Factor V (FV) present in platelet -granules has a significant but incompletely understood role in hemostasis. This report demonstrates that a fraction of platelets express very high levels of surface-bound, -granule FV on simultaneous activation with 2 agonists, thrombin and convulxin, an activator of the collagen receptor glycoprotein VI. This subpopulation of activated platelets represents 30.7% ± 4.7% of the total population and is referred to as convulxin and thrombin–induced-FV (COAT-FV) platelets. COAT-FV platelets are also observed on activation with thrombin plus collagen types I, V, or VI, but not with type III. No single agonist examined was able to produce COAT-FV platelets, although ionophore A23187 in conjunction with either thrombin or convulxin did generate this population. COAT-FV platelets bound annexin-V, indicating exposure of aminophospholipids and were enriched in young platelets as identified by the binding of thiazole orange. The functional significance of COAT-FV platelets was investigated by demonstrating that factor Xa preferentially bound to COAT-FV platelets, that COAT-FV platelets had more FV activity than either thrombin or A23187–activated platelets, and that COAT-FV platelets were capable of generating more prothrombinase activity than any other physiologic agonist examined. Microparticle production by dual stimulation with thrombin and convulxin was less than that observed with A23187, indicating that microparticles were not responsible for all the activities observed. These data demonstrate a new procoagulant component produced from dual stimulation of platelets with thrombin and collagen. COAT-FV platelets may explain the unique role of -granule FV and the hemostatic effectiveness of young platelets.

The effect of replacing the ␥-carboxyglutamic acid domain of activated protein C (APC) with that of prothrombin on the topography of the membrane-bound enzyme was examined using fluorescence resonance energy transfer. The average distance of closest approach (assuming 2 ‫؍‬ 2/3) between a fluorescein in the active site of the chimera and octadecylrhodamine at the membrane surface was 89 Å, compared with 94 Å for wildtype APC. The ␥-carboxyglutamic acid domain substitution therefore lowered and/or reoriented the active site, repositioning it close to the 84 Å observed for the APC⅐protein S complex. Protein S enhances wild-type APC cleavage of factor Va at Arg 306 , but the inactivation rate of factor Va Leiden by the chimera alone is essentially equal to that by wild-type APC plus protein S. These data suggest that the activities of the chimera and of the APC⅐protein S complex are equivalent because the active site of the chimeric protein is already positioned near the optimal location above the membrane surface to cleave Arg 306 . Thus, one mechanism by which protein S regulates APC activity is by relocating its active site to the proper position above the membrane surface to optimize factor Va cleavage.

To determine the structural basis of phosphatidylethanolamine (PE)-dependent activated protein C (APC) activity, we prepared a chimeric molecule in which the Gla domain and hydrophobic stack of protein C were replaced with the corresponding region of prothrombin. APC inactivation of factor Va was enhanced 10 -20-fold by PE. Protein S enhanced inactivation 2-fold and independently of PE. PE and protein S had little effect on the activity of the chimera. Factor Va inactivation by APC was approximately 5-fold less efficient than with the chimera on vesicles lacking PE and slightly more efficient on vesicles containing PE. The cleavage patterns of factor Va by APC and the chimera were similar, and PE enhanced the rate of Arg 506 and Arg 306 cleavage by APC but not the chimera. APC and the chimera bound to phosphatidylserine:phosphatidylcholine vesicles with similar affinity (K d Ϸ 500 nM), and PE increased affinity 2-3-fold. Factor Va and protein S synergistically increased the affinity of APC on vesicles without PE to 140 nM and with PE to 14 nM, but they were less effective in enhancing chimera binding to either vesicle. In a factor Xa one-stage plasma clotting assay, the chimera had ϳ5 times more anticoagulant activity than APC on PE-containing vesicles. Unlike APC, which showed a 10 fold dependence on protein S, the chimera was insensitive to protein S. To map the site of the PE and protein S dependence further, we prepared a chimera in which residues 1-22 were derived from prothrombin and the remainder were derived from protein C. This protein exhibited PE and protein S dependence. Thus, these special properties of the protein C Gla domain are resident outside of the region normally hypothesized to be critical for membrane interaction. We conclude that the protein C Gla domain possesses unique properties allowing synergistic interaction with factor Va and protein S on PE-containing membranes.

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In this study, we test the hypothesis that prothrombin levels may modulate activated protein C (APC) anticoagulant activity. Prothrombin in purified systems or plasma dramatically inhibited the ability of APC to inactivate factor Va and to anticoagulate plasma. This was not due solely to competition for binding to the membrane surface, as prothrombin also inhibited factor Va inactivation by APC in the absence of a membrane surface. Compared with normal factor Va, inactivation of factor Va Leiden by APC was much less sensitive to prothrombin inhibition. This may account for the observation that the Leiden mutation has less of an effect on plasma-based clotting assays than would be predicted from the purified system. Reduction of protein C levels to 20% of normal constitutes a significant risk of thrombosis, yet these levels are observed in neonates and patients on oral anticoagulant therapy. In both situations, the correspondingly low prothrombin levels would result in an increased effectiveness of the remaining functional APC of ≈5-fold. Thus, while the protein C activation system is impaired by the reduction in protein C levels, the APC that is formed is a more effective anticoagulant, allowing protein C levels to be reduced without significant thrombotic risk. In situations where prothrombin is high and protein C levels are low, as in early stages of oral anticoagulant therapy, the reduction in protein C would result only in impaired function of the anticoagulant system, possibly explaining the tendency for warfarin-induced skin necrosis.

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Activated protein C (APC) anticoagulant activity and the ability to be inhibited by autoantibodies associated with thrombosis are strongly augmented by the presence of phosphatidylethanolamine (PE) and phospholipid oxidation. ␤ 2 -glycoprotein I (␤ 2 -GPI) is a major antigen for antiphospholipid antibodies present in patients with the antiphospholipid syndrome. We therefore investigated whether anti-␤ 2 -GPI monoclonal antibodies (mAbs) could inhibit APC with similar membrane specificity. Five mouse mAbs that reacted with different epitopes on ␤ 2 -GPI were examined. Each inhibited the PE-, phospholipid oxidation-dependent enhancement of APC anticoagulant activity and required antibody divalency. A chimeric APC that retains anticoagulant activity but is relatively unaffected by protein S, PE, or oxidation was not inhibited by the antibodies. In purified systems, anti-␤ 2 -GPI mAb inhibition of factor Va inactivation was greater in the presence of protein S and required ␤ 2 -GPI. Surprisingly, although the mAbs did increase ␤ 2 -GPI affinity for membranes, PE and oxidation had little influence on the affinity of the ␤ 2 -GPI antibody complex for the membrane vesicles. We conclude that antibodies to ␤ 2 -GPI inhibit APC function specifically and contribute to a hypercoaguable state by disrupting specific protein-protein interactions induced by oxidation of PE-containing membranes. (Blood. 2005;106:1629-1635)

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