Protamine Sulfate Interacts with Partially Activated Forms of Factor V Which Contributes to Its Paradoxical Anticoagulant Effect

Unfractionated heparin (UFH) is a highly negatively charged molecule commonly used to prevent blood clotting in different clinical situations including cardiac and vascular surgeries. Its anticoagulant effect can be rapidly reversed or neutralized with protamine sulfate, a positively charged polypeptide that forms a stable interaction with UFH. Paradoxically, in vitro and in vivo studies have reported that protamine can also elicit an anticoagulant effect. Examination into this phenomenon (Ni Ainle, 2009; Bollinger, 2010) suggests that the anticoagulant activity of protamine relates to the inhibition of factor V (FV) activation and prothrombinase generation, but further mechanistic insights are lacking. FV circulates in blood as an inactive procofactor protein. Proteolytic cleavage of its central B-domain by thrombin converts it into an active cofactor (FVa) for factor Xa (FXa) in the prothrombinase complex. Our laboratory has established that the basic region (BR; residues 963-1008) and acidic region 2 (AR2; residues 1493-1537) within the B-domain are the minimal autoinhibitory sequences that keep FV inactive. Deletion of the BR yields a protein with cofactor-like properties in the absence of proteolysis, and the addition of the BR fragment in trans inhibits FV procoagulant function. Under normal physiological situations, naturally occurring ligands that mimic BR could re-engage forms of FV that harbor AR2 but lack BR (generically termed, FV_AR). For example, it has been found that the C-terminus of TFPIα is homologous to FV BR and binds to physiologically relevant forms of FV_AR including platelet FV as well as FV-short. FV-short represents less than 2% of plasma FV and is a splicing isoform initially found in the FV-East Texas family. It lacks most of the B-domain including BR and binds with high affinity to TFPIα. It is constitutively active, however the interaction with TFPIα converts it to a procofactor-like state, impairing its ability to bind to FXa and get proteolytically processed by thrombin. Due to its basic nature and observed anticoagulant effects, we hypothesized that protamine binds to the AR2 of FV_AR species and inhibits cofactor function. We purified plasma derived FV (pd-FV), recombinant FVa, FV-short and FV-shortQQ (a thrombin resistant mutant) and employed functional assays to characterize the interaction with protamine. Using purified component assays we found that FV-short and FV-shortQQ activities are comparable to FVa when assembled in prothrombinase. However, protamine had a major inhibitory effect on FV-short cofactor function, but not FVa. Further, using fluorescence anisotropy measurements, we found that protamine could displace labeled BR bound to FV-short, indicating that the two molecules compete for similar binding sites on FV-short. In addition, western blotting analysis showed that protamine impaired proteolytic processing of FV species by thrombin or FXa. When added to pooled normal human plasma (NHP) or FV-deficient plasma (FV-DP) reconstituted with pd-FV or FV-short or the QQ variant, protamine caused a dose-dependent anticoagulant response on thrombin generation (TG). The inhibition was greater with FV-shortQQ, while protamine did not affect TG when FVa was employed. The addition of protamine (10 μg/ml) to heparinized (0.2 U/ml) plasma restored TG in NHP and FV-DP reconstituted with FV-short or FVa. Addition of higher concentrations of protamine to heparinized plasma resulted in the progressive reduction of TG for NHP and FV-short plasma, with no further effects on plasma reconstituted with FVa. We conclude that protamine, through molecular mimicry of FV BR and the C-terminal region of TFPIα, binds forms of FV that have an available AR2 such as FV-short. This interaction not only decreases procoagulant function of FV_AR species and inhibits the formation of the prothrombinase complex but also delays their further processing by thrombin or FXa. These results suggest that, under physiological conditions, protamine exerts its anticoagulant effect by interacting with physiological relevant FV_AR species. These include platelet-derived FV, partially cleaved FV, and FV-short. This study provides a molecular mechanism for the adverse anticoagulant effects of protamine and highlights the importance of physiologically relevant FV_AR species.