Antithrombotic Protein C

The protein C anticoagulant system plays a critical regulatory role in blood coagulation and thrombosis. The protein C pathway is an antithrombotic system that inhibits blood coagulation by proteolytically cleaving and inactivating clotting factors Va and VIIIa, two cofactors that are essential for clot formation. Protein C is activated on the endothelial cell surface by thrombin-thrombomodulin-endothelial protein C receptor complex to activated protein C (APC). APC inactivates factor Va in a complex manner cleaving factor Va proteolytically in at least two and possibly at three sites. Heterozygous protein C deficiency is an autosomally inherited disorder and is associated with venous thrombosis. Protein C deficiencies are classified into two categories, Type I and Type II deficiency, based on activity and antigen level of the protein. Infants with homozygous protein C deficiency have an increased risk for purpura fulminans or venous thrombosis shortly after birth and die within weeks in the absence of aggressive anticoagulant therapy. An arginine (R) 352 to tryptophan (R352W) mutation in protein C was detected in a patient with venous thrombosis and type II deficiency (Greengard et al., 1994). R352 is located in a loop of protein C near the catalytic site and in a positively-charged exosite. Because of the clinical relevance and the close location of R352 to the catalytic triad (eight amino acids removed from Ser 360) it becomes an important candidate for biochemical investigation. In this application I propose to examine the structural and functional consequence of replacing this arginine residue with tryptophan at position 3 52 of protein C. Another mutation of arginine to alanine (R352A) has been constructed and will be studied to analyse the effect of different charge and size of the amino acid. Since R352A protein C mutation has not been described in an individual and R352W protein C-plasma antigen levels in the protein C deficient patient were insufficient for in vitro biochemical analyses, these mutant proteins will be recombinantally overexpressed using mammalian cell tissue culture to get a sufficient amount of the mutant proteins. The mutants will be constructed by site directed mutagenesis and then expressed in K293 kidney cells (Kunkel et al., 1987). Their enzymatic and anticoagulant activities will be compared to the recombinant wild type (rwt) protein C and plasma-derived protein C.