PubMed Journals: Biochemistry
Source: PMID: 10828960
⇦ ⇨ Biochemistry. 2000 May 30;39(21):6459-65.
Monoclonal antibody light chain with prothrombinase
Thiagarajan P(1), Dannenbring R, Matsuura
K, Tramontano A, Gololobov G, Paul S.
(1) Departments of Internal Medicine and
Pathology and Laboratory Medicine, Center
for Chemical Immunology, University of Texas-Houston
Medical School, 77030, USA.
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
microM; k(cat) of 2.62 x 10(-)(2)/min).
Four cleavage sites in prothrombin were
identified by N-terminal sequencing of the fragments:
Arg(284)-Thr(285), and Arg(393)-Ser(394).
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.
PMID: 10828960 [Indexed for MEDLINE]