Date of Award

Spring 1991

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Thomas M Laue

Abstract

The effect of Ca$\sp{2+}$ on the activation of protein C by thrombin bound to the elastase fragment of thrombomodulin (e1TM) is complex. The rate of activation increases from 0 at 0 mM Ca$\sp{2+}$ to a maximum at 0.3 mM Ca$\sp{2+}$ and decreases 25-fold upon further addition of Ca$\sp{2+}$ (Kurosawa et al., 1987). The results reported here demonstrate that thrombin, protein C and e1TM form a complex in the absence of Ca$\sp{2+}$ that falls apart with the addition of Ca$\sp{2+}$. This observation, in conjunction with the above mentioned kinetic studies, suggests that recognition of protein C by thrombin does not require Ca$\sp{2+}$ but that protein C activation requires a Ca$\sp{2+}$-induced conformational change; probably in protein C. The technique of sedimentation equilibrium is used to examine the effect of Ca$\sp{2+}$ on the interaction of this enzyme complex made up of three proteins. These proteins studied individually do not significantly self-associate. In all experiments, thrombin was either inhibited with a tripeptide chloromethyl ketone inhibitor (CK), or with diisopropylflourophosphate (DFP). Thrombin and e1TM form a strong 1:1 complex that is relatively unperturbed by the addition of Ca$\sp{2+}$. Thrombin and protein C form two complexes of 1:1 and 1:2 thrombin:protein C stoichiometry as suggested by the association model that fits the data best. The interactions between thrombin and protein C are strongest in 0 mM Ca$\sp{2+}$, but become much weaker with the addition of Ca$\sp{2+}$. Thrombin, protein C and e1TM form a single 1:1:1 complex that behaves similarly to the thrombin/protein C mixture upon the addition of Ca$\sp{2+}$. Studies with the 42 amino acid Gla ($\gamma$-carboxyglutamic acid) domain peptide and Gla-domainless protein C indicate that, at low Ca$\sp{2+}$ concentrations, the interaction of protein C with thrombin and e1TM may be mediated primarily through this Gla domain. The interaction of activated protein C with thrombin alone, and thrombin with e1TM is also examined. Surprisingly, there is little difference in the interaction of activated protein C as compared to protein C. This is true regardless of whether the thrombin inhibitor is CK which may block some of the substrate recognition site, or DFP, which blocks only the catalytic site. As suggested by these results, activated protein C is a good inhibitor of protein C activation.

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