Action of fibrin-stabilizing factor on cold-insoluble globulin and alpha2-macroglobulin in clotting plasma

Experiments were performed to investigate whether proteins other than fibrin are substrates for activated fibrin-stabilizing factor (FSF, blood coagulation Factor XIII, plasma transglutaminase) in clotting whole plasma. Three fluorescently labeled polypeptides were identified in serum prepared by clotting normal, but not FSF-deficient, plasma in the presence of the fluorescent amine, N-(5-aminopentyl)-5-dimethyl-aminonaphthalene-1-sulfonamide (dansylcadaverine). The major labeled polypeptide had a Mr (estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate) of 1.6 times 10(5) and was found in the protein fraction precipitated by 33 to 50% saturated ammonium sulfate. The second had a Mr of 2.0 times 10(5), was found in the protein fraction insoluble in 33% saturated ammonium sulfate, and was precipitated by gamma-globulin directed against cold-insoluble globulin. The third had a Mr of 1.1 times 10(5) and was precipitated by 33 to 50% saturated ammonium sulfate. All three polypeptides were found in the first protein peak when labeled serum was chromatographed on Sephadex G-200. The immunoprecipitin arc containing alpha2-macroglobulin was fluorescent when labeled serum was analyzed by immunoelectrophoresis. These results indicate that alpha2-macroglubulin, cold-insoluble globulin, and an unidentified third protein with a subunit of Mr = 1.1 times 10(5) are transamidated by FSF in clotting plasma. The concentration of cold-insoluble globulin was decreased in serum formed at 37 degrees from normal, but not from FSF-deficient, plasma. The depletion of cold-insoluble globulin in normal serum was partially blocked by clotting in the presence of dansylcadaverine and completely blocked by clotting in the absence of calcium ions. Sera formed at 2 degrees from both normal and FSF-deficient plasma contained less cold-insoluble globulin than plasma. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of clots formed at 2 degrees demonstrated cross-linking of cold-insoluble globulin to fibrin in the normal, but not the FSF-deficient, sample. The serum concentration of alpha2-macroglobulin was the same as the plasma concentration irrespective of the conditions of clotting. Thus, the experiments suggest that FSF catalyzes the cross-linking of cold-insoluble globulin (but not alpha2-macroglobulin) to fibrin in clotting plasma.