MAPPING OF FUNCTIONAL DOMAINS OF HUMAN HIGH MOLECULAR WEIGHT (HMW) AND LOW MOLECULAR WEIGHT (LMW) KININOGENS BY USING MURINE MONOCLONAL ANTIBODIES (MAbs)

<jats:p>It has been widely known that HMW and LMW kininogens are the large potential sources of kinin in human blood, and that HMW kininogen also functions as a cofactor in the contact activation of blood coagulation. Recently, it has been demonstrated that the heavy chains of kininogens strongly inhibited a number of cysteine proteinases such as calpains, cathepsins, papain and ficin. We made an attempt at mapping of functional domains on the molecules of both kininogens by using MAbs.</jats:p><jats:p>Thirty four MAbs raised against human HMW and LMW kininogens were screened by ELISA. By using HMW kininogen, kinin-free HMW kininogen, kinin and fragment 1.2 (fr 1.2)-free HMW kininogen, LMW kininogen, kinin-free LMW kininogen, heavy chain of LMW kininogen and light chains of both kininogens, the MAbs were characterized and.classified into four groups; [1] 20 MAbs reacted with heavy chain, a common region of HMW and LMW kininogens. These MAbs possessed the specificity for domain 1 (2 MAbs), domain 2 (2 MAbs), domain 3 (7 MAbs), and both domains 2 and 3 (7 MAbs) of the heavy chain; [2] 7 MAbs recognized the fr 1.2, a unique histidine-rich region; [3] 5 MAbs reacted with the light chain of HMW kininogen; [4] 2 MAbs recognized the light chain of LMW kininogen.</jats:p><jats:p>Two MAbs, designated HKG H7 and H12, effectively inhibited the cysteine proteinase inhibitor activity of HMW and LMW kininogens and the others did not affect it. Further, the MAbs, which recognized the fr 1.2 or light chain of HMW kininogen, suppressed the clotting activity. Especially, 2 MAbs, named HKG L2 and L5, effectively suppressed the clotting activity of HMW kininogen. The former, which neutralized about 70% of the clotting activity, reacted specifically with fr 1.2 region of HMW kininogen, and the latter, which neutralized more than 90% of it, recognized the light chain of HMW kininogen. In the results of competition ELISA, fr 1.2 specific MAbs could be classified into 5 kinds of MAbs for recognition sites, and the light chain (HMW kininogen)-specific MAbs also could be classified into 3 kinds of MAbs. Further, 2 light chain (LMW kininogen)-specific MAbs were thought to recognize an identical antigenic site.</jats:p>