In protein S Heerlen, an S-to-P (single-letter amino acid codes) mutation at position 460 results in the loss of glycosylation of N458. This polymorphism has been found to be slightly more prevalent in thrombophilic populations than in normal controls, particularly in cohorts of patients having free protein S deficiency. This suggests that carriers of the Heerlen allele may have an increased risk of thrombosis. We have now characterized the expression in cell cultures of recombinant protein S Heerlen and investigated the anticoagulant functions of the purified recombinant protein in vitro. Protein S Heerlen was synthesized and secreted equally well as wild-type protein S by transiently transfected COS-1 cells. The recombinant protein S Heerlen interacted with conformation-dependent monoclonal antibodies and bound C4b-binding protein to the same extent as wild-type protein S. Protein S Heerlen displayed reduced anticoagulant activity as cofactor to activated protein C (APC) in plasma-based assays, as well as in a factor VIIIa-degradation system. In contrast, protein S Heerlen functioned equally well as an APC cofactor in the degradation of factor Va as wild-type protein S did. However, when recombinant activated factor V Leiden (FVa:Q506) was used as APC substrate, protein S Heerlen was found to be a poor APC cofactor as compared with wild-type protein S. These in vitro results suggest a possible mechanism of synergy between protein S Heerlen and factor V Leiden that might be involved in the pathogenesis of thrombosis in individuals carrying both genetic traits. (Blood. 2000;96:523-531)