HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Protein S Gla-domain mutations causing impaired Ca2-induced phospholipid binding and severe functional protein S deficiency

mutations in the exon that encodes the vitamin K–dependent Gla domain of pro-tein S (Gly11Asp and Thr37Met) in kin-dred with phenotypic protein S deficiency and thrombosis. In studies using recom-binant proteins, substitution of Gly11Asp did not affect production of protein S but resulted in 15.2-fold reduced protein S activity in a factor Va inactivation assay. Substitution of Thr37Met reduced expres-sion by 33.2 % (P <.001) and activity by 3.6-fold. The Gly11Asp variant had 5.4-fold reduced affinity for anionic phospho-lipid vesicles (P <.0001) and decreased affinity for an antibody specific for the Ca2-dependent conformation of the pro-tein S Gla domain (HPS21). Examination of a molecular model suggested that this could be due to repositioning of Gla29. In contrast, the Thr37Met variant had only a modest 1.5-fold (P <.001), reduced affini-ties for phospholipid and HPS21. This mutation seems to disrupt the aromatic stack region. The proposita was a com-pound heterozygote with free protein S antigen levels just below the lower limit of the normal range, and this is now attrib-uted to the partial expression defect of the Thr37Met mutation. The activity levels were strongly reduced to 15 % of normal, probably reflecting the functional deficit of both protein S variants. Her son (who was heterozygous only for Thr37Met) had borderline levels of protein S antigen and activity, reflecting the partial secretion and functional defect associated with this mutation. This first characterization of natural protein S Gla-domain variants highlights the importance of the high affinity protein S–phospholipid interac-tion for its anticoagulant role. (Blood