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Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1693-1700
From the Institutes of Thrombosis and Hemostasis, Neufeld Cardiac
Research Institute, and Clinical Epidemiology, Sackler School of
Medical, Sheba Medical Center, Tel-Hashomer, Israel; and the Whitaker
Cardiovascular Institute and Evans Department of Medicine, Boston
University School of Medicine, Boston, MA.
The recombinant fragment of von Willebrand factor (vWF) spanning
Ala444 to Asp730 and containing an Arg545Cys mutation (denoted AR545C)
has antithrombotic properties that are principally a consequence of its
ability to inhibit platelet adhesion to subendothelial matrix.
Endothelial-derived nitric oxide (NO) can also inhibit platelet
function, both as a consequence of inhibiting adhesion as well as
activation and aggregation. Nitric oxide can react with thiol
functional groups in the presence of oxygen to form S-nitrosothiols,
which are naturally occurring NO derivatives that prolong the
biological actions of NO. Because AR545C has a single free cysteine
(Cys545), we attempted to synthesize the S-nitroso-derivative of AR545C
and to characterize its antiplatelet effects. We successfully
synthesized S-nitroso-AR545C and found that it contained 0.96 mol S-NO
per mole peptide. S-nitroso-AR545C was approximately 5-fold more potent
at inhibiting platelet agglutination than was the unmodified peptide
(IC50 = 0.02 ± 0.006 µmol/L v 0.1 ± 0.03 µmol/L, P = .001). In addition and by contrast,
S-nitroso-AR545C was a powerful inhibitor of adenosine
diphosphate-induced platelet aggregation (IC50 = 0.018 ± 0.002 µmol/L), while AR545C had no effect on aggregation. These
effects were confirmed in studies of adhesion to and aggregation on
extracellular matrix under conditions of shear stress in a cone-plate
viscometer, where 1.5 µmol/L S-nitroso-AR545C inhibited platelet
adhesion by 83% and essentially completely inhibited aggregate
formation, while the same concentration of AR545C inhibited platelet
adhesion by 74% and had significantly lesser effect on aggregate
formation on matrix (P This article has been cited by other articles:
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| Copyright © 1999 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
.004 for each parameter by
ANOVA). In an ex vivo rabbit model, we also found that
S-nitroso-AR545C had a more marked and more durable inhibitory effect
on botrocetin-induced platelet aggregation than did AR545C, and these
differences were also reflected in the extent and duration of effect on
the prolongation of the bleeding time in these animals. These data show
that S-nitroso-AR545C has significant and unique antiplatelet effects,
inhibiting both adhesion and aggregation, by blocking platelet GPIb
receptor through the AR545C moiety and elevating platelet cyclic
3',5'-guanosine monophosphate through the -SNO
moiety. These observations suggest that this NO-modified fragment of
vWF may have potential therapeutic benefits as a unique antithrombotic agent.
