Sappearance of multiple FVa degradation intermediates as well as proth…

Sappearance of multiple FVa degradation intermediates as well as prothrombinase activity changes, with experiments done multiple times, with plasma proteins derived from multiple preparations. Current coagulation models that incorporate the protein C pathway [69-79] provide an often simplistic one step inactivation reaction of FVa by APC, without incorporating the feedback inhibition of the APC Va1-306 VaLC complex, the ability of partially proteolyzed FVa species to form catalytically active prothrombinase species, and the formation of FVa T species. They either do not provide empirical data verifying their model construct or rely on a single global output to validate the entire model construct. Wagenvoord et al. have outlined the limitations of mathematical models of complex reaction networks validated by a single analyte [80]. This study highlights the complexity of theHuman prothrombin and Factor X were isolated according to methods as described [81]. Factor Xa was prepared as previously described using Russel's Viper Venom Factor Xa Activator [82,83]. Human Factor V was purified from citrated plasma according to previously described procedures [83]. Activated human protein C was purchased from Haematologic Technologies, Essex Junction, VT. 1,2Dioleolyl-sn-Glycero-3-Phospho-L-Serine (PS) and 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (PC) were purchased from Avanti Polar Lipids, Inc (Alabaster, AL) and phospholipid vesicles (PC:PS) composed of 75 PC and 25 PS were prepared as described [60,84]. Spectrozyme TH and recombinant hirudin were purchased from American Diagnostica, Inc (Greenwich, CT) and EDTA was purchased from Sigma (St Louis, MO). D-Phe-Pro-ArgCH2Cl (FPRck) was prepared in house. Monoclonal 2-Bromo-4-fluoro-5-methylbenzoic acid anti-fV (HFV#17) was obtained from the Biochemistry Antibody Core Laboratory (University of Vermont) and a goat anti-mouse IgG conjugated to HRP was purchased from Southern Biotech (Birmingham, AL). Active site blocked FXa (FXa*) was produced according to the previously published method [85]. Multiple preparations of FV/FVa and different lots of APC were utilized for these studies. Preparations of Factor Va were made fresh prior to all experiments. Factor V (1.0 M) in 0.2 M HEPES, 0.15 M NaCl, 0.1 PEG-8000, 2.0 mM CaCl2, pH 7.4 (HBS-PEG-Ca) was activated with 10 nM human thrombin for 10 min at 37 . Thrombin activation was stopped by addition of 12 nM recombinant hirudin and placing the sample on ice. Factor Va preparations were used within 4 hours. The partially proteolyzed species of factor Va cleaved only at Arg506 was generated by incubating FVa (750 nM) in HBS-PEG-Ca with 5.0 nMBravo et al. BMC Systems Biology 2012, 6:45 http://www.biomedcentral.com/1752-0509/6/Page 17 ofFigure 10 Reaction Scheme of APC Inactivation of FVa in the Presence of Either FXa PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/9638576 or Prothrombin. Schematic of reactions and interactions involved in the APC inactivation of FVa when in the presence of either FXa (highlighted in blue and red) or prothrombin (highlighted in green). The interaction highlighted in red was identified in this study through the validation of the mathematical model describing the APC inactivation PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17591728 of FVa. Formation of the prothrombinase plus prothrombin species (Table 3, Eqns 19?2) tert-Butyl 2,2-difluoro-3-(methacryloyloxy)pentanoate and their dissociation (Table 3, Eqns 23?4) are not shown in this figure for clarity.APC for 20 minutes at 37 , after 20 minutes an additional 5.0 nM APC was added. Following the inactivation reaction, the FVa506 containing reaction was placed on ice and i.