Generation of anaphylatoxin activity related to C3a, by treatment of human serum with the nitrogen nucleophile N2H4 or the chaotrope KSCN.

Abstract

The present study is concerned with the proteolytic processing of complement component C3 in normal human serum treated with N2H4 or KSCN in the presence of EDTA. Upon incubation with these agents, C3 is first converted to the thiolester-cleaved form (C3i) and thereafter fragmented by factor I. In consecutive, relatively slow steps, spasmogenic and platelet-aggregating activity is released. The active principle shows characteristics of C3a. First, the pretreated sera deactivate guinea pig ileum and platelets towards the action of C3ahog, but not C5a-desArghog. Second, the activity is only stable under conditions causing inhibition of serum carboxypeptidase N, such as in the presence of EDTA or of MERGETPA (DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid). Third, the molecular weight determined by gel filtration is in agreement with that of C3a. The release of C3a activity requires conversion of C3 to C3i, as well as the complement-independent generation of proteolytic activity in the pretreated sera. The enzyme releasing C3a activity is a serine esterase probably identical with Hageman factor, kallikrein, or another protease related to the contact system.