Protamine Inhibits Plasma Carboxypeptidase N, the Inactivator of Anaphylatoxins and Kinins

Abstract

Protamine given to neutralize heparin after extracorporeal circulation can trigger a catastrophic reaction in some patients. While searching for a biochemical basis for this reaction, protamine was tested as an inhibitor of human plasma carboxypeptidase N (CPN) or kininase I, the inactivator of anaphylatoxins and kinins. Human plasma and CPN purified from human plasma, (Mr = 280 K) or its isolated active subunit (Mr = 48 K) were the sources of enzyme. The hydrolysis of furylacryloyl (FA)-Ala-Lys was measured in a UV spectrophotometer and that of bradykinin and the synthetic C-terminal octapeptide of anaphylatoxin C3a (C3a8) by high performance liquid chromatography. Protamine inhibited the hydrolysis of FA-Ala-Lys by CPN, (IC50 = 3.2 .times. 10-7 M); added human serum albumin (30 mg/ml) increased the IC50 to 7 .times. 10-6 M. When plasma was the source of CPN, the IC50 was 2 .times. 10-6 M. Protamine more effectively inhibited the hydrolysis of bradykinin and C3a8. The IC50 for protamine was 5 .times. 10-8 M with CPN and bradykinin, 7 .times. 10-8 M with CPN and C3a8 and with the 48 K subunit and bradykinin it was 7 .times. 10-8 M of protamine. Heparin competes with CPN for protamine, because in high concentration (18 U/ml) it reverses the inhibition by protamine. Protamine did not inhibit angiotensin I converting enzyme (kininase II) or the endopeptidase 24.11 (enkephalinase). Kinetic studies showed the mechanism of protamine inhibition to be partially competitive; about 10-20% of the hydrolysis of bradykinin by CPN was not inhibited by protamine. Thus, by blocking the inactivation of mediators released in shock, protamine inhibition of CPN may be partially responsible for the catastrophic reaction observed to occur in some patients.