Abstract
In normal human plasma two forms of kininogen exist, low molecular weight kininogen (LMWK) and high molecular weight kininogen (HMWK). When these proteins are cleaved they are found to have a common heavy chain and bradykinin, but each has a unique light chain. Monoclonal antibodies to the heavy and light chains of HMWK have been developed, and the effects of each on the function of this protein are defined. Initial studies showed that an antibody, C11C1, completely neutralized the coagulant activity of plasma HMWK whereas another antibody, 2B5, did not. On a competitive enzyme-linked immunosorbent assay (CELISA) the C11C1 antibody was consumed by kininogen antigen in normal plasma but not by kininogen antigen in HMWK-deficient plasma. On immunoblot, the C11C1 antibody recognized one kininogen protein in normal plasma and did not recognize any kininogen antigen in HMWK-deficient plasma. These combined studies indicated that the C11C1 antibody was directed to an epitope on the unique 46-kDa light chain of HMWK. In contrast, the 2B5 antibody on a CELISA was consumed by kininogen antigen in both normal plasma and HMWK-deficient plasma but not by total kininogen-deficient plasma. On immunoblot, the 2B5 antibody recognized both kininogens in normal plasma but only LMWK in HMWK-deficient plasma. These combined studies indicated that the 2B5 antibody was directed to the common 64-kDa heavy chain of the plasma kininogens. Utilizing direct binding studies or competition kinetic experiments, the 2B5 and C11C1 antibodies bound with high affinity (1.71 and 0.77 nM, respectively) to their antigenic determinants on the HMWK molecule. The 2B5 antibody did neutralize the ability of HMWK to inhibit platelet calpain. These studies with monoclonal antibodies directed to each of the HMWK chains indicate that HMWK is a bifunctional molecule that can serve as a cofactor for serine zymogen activation and an inhibitor of cysteine proteases.
Highlights
In normal human plasma two forms of kininogen exist, low molecular weight kininogen (LMWK) and high molecular weight kininogen(HMWK)
On a competitive enzyme-linked immunosorbent assay (CELISA)the C11C1antibody wasconsumed by kininogen antigen in normal plasma but not by kininogen antigen in HMWK-deficient plasma
The 2B5 antibody did neutralitzhee ability of HMWK toinhibitplatelet calpain. These studies withmonoclonal antibodies directed toeach of the HMWK chains indicate that HMWK is a bifunctional molecule that can serveas a cofactor for serine zymogen activation and an inhibitor of cysteine proteases
Summary
The 2B5 and CllCl antibodies were each found to be IgGl subtype, K light chain. Purified CllCl antibody neutralized the coagulant activity of plasma HMWKasa function of concentration, and at high concentration the inhibition was complete (datanot shown). Since Fitzgerald plasma does not contain HMWK but does contain 0.98 PM LMWK, the 2B5 antibody appeared to be directed to the heavy chain of the plasma kininogens This assessment was further evaluated by immunoblot studies on these proteins in plasma. This band represents LMWK since this protein is the only form of kininogen in Fitzgerald plasma These experiments indicated that the 2B5 antibody was directed toward LMWK and theheavy chain of HMWK, the kininogen antigen common in bothnormal and Fitzgerald plasmas. When applied to the affinity column, the unbound protein, which contained all the coagulant activity, consisted of two bands at 56 and 46 kDa (Fig.2), representing the light chain of HMWK and itsproteolytic derivative. Since Fitzgerald plasma does not contain HMWK and the CllCl antibody neutralized the coagulant activity of normal plasma, this antibody appeared to be directed to the light
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