ENHANCEMENT OF HUMAN PROTEIN-C FUNCTION BY SITE-DIRECTED MUTAGENESIS OF THE GAMMA-CARBOXYGLUTAMIC ACID DOMAIN

This study reports properties of site-directed mutants of human protei n C that display enhanced calcium and/or membrane binding properties. Mutants containing the S11G modification all showed increased affinity for membranes at saturating calcium concentration. Ser-ll is unique t o human protein C, whereas all other vitamin R-dependent proteins cont ain glycine, This site is located in a compact region of the protein, close to a suggested membrane contact site. Additional changes of H10Q or S12N resulted in proteins with lower calcium requirement for membr ane contact but without further increase in membrane affinity at satur ating calcium. Mutations Q32E and N33D did not, by themselves, alter m embrane affinity to a significant degree. These mutations were include d in other mutant proteins and may contribute somewhat to higher funct ion in these mutants. This family of mutants helped discriminate event s that are necessary for protein-membrane binding. These include calci um binding to the free protein and subsequent protein-membrane contact . Depending on conditions of the assay used, the mutants displayed inc reased activity of the corresponding activated protein C (APC) derivat ives, The degree of enhanced activity (up to 10-fold) was dependent on the concentration of phospholipid and quality of phospholipid (+/- ph osphatidylethanolamine) used in the assay. This was expected, because APC is active in its membrane-associated form, which can be regulated by changes in either the protein or phospholipid. As expected, the lar gest impact of the mutants occurred at low phospholipid concentration and in the absence of phosphatidylethanolamine. The anticoagulant acti vity of all proteins was stimulated by protein S, with the greatest im pact on the enhanced mutants. Whereas plasma containing Factor V:R506Q was partially resistant to all forms of APC, the enhanced variants we re more active than normal APC, Protein C variants with enhanced funct ion present new reagents for study of coagulation and may offer improv ed materials for biomedical applications.