arginyl-glycyl-aspartyl-valine and Thrombosis

Resistance and nonresponse to aspirin dramatically decreases its therapeutic efficacy. To overcome this issue, a small-molecule thrombus-targeting drug delivery system, aspirin-Arg-Gly-Asp-Val (A-RGDV), is developed by covalently linking Arg-Gly-Asp-Val tetrapeptide with aspirin. The 2D ROESY NMR and ESI-MS spectra support a molecular model of an A-RGDV tetramer. Transmission electron microscopy images suggest that the tetramer spontaneously assembles to nanoparticles (ranging from 5 to 50 nm in diameter) in water. Scanning electron microscopy images and atomic force microscopy images indicate that the smaller nanoparticles of A-RGDV further assemble to bigger particles that are stable in rat blood. The delivery investigation implies that in rat blood A-RGDV is able to keep its molecular integrity, while in a thrombus it releases aspirin. The in vitro antiplatelet aggregation assay suggests that A-RGDV selectively inhibits arachidonic acid induced platelet aggregation. The mechanisms of action probably include releasing aspirin, modifying cyclic oxidase, and decreasing the expression of GPIIb/IIIa. The in vivo assay demonstrates that the effective antithrombotic dose of A-RGDV is 16700-fold lower than the nonresponsive dose of aspirin.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Cyclooxygenase 1; Dose-Response Relationship, Drug; Fibrinolytic Agents; Male; Nanocapsules; Oligopeptides; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Rats; Rats, Wistar; Thrombosis; Treatment Outcome

In this study, a new class of phenolic tetrahydro-beta-carboline RGD peptidomimetic conjugates was designed and synthesized. The radical scavenging activities of these newly synthesized compounds 12a-c were evaluated in PC12 cell survival assays. The NO scavenging activities of these compounds were confirmed in the acetylcholine-induced vasorelaxation assay. Compounds 12a-c were efficacious in a rat arterial thrombosis model, and were active in ADP- or PAF-induced in vitro platelet aggregation assays, which suggests these compounds also possess anti-thrombotic activity. The beneficial effects of dual-acting agent 12c were demonstrated on the ischemia-reperfusion induced cardiac infarct size and oxidative change in an in vivo rat model.

Topics: Animals; Carbolines; Cell Survival; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Design; Fibrinolytic Agents; Free Radical Scavengers; Molecular Structure; Oligopeptides; PC12 Cells; Phenol; Platelet Aggregation; Platelet Aggregation Inhibitors; Rats; Thrombosis

A new approach to construct a single dual-acting agent is described. Compounds 6a-c are potent free radical scavengers as demonstrated by the EC(50) values in PC12 cell survival assay in term of NO, H(2)O(2), and ()OH scavenging activity. The Ach-induced vaso-relaxation assay further confirms the potent NO scavenging activity of compounds 6a-c. In addition, 6a-c are efficacious in a rat arterial thrombosis, and are active in ADP- or PAF-induced in vitro platelet aggregation assay, suggesting that compounds 6a-c also possess anti-thrombotic activities. Since both free radical and thrombogenesis are important risk factors in myocardial ischemic/reperfusion injuries, these dual-acting agents having both free radical scavenging and antithrombolic activities may potentially be beneficial toward their treatment.

Topics: Adenosine Diphosphate; Animals; Carbolines; Drug Design; Fibrinolytic Agents; Free Radical Scavengers; Hydrogen; Molecular Structure; Oligopeptides; PC12 Cells; Phenol; Platelet Activating Factor; Platelet Aggregation; Rabbits; Rats; Thrombosis

Fibrin forms on and binds to the extracellular matrix of endotoxin-stimulated endothelium when exposed to flowing non-anticoagulated blood. These processes have been investigated by employing a human ex vivo perfusion model, a synthetic peptide Arg-Gly-Asp-Val and a monoclonal anti-tissue factor antibody which inhibits tissue factor/FVIIa-induced coagulation. Procoagulant extracellular matrix on plastic cover-slips was prepared from cultures of endotoxin-stimulated human endothelium following brief exposure to 0.1 M NH4OH. Non-anticoagulated blood was drawn directly from an antecubital vein by a pump at venous (100/s) and arterial (650/s) wall shear rates over the matrix-coated cover-slips positioned in parallel-plate perfusion chambers. Deposition of fibrin and platelets on the matrix was quantified by morphometry. Preincubation of the matrix with Arg-Gly-Asp-Val inhibited fibrin deposition by 80-90% at both venous (P < 0.001) and arterial shear (P < 0.05). However, the peptide had no effect on the clotting time in a modified one-stage clotting assay where coagulation was initiated by lysed endotoxin-stimulated endothelial cells, indicating that the peptide interfered with the binding of fibrin to the matrix in the perfusion model. Preincubation of the matrix with the anti-tissue factor antibody, which blocked the coagulant activity ( > 95%, P < 0.01) in the modified coagulation assay, also inhibited fibrin deposition on the matrix by 90-95% (P < 0.01) at both shear rates. In the absence of either inhibitor, platelets adhered preferentially to the fibrin meshwork, and more so at arterial shear. Platelet thrombus formation on the fibrin coat was in particular pronounced at arterial shear. Thus, it appears that the extracellular matrix of endotoxin-stimulated endothelium initiates coagulation predominantly through tissue factor/FVIIa and that the resulting fibrin meshwork forming on the surface induces rapid platelet thrombus formation. The inhibitory effect of Arg-Gly-Asp-Val on the binding of fibrin to the matrix may indicate the presence of specific matrix fibrinogen/fibrin binding site(s) with a recognition sequence of Arg-Gly-Asp.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Blood Coagulation Tests; Endothelium, Vascular; Extracellular Matrix; Humans; Molecular Sequence Data; Oligopeptides; Platelet Adhesiveness; Thromboplastin; Thrombosis