Structural pathology and functional analysis of vitamin K-dependent protein S

Sammanfattning: Protein S deficiency is an autosomal dominant trait affecting around 10% of families thrombophilic families. The high affinity interaction of approximately 60% of protein S with C4b -binding protein (C4BP) has raised a complicated situation for the diagnosis of deficiency states. Because only free protein S functions as cofactor in the protein C anticoagulant, it is important to measure free protein S level precisely. In this study a free protein S assay was developed using its natural ligand C4BP to capture free protein S from plasma for routine clinical purpose. Although interference by C4BP is so far considered as the major hindrance for developing ELISA, the new approach has utilized this phenomenon of high affinity interaction between protein S and C4BP, thereby reversing the adverse effect of C4BP to one of good use. (1) Mutations in a variety of human genes are now known to predispose to venous thrombosis. Characterization of the wide spectrum of gene mutations causing thrombosis may allow us to relate specific gene lesions to the probability of thromboembolism as well as to the severity of thrombotic episodes, beside providing the molecular mechanism of deficiency states. To establish the relationship between genetic abnormalities of protein S and their phenotypic expressions, four naturally occurring missense mutations were chosen to analyze thier in vitro secretion profiles and functional characteristics, which illustrated the importance of in vitro experimental characterization in each and individual cases of naturally occurring missense mutations before marking them as the underlying genetic defect. (2) Since early nineties, the suggestion about the synergistic effect between two thrombotic risk factors when associated in one patient has been lacking a biochemical basis. The now observed deficient APC-cofactor activity of protein S Heerlen in the degradation of FVa Leiden suggests a possible synergistic pathogenic mechanism between these two genetic traits resulting in increased risk of thrombosis. (3) In addition, using two monoclonal antibodies as probes the structure-function relationship studies on protein S and APC interaction were carried out. R49, Q52 in TSR was found to be part of the epitope of monoclonal HPS 67 and K97, T103 in EGF1 for monoclonal HPS 54. These data implicated indirectly as the key amino acids for APC interaction, based on the fact that these two antibodies could completely block the APC-cofactor activity of protein S. The observation that HPS 67 did not inhibit phospholipid binding of protein S has implications for the possible orientation of protein S on the membrane surface, suggesting that TSR is free to interact with membrane-bound APC. (4) Also, evaluation of the importance of amino acid residues 447-460 in protein S for binding to C4BP was performed. One amino acid Y456 was found to be important residue for C4BP interaction in this region. However, blocking this region by monoclonal antibody HPS 34 was not sufficient enough to inhibit protein S-C4BP interaction completely, suggesting that the interaction site constitute a fairly large binding surface.