MOLECULAR MECHANISM FOR FAMILIAL PROTEIN-C DEFICIENCY AND THROMBOSIS IN PROTEIN C-VERMONT (GLU(20)-]ALA AND VAL(34)-]MET)

The role of two protein C gamma-carboxyglutamic acid domain mutations in familial thrombosis, protein C-Vermont (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 mutati ons (Glu(20) --> Ala and Val(34) --> Met) and the naturally occurring double mutation mere created by site directed mutagenesis and were exp ressed in human kidney 293 cells. Purified recombinant protein C with the mutation glutamate to alanine at position 20 is defective in the a ssays 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 Glu(20) mutants by thrombin-thr ombomodulin was not enhanced by phospholipid vesicles and showed a dif ferent calcium dependence compared with the wild type, suggesting that Gla(20) is important in the interaction of the protein C Gla domain w ith a phospholipid-mediated site on the thrombomodulin molecule. Glu(2 0)-substituted protein C is not inhibited by calcium ion in its intera ction with the calcium-dependent monoclonal antibody H-11, suggesting that this mutation has lost the calcium-induced, lipid-independent con formational transition of the protein C Gla domain. These data indicat e that the loss of Gla(20) causes the major familial dysfunction of pr otein C to associate with phospholipid as well as to undergo Ca2+-depe ndent, lipid-independent conformational changes and are consistent wit h the importance of Gla(20) in both external and internal Ca2+ binding based upon the x-ray-derived structure of the homologous Gla domain i n bovine prothrombin.