Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C.

Abstract

The isolation from rabbit lung of a cofactor for thrombin-catalyzed Protein C activation is described. The lung is perfused to remove blood, homogenized, and extracted with Triton X-100. The cofactor is purified from the extract with two chromatographic columns of diisopropylphosphorothrombin-agarose. The purified cofactor is a protein with an apparent molecular weight on sodium dodecyl sulfate (SDS)-acrylamide gels of 68,000 k 5,000 before disulfide bond reduction and 74,000 k 6,000 after disulfide bond reduction. The cofactor activity is not inactivated permanently by SDS (l%), 8 M urea, or 6 M guanidinium C1, is stable to boiling for greater than 15 min, and is stable from pH 2.0-10. Stability is determined by assay immediately after dilutions of the cofactor from the perturbant. The cofactor activity is destroyed by pepsin or P-mercaptoethanol. The stability of the cofactor in SDS and SDS and 8 M urea solutions allows correlation of the protein species visualized by protein stain with the cofactor activity determined by assay of the extracted gel slices after electrophoresis with these denaturants. The cofactor activity also co-migrates with the major protein species on alkaline disc gel electrophoresis in the presence of Lubrol PX. Neither the cofactor activity nor the protein enters the gel in the absence of Lubrol. From 10 perfused rabbit lungs, 0.7-1.5 mg of cofactor is isolated with an overall yield of 10-20%. The purified cofactor requires thrombin to activate Protein C. The rate of activation of Protein C at constant thrombin is saturable with respect to cofactor. The isolated cofactor increases the rate of Protein C activation 1000-fold. Expression of cofactor activity requires Caz+ or other specific multivalent ions. The relationship between Ca2+ concentration and the initial rate of Protein C activation is a simple hyperbola with half-maximal stimulation at 0.2 k 0.1 m~ Ca2+. Other functional ions in order of effectiveness (in parentheses) are T b 3 + (71%), Mnz+ (52%), Sr2+ (36%), Cr2+ (23%), Co2+ (23%), Cd2+ (16%), and Gd3+ (12%). M 6 + , Ni”, Zn2+, Cu2+, and Ba2+ were less than 5% as active as Ca”.