Insights on monoclonal antibodies to kininogens' heavy chain which influence kininogens' binding to platelets.
Purified domains of low molecular weight kininogen (LK) can be used directly to determine the epitopes of monoclonal antibodies (mAbs) that have been shown to influence kininogen function. LK, purified from plasma by carboxymethyl-papain-Sepharose 4B affinity chromatography and kaolin adsorption, was digested by trypsin and chymotrypsin. The domains of LK were then separated by gel filtration followed by carboxymethyl-papain-Sepharose 4B affinity chromatography. Using the purified domains of LK's heavy chain, the regions on kininogens' heavy chain which various monoclonal antibodies are directed to were determined by enzyme-linked immunosorbent assay and immunoblotting. MAb 2B5 which neutralized kininogens' ability to inhibit calpain cross-reacted with domains 2 and 3. MAb HKH8 which reacted with kininogens' domain 1 and 2 was found to inhibit 125I-HK binding to platelets. At two-fold molar excess, mAb HKH8 was a better inhibitor of 125I-HK binding to platelets than higher concentrations, where the antibody was shown to cause increased binding to platelets. Alternatively, HKH8 F(ab')2 completely inhibited 125I-HK binding to platelets even at high concentrations of antibody. These studies indicate that purified domains of kininogens' heavy chain can be used to rapidly localize epitopes for antibodies. Further, mAb HKH8 should be a valuable probe to understand the mechanisms of kininogens' binding to platelets.
Jiang
Y
Y
Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA.
Nawarawong
W
W
Meloni
F J
FJ
Schmaier
A H
AH
D000327:Adsorption; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D007616:Kaolin; D007700:Kinetics; D007704:Kininogens / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D008970:Molecular Weight; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D007704:Kininogens; D007616:Kaolin