TY - JOUR
T1 - Monoclonal antibody light chain with prothrombinase activity
AU - Thiagarajan, Perumal
AU - Dannenbring, Robert
AU - Matsuura, Kinji
AU - Tramontano, Alfonso
AU - Gololobov, Gennady
AU - Paul, Sudhir
PY - 2000/5/30
Y1 - 2000/5/30
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0034732941&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0034732941&partnerID=8YFLogxK
U2 - 10.1021/bi992588w
DO - 10.1021/bi992588w
M3 - Article
C2 - 10828960
AN - SCOPUS:0034732941
VL - 39
SP - 6459
EP - 6465
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 21
ER -