Direct anticoagulant activity of protein S-C4b binding protein complex in Heerlen heterozygotes and normals*

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Abstract
Plasma protein S normally circulates free (40%) or complexed with C4b-binding protein (PS-C4BP); only free protein S is a cofactor for activated protein C during factor (F) Va inactivation. Protein S-Heerlen lacks a carbohydrate group, leading to low plasma free protein S levels, but normal levels of PS-C4BP. Because protein S-Heerlen is not associated with thrombosis, we investigated whether PS-C4BP is directly anticoagulant in plasma and whether PS-Heerlen-C4BP has enhanced direct anticoagulant activity. An assay for protein S direct activity was applied to Heerlen-heterozygous plasmas. Free and complexed protein S were repeatedly isolated from normal and Heerlen-heterozygous plasmas and tested for direct anticoagulant activity in prothrombinase assays and in plasma. Heerlen-heterozygous plasmas were deficient in free and total protein S antigen but had normal to high protein S direct anticoagulant activity. Purified Heerlen-heterozygous PS-C4BP was 7-fold more potent than normal PS-C4BP in inhibiting full prothrombinase activity, and 22-fold more potent in inhibiting prothrombin activation in the absence of FVa; it also specifically prolonged plasma clotting times 14-fold more than normal PS-C4BP. Heerlen-heterozygous PS-C4BP did not compete for limiting phospholipids any better than normal PS-C4BP. However, ligand blots and surface plasmon resonance studies showed that Heerlen-heterozygous PS-C4BP bound more avidly to FXa than did normal PS-C4BP (apparent Kd = 4.3 nm vs. 82 nm). Plasma-derived PS-C4BP has direct anticoagulant activity in plasma and in purified systems. Enhanced direct activity of PS-Heerlen-C4BP may compensate for low free protein S levels and low cofactor activity in individuals with protein S-Heerlen.