APC cofactor function and we recently hypothesized that they may be part of a FVa interaction site. 46,50,51 This is in agreement with a previously published study, identifying Gla-TSR residues 37-50 (light green) being involved in FVa binding. 56 The epidermal growth factor 2 (EGF2) domain has also been shown to be essential for the APC cofactor function of protein S, suggesting its involvement in important protein-protein interactions.…”

“…Specifically, Arg306 cleavage was severely reduced. In a recent follow-up study, 46 we showed that this was in part due to reduced affinity toward negatively charged phospholipid membranes.…”

“…Together, this results in an overall 6-to 8-fold reduction in enhancement of APC-mediated inactivation of FVa by C4BP-bound compared to free protein S. 19 This is likely explained by the reduced ability of C4BP-bound protein S to be incorporated into the tri-molecular FVa inactivation complex. 46 The SHBG-like region, which interacts with C4BP, has previously been suggested to be important for the enhancement of APC and to contain an interaction site for FVa ( Figure 2). [47][48][49] It is therefore possible that C4BP blocks a protein S-FVa interaction site.…”

“…Using a flow cytometry approach and fluorescently labelled/inactive APC, our study confirmed that FVa is involved in the recruitment of APC to the phospholipid surfaces. 46 While protein S enhanced the binding of APC to negatively charged membranes by a modest ~2-fold, the affinity was enhanced >14-fold in the presence of both protein S and FVa ( Figure 3B). It is well known that C4BP-bound protein S is an inefficient cofactor of APC.…”

“…A, Several studies have shown that protein S and FVa enhance APC binding to a negatively charged phospholipid membrane, where it forms a FVa inactivation complex. 32,45,46 B, We recently showed that protein S enhances the APC affinity for phospholipid membranes by ~2-fold while protein S and FVa synergistically enhance the APC-phospholipid binding by >14-fold 46 suggested that both residues may be involved in the interaction with FVa. This is supported by a paper published by Heeb et al who identified protein S residues 37-50 as a potential binding site for FVa.…”