Natural Mutations in the 4th EGF-Like Domain of Protein S Alter GLA and SHBG Domain Interactions.

We report the characterization of two rare natural variants of protein S (PS) produced by a missense mutation (PSN217S) and an in-frame deletion (PSDelI203D204), originating from a splicing mutation (IVSG–2A/T). Both defects affect the EGF4 domain, a module with a poorly defined functional role, and were found in the heterozygous condition in thrombotic patients with type III (PSN217S) or type II (PSDelI203D204) deficiencies, and in several members of their families. The interaction between C4bBP and the recombinant PS217S (A) and PSDelI203D204 (B) variants, studied through binding assay, was reduced but not abolished (A, Kd = 47.2 ± 3.7 nM and B, Kd = 107.7 ± 14.0 nM) as compared to rPSwt (Kd = 8.2 ± 1.0 nM). Interaction with phospholipid vesicles (PC/PS/PE 60 : 20 : 20) was also markedly reduced (A, Kd = 175.5 ± 53.1 nM; B, Kd = 235.7 ± 30.8 nM) as compared to rPSwt (Kd = 15.2 ± 0.9 nM). These data suggest extended conformational changes in both rPS variants, confirmed by reduced interaction with a GLA domain conformation-specific antibody (A, Kd = 2.76 ± 0.23 nM; B, Kd = 3.83 ± 0.39 nM and rPSwt Kd = 0.37 ± 0.04 nM) and an EGF1 conformationspecific antibody (A, Kd = 3.85 nM, B, Kd = 4.83 nM and rPSwt Kd = 0.68 nM). Anticoagulant APC cofactor activity was markedly reduced for both PS variants (A, 10% and B, 8% of rPSwt) and APC-independent activity, measured in a prothrombinase assay, was decreased (A, 41.2 ± 10.0%; B, 37.8 ± 4.6% of rPSwt). These data provide examples of extended conformational changes, with a similar functional effect, produced by different mutation types in one domain (EGF4) and transmitted through intra-molecular communication of in tandem EGF-like modules to the GLA domain. They also provide the molecular bases for peculiar PS deficiencies and their thrombophilic nature.