Detection of in vitro and in vivo cleavage of high molecular weight kininogen in human plasma by immunoblotting with monoclonal antibodies

twobands showed coagulant activity. Six murine monoclonal antibodies (Mabs) against HMWK were produced and purified. In immunoblotting studies. three Mabs bound to the isolated alkylated heavy chain and one to the alkylated light chain of HMWK. whereas the remainingtwo bound only to the single-chain or unreduced two-chain molecule. None of the Mabs inhibited the clotting activity of HMWK or its binding to kaolin. Two of the Mabs, one directed against the light chain and one against the heavy chain. were used as specific probes A POTENT vasoactive peptide, bnadykinin, is liberated in plasma from two precursor proteins, high- and low-mob wt kininogens (HMWK and LMWK), by the action of specific enzymes termed � Human HMWK and LMWK are single-chain glycoproteins, with mol wts of � I 1 0,000 to I 20,000 and 50,000 to 70,000, respectively. Each protein consists of three domains: an amino terminal heavy chain, a bradykinin moiety, and a carboxyb terminal bight chain.3’4 Based on cloning and sequence analysis of cDNAs for human high- and bow-mol-wt prekininogens, a complete identity in the amino acid sequence has been determined for a barge portion of the two molecules, including the heavy chain, the bradykinin sequence, and the first part of the carboxyl terminal region; the two proteins are transcribed from the same gene.5 The structural homology accounts for the recently discovered thiob protease inhibitory activity common to both proteins.� The unique structure of the light chain of HMWK, however, provides this protein with the function of accelerating the reactions of the contact phase of blood coagulation, whereby HMWK acts as a nonenzymatic cofacton by facibitating the proteobytic activations of factor XII, prekallikrein, and factor XI on a negatively charged surface.9”#{176} One of the activation products, kalliknein, potently cleaves HMWK at two specific sites to liberate bradykinin and to form a kinin-free protein.”’3 The cleavage occurs within a critical disulfide bridge so that single-chain HMWK is converted to a two-chain molecule, in which a heavy chain of -66,000 mob wt is disubfide-linked to a light chain of -55,000 mob wt. The light chain is further cleaved by kallikrein to a smaller chain of 45,000 mob wt.’3 The pathophysiologic role of plasma HMWK still remains largely unclear. The hereditary deficiency of this protein is not associated with a bleeding disorder.’4 Because bradykinin possesses a number of actions potentially involved in inflammation reactions, including vasodilation, increase in vascular permeability, and pain generation, a robe for HMWK and the other proteins of the contact activation system in the to study HMWK in plasma samples using an immunoblotting technique. The anti-light chain Mab identified two distinct bands (-120.000 and -.105.000 mol wt) in normal human plasma. but not in plasma from patients with hereditary HMWK deficiency. The anti-heavy chain Mab detected two additional bands (-60.000 and -54.000 mol wt) corresponding to low-mol-wt kininogen (LMWK) in normal plasma. A sensitive and specific quantitative immunoblotting assay of HMWK antigen in plasma was developed. Moreover, the immunoblotting technique with the anti-light chain Mab was used to detect the cleavage of HMWK in plasma samples after in vitro or in vivo activation of the contact system. The anti-light chain Mab demonstrated in vivo activation and cleavage of HMWK during an angioedema attack in a patient with hereditary angioedema and Cl -inhibitor deficiency. S 1986 by Grune & Stratton, Inc. pathogenesis of several pathological conditions has been suggested.’4 Evidence of HMWK cleavage in vivo, however, has been indirect only and has involved functional and/on immunological measurements of plasma levels of the protein or its released product, bnadykinin. We have prepared a series of monoclonab antibodies (Mabs) against HMWK and describe here their use as specific qualitative and quantitative probes for HMWK in plasma samples subjected to immunobbotting.