The Ser460Pro mutation in recombinant protein S Heerlen does not affect its APC-cofactor and APC-independent anticoagulant activities

Abstract

Protein S is a vitamin K-dependent plasma protein that functions as an APC-cofactor, but also exhibits anticoagulant activity in the absence of APC. The Heerlen polymorphism of protein S is characterized by a Ser460Pro substitution and lacks glycosylation at Asn458. It is associated with decreased protein S levels due to selective deficiency of free protein S Heerlen. To understand the lack of thrombotic complications associated with the protein S Heerlen mutation, we compared recombinant protein S Heerlen, wild type (wt) protein S and plasma-derived protein S. wt-Protein S and protein S Heerlen each bound 1:1 to C4BP with dissociation constants of 0.27 and 0.33 nM, respectively. Both wt-protein S and protein S Heerlen, either free or in complex with C4BP, were equally active as prothrombinase inhibitors in the absence of APC. All three protein S preparations stimulated APC-catalyzed inactivation of normal FVa, FVa Leiden and FVIIIa to the same extent. If extrapolated to plasma, it is not likely that the decreased free protein S levels in carriers of the protein S Heerlen mutation are compensated by an increased anticoagulant activity of protein S Heerlen-C4BP complexes. It is possible that an unrecognized plasma factor selectively enhances the anticoagulant activity of protein S Heerlen. If not, the reduction of free protein S levels in heterozygous protein S Heerlen-carriers combined with (low) normal total protein S levels apparently minimally affects the total anticoagulant activity of protein S (APC-cofactor and APC-independent activity) and hence is not associated with increased risk of venous thrombosis.

Protein C and protein S deficiencies: similarities and differences between two brothers playing in the same game

“…However, this hypothesis was not confirmed by another study. The cause of the decrease in free PS concentrations associated with PS Heerlen has not been clarified, but most likely is a consequence of increased clearance (84).…”

In Vivo Clearance of Human Protein S in a Mouse Model

“…To facilitate complex formation, protein S (0.3 mol/L) and C4BP␣␤ (0.6 mol/L) were incubated for 45 minutes before injection. Because the concentration of both proteins is Ͼ300-fold above the K D value (Ͻ1 nmol/L), 24 these conditions result in Ͼ95% of protein S being in complex with C4BP␣␤. Furthermore, concentrations of C4BP␣␤ were chosen to be Ն4-fold below those that saturate the clearance system.…”

“…Protein S-Heerlen is found in Ϸ0.5% of the population 20 and is associated with reduced levels of free protein S in heterozygous or homozygous individuals. 21,22 Expression studies demonstrated that recombinant protein S-S460P is normally secreted, 21 indicating that other mechanisms should be in- 20,21,24,25 The notion that protein S-S460P is similar to wild-type (wt)-protein S in terms of secretion efficiency and binding to C4BP␣␤ prompted us to investigate the previously proposed hypothesis that protein S-Heerlen is cleared from the circulation more rapidly than normal protein S (Dr R.M. Bertina, XVI ISTH Congress, Florence, Italy, 1997).…”

“…No protein S antigen could be detected in these C4BP␣␤ preparations in an immunosorbent assay, whereas the ability to bind protein S was fully retained. Purification of recombinant C4BP␣, recombinant wt-protein S, and recombinant protein S-S460P has been described previously 24,26,27 (see also online supplemental data). …”

“…23 However, protein S-S460P and protein S were similar in their interaction with C4BP␣␤ in various other reports. 20,21,24,25 The notion that protein S-S460P is similar to wild-type (wt)-protein S in terms of secretion efficiency and binding to C4BP␣␤ prompted us to investigate the previously proposed hypothesis that protein S-Heerlen is cleared from the circulation more rapidly than normal protein S (Dr R.M. Bertina, XVI ISTH Congress, Florence, Italy, 1997).…”