Monoclonal antibody light chain with prothrombinase activity

Prothrombin is the precursor of thrombin, a central enzyme in coagulation. Autoantibodies to prothrombin are associated with thromboembolism, but the mechanisms by which the antibodies modulate the coagulation processes are n ot understood. We screened a panel of 34 monoclonal antibody light chains i solated from patients with multiple myeloma for prothrombinase activity by an electrophoresis method. Two light chains with the activity were identifi ed, and one of the light chains was characterized further. The prothrombina se activity eluted from a gel-filtration column run in denaturing solvent ( 6 M guanidine hydrochloride) at the characteristic positions of the light c hain dimer and monomer. A constant level of catalytic activity was observed across the width of the light chain monomer peak, assessed as the cleavage of IEGR-methylcoumarinamide, a peptide substrate corresponding to residues 268-271 of prothrombin. Hydrolysis of this peptide by the light chain was saturable and consistent with Michaelis-Menten-Henri kinetics (K-m 103 mu M ; k(cat) of 2.62 x 10(-2)/min). Four cleavage sites in prothrombin were ide ntified by N-terminal sequencing of the fragments: Arg(155)-Ser(156), Arg(2 71)-Thr(272), Arg(284)-Thr(285), and Arg(393)-Ser(394). The light chain did not cleave radiolabeled albumin, thyroglobulin, and annexin V under condit ions that readily permitted detectable prothrombin cleavage. Two prothrombi n fragments (M-r 55 000 and 38 000), were isolated by anion-exchange chroma tography and were observed to cleave a thrombin substrate, tosyl-GPR-nitroa nilide. Conversion of fibrinogen to fibrin was accelerated by the prothromb in fragments generated by the light chain. These finding suggest a novel me chanism whereby antibodies can induce a procoagulant state, i.e., prothromb in activation via cleavage of the molecule.