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Inactivation of factor VIII by activated protein C and protein S
Insights
Activated protein C inactivates Factor VIII by cleaving its heavy chains, a process accelerated by protein S. Factor IX inhibits this inactivation, indicating Factor VIII and Factor Va compete for activated protein C.
Area of Science:
- Biochemistry
- Hematology
- Molecular Biology
Background:
- Factor VIII is a crucial blood clotting protein.
- Activated protein C (APC) is a key regulator of coagulation.
- Understanding APC's regulation of Factor VIII is vital for hemostasis research.
Purpose of the Study:
- To elucidate the mechanism of Factor VIII inactivation by APC.
- To investigate the role of cofactors like protein S and factor IX in this process.
- To determine the substrate specificity of APC.
Main Methods:
- Analysis of Factor VIII cleavage products using SDS-PAGE.
- Investigating the effect of protein S and factor IX on Factor VIII inactivation kinetics.
- Assessing the competition between Factor VIII and Factor Va for APC.
Main Results:
- APC inactivates Factor VIII through multiple cleavages of its heavy chains, producing specific peptides.
- Protein S significantly accelerates APC-mediated Factor VIII cleavage.
- Factor IX dose-dependently inhibits Factor VIII inactivation by competing for APC, similar to Factor Va.
Conclusions:
- APC inactivates Factor VIII via sequential cleavages primarily in the heavy chain.
- Protein S enhances APC's activity on Factor VIII.
- Factor VIII and Factor Va are competitive substrates for APC, highlighting complex regulatory interactions in coagulation.
Abstract:
Factor VIII was inactivated by activated protein C in the presence of calcium and phospholipids. Analysis of the activated protein C-catalyzed cleavage products of factor VIII indicated that inactivation resulted from the cleavage of the heavy chains. The heavy chains appeared to be converted into 93- and 53-kDa peptides. Inactivation of factor VIII that was only composed of the 93-kDa heavy chain and 83-kDa light chain indicated that the 93-kDa polypeptide could be degraded into a 68-kDa peptide that could be subsequently cleaved into 48- and 23-kDa polypeptides. Thus, activated protein C catalyzed a minimum of four cleavages in the heavy chain. Activated protein C did not appear to alter the factor VIII light chain. The addition of protein S accelerated the rate of inactivation and the rate of all of the cleavages. The effect of protein S could be observed on the cleavage of the heavy chains and on secondary cleavages of the smaller products, including the 93-, 68-, and 53-kDa polypeptides. The addition of factor IX to the factor VIII-activated protein C reaction mixture resulted in the inhibition of factor VIII inactivation. The effect of factor IX was dose dependent. Factor VIII was observed to compete with factor Va for activated protein C. The concentration dependence of factor VIII inhibition of factor Va inactivation suggested that factor VIII and factor Va were equivalent substrates for activated protein C.