Monoclonal antibody light chain with prothrombinase activity

Perumal Thiagarajan, Robert Dannenbring, Kinji Matsuura, Alfonso Tramontano, Gennady Gololobov, Sudhir Paul

Research output: Contribution to journalArticle

92 Scopus citations

Abstract

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 not understood. We screened a panel of 34 monoclonal antibody light chains isolated from patients with multiple myeloma for prothrombinase activity by an electrophoresis method. Two light chains with the activity were identified, and one of the light chains was characterized further. The prothrombinase activity eluted from a gel- filtration column run in denaturing solvent (6 M guanidine hydrochloride) at the characteristic positions of the light chain 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 μM; k(cat) of 2.62 x 10-2/min). Four cleavage sites in prothrombin were identified by N-terminal sequencing of the fragments: Arg155-Ser156, Arg271-Thr272, Arg284-Thr285, and Arg393-Ser394. The light chain did not cleave radiolabeled albumin, thyroglobulin, and annexin V under conditions that readily permitted detectable prothrombin cleavage. Two prothrombin fragments (M(r) 55 000 and 38 000), were isolated by anion-exchange chromatography and were observed to cleave a thrombin substrate, tosyl-GPR-nitroanilide. Conversion of fibrinogen to fibrin was accelerated by the prothrombin fragments generated by the light chain. These finding suggest a novel mechanism whereby antibodies can induce a procoagulant state, i.e., prothrombin activation via cleavage of the molecule.

Original languageEnglish (US)
Pages (from-to)6459-6465
Number of pages7
JournalBiochemistry
Volume39
Issue number21
DOIs
StatePublished - May 30 2000
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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    Thiagarajan, P., Dannenbring, R., Matsuura, K., Tramontano, A., Gololobov, G., & Paul, S. (2000). Monoclonal antibody light chain with prothrombinase activity. Biochemistry, 39(21), 6459-6465. https://doi.org/10.1021/bi992588w