Protein S Regulates Factor IXa/VIIIa Activity Independent of Activated Protein C

Abstract 2196

Protein S (PS) is a vitamin-K-dependent plasma protein that plays an important role in the feedback regulation of thrombin generation. PS is a well recognized anticoagulant protein, the absence of which causes venous and arterial thrombosis. While PS has been studied for over three decades, the precise role this protein plays in attenuating the hemostatic response is far from clear. PS was initially identified as a cofactor for activated protein C (APC). Interestingly, recent reports suggest PS may function in an APC-independent manner as it has been suggested to inhibit prothrombinase complex formation and act as a stimulator of factor Xa-TFPI (tissue factor pathway inhibitor) complex.

Based on these new data, we have begun to examine possible roles of PS outside of the PC pathway which contribute to the regulation of thrombin generation. We recently obtained exciting preliminary data which suggests that PS plays an important role in the inhibition of Xa generation by the intrinsic Xase complex. Our central hypothesis is that PS binds to the enzyme FIXa in the presence of Phosphatidyl serine (PhosSer) containing membranes and inhibits FXa generation in the presence and absence of VIIIa.

We did clotting assays with FIX deficient plasma both with and without anti PS antibody. Clotting initiated with 0.1 nM FIXa shows a must faster clotting in the presence of anti PS antibody (57 seconds compared to 107 seconds without the incubation with anti PS antibody) (see Table 1). However, there is no difference in clotting times when the clotting is initiated with FVIIa in the presence and absence of anti PS antibody. Our results showing PS is only able to alter FIXa initiated clotting indicates that PS inhibits the FXa generation by FIXa/FVIIIa in plasma.

Table 1: Clotting of FIX deficient plasma initiated with FIXa (A) & initiated with FVIIa (B)

We determined the binding of PS to factor IXa by determining the change in anisotropy and fluorescence of DEGR(5 (dimethylamino)1-naphthalenesulfonyl]-glutamylglycylarginyl chloromethyl ketone)-IXa. The K_d was ~ 1 nM in both cases suggesting a tight binding of PS with factor IXa.

We also determined the activation of FX by FIXa in the presence of FVIIIa and 50 μM Phosphatidylserine membrane in the presence of PS. We used physiological concentrations of FX, PS,

0.1 nM FIXa, 5 nM FVIIIa and monitored the rate of FXa generation by using S2222 (chromogenic substrate). There was ~ 95 % inhibition in the rate of FXa generation by FIXa in the

0.2 presence of FVIIIa. Our results clearly indicate that PS inhibits FXa generation by factors IXa/VIIIa.

We believe that these new findings have major implications for better understanding how FXa generation is regulated during the initial phases of coagulation and may shed light on a major outstanding question in the field relating to how a key anticoagulant, PS contributes to the down regulation of the haemostatic response.