Protein S K196E mutation reduces its cofactor activity for APC but not for TFPI

Abstract

BackgroundProtein S (PS) is an anticoagulant molecule that functions as a cofactor for activated protein C (APC) in the inactivation of activated coagulation factors Va (FVa) and VIIIa. It also serves as a cofactor for tissue factor pathway inhibitor (TFPI) in the efficient inhibition of factor Xa (FXa). The Lys196‐to‐Glu (K196E, Tokushima) mutation in the EGF‐2 domain of PS is a genetic risk factor for venous thromboembolism (VTE) in the Japanese population.ObjectivesTo investigate the molecular basis of the thrombophilic phenotype of Japanese patients carrying the PS K196E mutation.MethodsWe expressed recombinant human PS wild‐type (PS‐K) and K196E‐mutant (PS‐E) in CHO cells, and purified them by Ni2+‐affinity and anion exchange column chromatography. We investigated the anticoagulant functions of PS‐K and PS‐E by measuring APC cofactor activity, TFPI cofactor activity, affinity for the β chain of complement component C4b‐binding protein (C4BP), and cleavage by thrombin.Results PS‐E had approximately 40% APC cofactor activity compared with PS‐K in a clotting‐based assay and a FVa inactivation assay. The TFPI cofactor activity of PS‐E in the FXa inactivation assay was equivalent to that of PS‐K in the absence and presence of coagulation factor V. The strengths of PS‐E and PS‐K binding to the β chain of C4BP were comparable, and both were equally cleaved by thrombin.ConclusionsThe PS K196E mutation increases the risk of VTE because of reduced APC cofactor activity but does not alter various other properties, including the TFPI cofactor activity.

Carrier frequencies of antithrombin, protein C, and protein S deficiency variants estimated using a public database and expression experiments

“…Antigen levels of AT and PC in culture media were measured using Matched‐Pair Antibody Sets for ELISA (Affinity Biologicals, Ancaster, ON, Canada). Antigen levels of PS were measured using an in‐house sandwich ELISA with a polyclonal rabbit antihuman PS primary antibody (Agilent Dako, Carpinteria, CA, USA) and horseradish peroxidase–labeled anti‐human PS secondary antibody (Affinity Biologicals) 4 . Although the protein S ELISA used can detect both C4b‐bound and free (unbound) protein S, all recombinant protein S was free in the present study.…”

“…The human PC expression vector ORF‐NM_000312‐pCMV‐SPORT6 was purchased from RIKEN BRC (Tsukuba, Japan). The human PS expression vector was previously described 4 . In total, we constructed 73 variants using a site‐directed mutagenesis technique and verified their sequences using an Applied Biosystems 3500xL genetic analyzer (Life Technologies, Rockville, MD, USA).…”

“…The human PS expression vector was previously described. 4 In total, we constructed 73 variants using a site-directed mutagenesis technique and verified their sequences using an Applied Biosystems 3500xL genetic analyzer (Life Technologies, Rockville, MD, USA). Two clones of each variant were selected for expression experiments and are described below.…”

Pleiotropic anticoagulant functions of protein S, consequences for the clinical laboratory. Communication from the SSC of the ISTH

“…Second, a mutation in the EGF2 domain known as PS Tokushima reduces the APC-cofactor activity of PS, but not its TFPIα-cofactor activity. 45 However, while many of the interaction sites involved in the two pathways are different, it is possible that some pleiotropic effects may still exist. For example, Gla-domain-mediated binding to phospholipids is involved in both APC-and TFPIα-dependent cofactor activities of protein S. 1,4 Thus, a qualitative PS defect may selectively decrease the APC-or the TFPIα-cofactor activity of PS, or pleiotropically affect both.…”

“…First, APC interacts with residues in the TSR‐EGF1‐EGF2 domains of PS, 8‐10 while TFPIα interaction sites have been mapped to the SHBG‐like region, and more specifically LG1, of PS 15 (see also Figure 1C). Second, a mutation in the EGF2 domain known as PS Tokushima reduces the APC‐cofactor activity of PS, but not its TFPIα‐cofactor activity 45 . However, while many of the interaction sites involved in the two pathways are different, it is possible that some pleiotropic effects may still exist.…”