SURFACE-INDUCED ALTERATIONS IN THE KINETIC PATHWAY FOR CLEAVAGE OF HUMAN HIGH-MOLECULAR-WEIGHT KININOGEN BY PLASMA KALLIKREIN

We have studied the cleavage of human high molecular weight kininogen (HK) by plasma kallikrein in the absence and presence of the surfaces, dextran sulfate (DxSO(4)) and sulfatides. Using a combination of SDS- polyacrylamide gel electrophoresis, Western blotting with polyclonal a ntibodies that specifically recognize the COOH terminus of the bradyki nin moiety, and high pressure liquid chromatography analyses of the cl eavage reaction, we have identified two intermediates in the formation of bradykinin from intact kininogen and demonstrated that alternative cleavage pathways are followed in the absence and presence of surface s. The COOH-terminal bradykinin cleavage occurred first both in the ab sence and presence of DxSO(4), producing a 103-kDa HK intermediate con sisting of disulfide-linked heavy and light chains that retained the k inin moiety. In the presence of DxSO(4), this was followed exclusively by the NH2-terminal bradykinin cleavage and release of kinin with no apparent change in molecular mass. Subsequently, a slower cleavage of an 8-kDa peptide from the amino terminus of the HK light chain occurre d to form a 95-kDa end product. In contrast to this sequential cleavag e pattern, NH2-terminal bradykinin and light chain cleavages occurred randomly in the absence of DxSO(4), resulting in the production of an additional 95-kDa intermediate that retained bradykinin but had lost t he 8-kDa peptide from the HK light chain. Comparison of the relative r ates of the three kallikrein cleavages in the absence and presence of DxSO(4) indicated that the surface enhanced the rates of both bradykin in cleavages 2-4-fold, but inhibited the light chain cleavage rate sim ilar to 10-fold, thereby accounting for the change from a partially ra ndom to a sequential cleavage pattern in the presence of the surface. Steady-state kinetic analysis revealed that DxSO(4) enhanced the K-cat /K-M for bradykinin release by the rate-limiting NH2-terminal bradykin in cleavage by similar to 2-fold due exclusively to an increase in K-c at. Sulfatides appeared to produce the same effects on the pattern of HK cleavages as DxSO(4). Blocking of the nonactive site, i.e. exosite, interaction between kallikrein and HK with excess prekallikrein or a synthetic peptide containing the region of HK that interacts with the kallikrein exosite significantly reduced the rate of bradykinin releas e as well as HK cleavages detected by SDS-polyacrylamide gel electroph oresis either in the absence or presence of DxSO(4), indicating that t he exosite interaction facilitates bradykinin cleavage.