The Preclinical Study Of A Novel Platelet Glycoprotein Ⅱb/Ⅲa Receptor Antagonist-A₀GDW

Fibrinogen-dependent cross-linking of glycoprotein(GP)Ⅱb/Ⅲa on activated platelets is the final mechanism leading to platelet-dependent thrombus formation. The binding of fibrino- gen toⅡb/Ⅲa is mediated by the Arg-Gly-Asp (RGD) recognition sequence. As platelet GPⅡb/Ⅲa receptor antagonist, synthetic peptides based RGD have been proved to be an effective antiplatelet drug. Based on the RGD sequence, we designed a kind of linear peptide: H2N(CH2)7-CO-Gly-Asp-Trp (A0GDW). In the preclinical study, the preparation technology and quality control method of A0GDW were set up. On the basis of all the work, the antithrombotic effective and safety of A0GDW were evaluated.A0GDW were synthesized by Fmoc-SPPS method. To synthesized A0GDW, the strategies are as followings: Fmoc-group as protective group forα-NH2;TBTU,DIEA and HOBt as coupling reagent; And ethanedithiol-phenol-thioanisole- H2O-TFA(2.5:5:5:5:82.5,V/V) as cleav- ing solution. The structure of A0GDW was identified to be correct by ESI-MS analysis. The separation and purification of A0GDW was carried out with reversed-phase liquid chromato- graphy(RPLC). The purity of A0GDW was 98.3%.A specific HPLC method with high sensitivity was established which has desirable separation results. The appearance, melting point, the stability and changes of the materials and content are also discussed.We used turbidmetric method to measure platelet aggregation. A0GDW remarkably inhibit- ed rabbit platelet aggregation induced by adenosine diphosphate (ADP), arachidonic acid (AA) or collagen (COLL) in vitro. A0GDW at 0.4mg/kg iv inhibited platelet aggregation induced by ADP in rabbits, taking effect immediately after A0GDW intravenous injection and lasting for 100min. To study the antithrombosis effect of A0GDW, we established two kinds of animal models. On the rabbit model of artery-vein bypass thrombus formation, A0GDW can abate the wet weight of thrombus in vivo. The result also showed that there were dose-dependent increases in inhibiting rate of the wet weight. On the model of rat carotid thrombosis induced by 20% FeCl3, A0GDW was intravenously infused for prevention before thrombosis. It was found that the thrombosis was significantly suppressed. The tail artery bleeding time was determined in mice through tail venous injection. As a presumed consequence of the inhibition of platelet function, A0GDW remarkably lengthened the bleeding time. But the bleeding time of A0GDW is much shorter than the same dosage of Tirofiban (the positive control). In addition, we testified the effect of A0GDW on...