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

Summary.  Background:  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. Objectives:  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. Methods:  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. Results:  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 n m vs. 82 n m ). Conclusions:  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.