Molecular mechanism for familial protein C deficiency and thrombosis in protein CVermont (Glu20-->Ala and Val34-->Met).

The role of two protein C gamma-carboxyglutamic acid domain mutations in familial thrombosis, protein CVermont (Bovill, E. G., Tomczak, J. A., Grant, B., Bhushan, F., Pillemer, E., Rainville, I.R., and Long, G. L. (1992) Blood 79, 1456-1465), was investigated. Two single mutations (Glu20-->Ala and Val34-->Met) and the naturally occurring double mutation were created by site-directed mutagenesis and were expressed in human kidney 293 cells. Purified recombinant protein C with the mutation glutamate to alanine at position 20 is defective in the assays of activated partial thromboplastin time, factor Va inactivation, and fibrinolysis. Mutation from valine to methionine at position 34 has only a minor effect. Activation of Glu20 mutants by thrombin-thrombomodulin was not enhanced by phospholipid vesicles and showed a different calcium dependence compared with the wild type, suggesting that Gla20 is important in the interaction of the protein C Gla domain with a phospholipid-mediated site on the thrombomodulin molecule. Glu20-substituted protein C is not inhibited by calcium ion in its interaction with the calcium-dependent monoclonal antibody H-11, suggesting that this mutation has lost the calcium-induced, lipid-independent conformational transition of the protein C Gla domain. These data indicate that the loss of Gla20 causes the major familial dysfunction of protein C to associate with phospholipid as well as to undergo Ca(2+)-dependent, lipid-independent conformational changes and are consistent with the importance of Gla20 in both external and internal Ca2+ binding based upon the x-ray-derived structure of the homologous Gla domain in bovine prothrombin.