RECOMBINANT RAT PROTEIN-C - COMPARATIVE-STUDIES OF STRUCTURE, FUNCTION AND SYNTHESIS WITH PLASMA PROTEIN-C

In order to elucidate the role of protein C (PC) in the rat, we expres sed, purified, and characterized recombinant rat PC. The purified reco mbinant rat PC was 70-90% two-chain (41 kDa heavy chain; 22 and 23 kDa light chain) and 10-30% single-chain (61 kDa). Amino acid analysis co nfirmed the presence of 10 moles Of gamma-carboxyglutamic acid residue s per mol of protein. For comparison, plasma rat PC was purified from a barium citrate precipitate using similar method. Plasma rat PC was a two-chain form (41 kDa heavy chain; 22 kDa light chain) with no detec table single-chain nor 23 kDa light chain. For determination of the in vitro secreted species, primary cultured rat hepatocytes were incubat ed for 6 h with methionine-free MEM containing vitamin K1, aprotinin, and [S-35]Methionine. The supernatant was immunoprecipitated and analy zed by SDS-PAGE followed by autoradiography. Approximately 90% of the PC radioactivity migrated as a two-chain molecule. These results indic ate that rat PC is secreted mainly as a two-chain molecule from the li ver. PROTAC-activated forms of recombinant rat PC, plasma rat PC, and plasma human PC hydrolyzed the S-2366 chromogenic substrate at the sam e rate. Recombinant rat PC was also activated by the thrombin-thrombom odulin complex at a rate similar to plasma rat PC. The anticoagulant a ctivities of the three activated PCs were examined in rat plasma. Both recombinant and plasma rat PC prolonged the activated partial thrombo plastin time in a dose-dependent manner, but plasma human PC was less effective. These results suggest that recombinant rat PC is applicable for in vivo thrombosis studies in the rat.