Unique antiplatelet effects of a novel S-nitrosoderivative of a recombinant fragment of von Willebrand factor, AR545C: in vitro and ex vivo inhibition of platelet function.
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 (IC(50) = 0.02 +/- 0. 006 micromol/L v 0.1 +/- 0.03 micromol/L, P =.001). In addition and by contrast, S-nitroso-AR545C was a powerful inhibitor of adenosine diphosphate-induced platelet aggregation (IC(50) = 0.018 +/- 0.002 micromol/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 micromol/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 </=.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.
Inbal
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A
Institute of Thrombosis and Hemostasis, Sackler School of Medical, Sheba Medical Center, Tel-Hashomer, Israel.
Gurevitz
O
O
Tamarin
I
I
Eskaraev
R
R
Chetrit
A
A
Novicov
I
I
Feldman
M
M
Varon
D
D
Eldar
M
M
Loscalzo
J
J
D000328:Adult; D000818:Animals; D004305:Dose-Response Relationship, Drug; D006801:Humans; D009569:Nitric Oxide; D009603:Nitroso Compounds / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry* / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D014841:von Willebrand Factor / Q000737:chemistry* / Q000494:pharmacology*
C112046:AR545C; D009603:Nitroso Compounds; D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; C121400:S-nitroso-AR545C; D014841:von Willebrand Factor; D009569:Nitric Oxide