Cleavage of human high-molecular weight kininogen by purified kallikreins and upon contact activation of plasma.
To study the digestion pattern of human high-molecular weight (mol wt) kininogen (HMWK) in plasma during contact activation we have prepared monoclonal antibodies (MoAbs) to the light-chain (LC) and the heavy-chain moiety of HMWK. One MoAb from each set was purified, and neither MoAb inhibited the clotting activity of HMWK. In enzyme-linked immunosorbent assay and immunoblotting experiments neither antibody bound to kininogen-deficient plasma. Digestion of purified HMWK with plasma kallikrein yielded, on reduced sodium dodecyl sulfate gels, two LC forms, at 62 and 49 kd, respectively. Digestion of HMWK with tissue kallikrein (TK) yielded mainly the 62-kd form. In immunoblot analyses of these digests, the anti-LC MoAb detected products at 62 and 49 kd respectively. With plasma kallikrein, the 62-kd species slowly shifted to 49 kd, and with TK, the 62-kd species accumulated with time. Anti-LC MoAb was also used as a probe in immunoblotting experiments to study the digestion pattern of HMWK in whole plasma activated with kaolin or dextran sulfate. In activated normal pooled plasma (NHP) and factor XI-deficient plasma, native HMWK (mol wt, 115 kd) was cleaved within five to ten minutes, and two LC forms at 62 and 49 kd were detected. In kaolin-activated prekallikrein (PK)-deficient plasma, the disappearance of the 115-kd form was relatively slow, and only the 62-kd form of LC was seen. HMWK was not cleaved when factor XII-deficient plasma was incubated with kaolin. LC-dependent coagulant activity paralleled the presence of LC bands seen in the immunoblots, and lower-mol wt fragments of LC were not identified. These data indicate that in activated NHP two forms of LC of HMWK (62 and 49 kd) are formed sequentially. Further, the LC-dependent coagulant activity remains detectable long enough to suggest that proteolytic inactivation of LC is too slow to be an important control mechanism.
D000911:Antibodies, Monoclonal / Q000276:immunology; D001920:Bradykinin / Q000096:biosynthesis; D016264:Dextran Sulfate; D003911:Dextrans; D006801:Humans; D007610:Kallikreins / Q000145:classification / Q000378:metabolism*; D007616:Kaolin; D007704:Kininogens / Q000276:immunology / Q000378:metabolism*; D008970:Molecular Weight
D000911:Antibodies, Monoclonal; D003911:Dextrans; D007704:Kininogens; D007616:Kaolin; D016264:Dextran Sulfate; D007610:Kallikreins; D001920:Bradykinin