The Study Of The Inhibition Of Recombinant Platelet Glycoprotein Ibα On Platelet Aggregation

The binding between vWF and GPIb, and the resulting platelet adhesion to injured vessels is very important for primary hemostasis - platelet plug formation.Thus, much research has been committed to discovering and producing ways of antithrombotic treatments via GpIb and vWF, the two targets. Currently a variety of different antithrombotic treatments options have been developed, including the use of anti-von Willebrand monoclonal antibodies, GPIb-binding snake venom proteins, aurintricarboxylicacid that binds to vWF, and recombinant von Willebrand fragments. All of these studies showed the potential feasibility of antithrombotic therapy based on inhibition of the interaction of GPIb. Despite the potential therapeutic advantages of targeting GPIb, this field has progressed slowly.Although recombinant fragments of the vWF molecule have been found the ability of binding to GP I b a and vWF, it delay but do not abolish thrombus formation. The pharmacological agents could be developed in combination with aspirin or with thrombolytic agents.Thus, taking vWF as an explorative target,the recombinant protein of vWF binding region of human GPIb a (GP302) was obtained, The binding activity of GP302 to the vWF in plasma was detected in vitro using ristocetin induced platelet aggregation assay.In order to identify the expression of platelet GPIba in mammalian cells, we expressed a fusion protein of GST-GP302 in E. coli.and obtained the corresponding polyclonal antibody. Firstly, in the current study the GP302 gene was inserted into pGEX-4T-1. The recombinant vector was identificated by restriction endonuclease digestion analysis. GST-GP302 fusion protein was expressed in E. coli via IPTG induction. Rabbits were immunized with renatured GST-GP302 three times. Antisera were collected on the 7th day after the third immunization. Antibody titers were detected by regular ELISA.The specificity binding between polyclonal antibody and platelet membrane GPIb a was identified by Western blot. The results showed that the GP302 gene had been exactly inserted into pGEX-4T-1. SDS-PAGEanalysis showed that the relative molecular mass(Mr) of the fusion protein was about 59kD. ELISA analysis proved that the titer of rabbit serum against GST-GP302 was 10"5. The polyclonal antibody specifically bound to purified platelet GPIb a . The polyclonal antibody of GP302 peptide segments provides a good method to identify platelet GPIba.Subsequently, in order to obtain the recombinant protein which have the vWF binding activity, A eukaryotic expression vector containing the GP302 cDNA, pSecTaq2/Hygro B-GP302, was constructed and CHO cells were tranfected by transfast?transfection reagent. The transformants were selected by Hygromycin B and the recombinant protein was detected by c-myc monoclonal antibody and rabbit anti-GP302 polycolonal antibody. Because there are six histidine residues in the C-terminal of the recombinant protein, the recombinant GP302 protein was purified by Ni-NTA agarose. Ristocetin is an inducer which can mediates platelet aggregation through adhesion of platelet membrane GPIba to plasma vWF. Therefore, the vWF binding activity of rGP302 has been assayed initially by ristocetin-induced platelet aggregation assay. The results showed that GP302 gene was exactly inserted into pSecTaq2/HygroB vector. The recombinant protein , which the ralative molecular mass (Mr) was about 33kD, was expressed in CHO cells and it could bind anti-GP302 polyclonal antibody and c-myc McAb. specifically.In ristocetin-induced platelet aggregation test rGP302 was able to bind to vWF in plasma and inhibit ristocetin-induced platelet aggregation by means of competitively inhibit the interaction between platelet membrane GPIb a and plasma vWF.In the current study, based on the significant function of the interaction of GPIba and vWF in arterial thrombosis, we have prepared soluble recombinant GPIba fragment and evaluated its vWF binding activity in vitro. The results suggested that recombinant GPIba fragment has the property of binding vWF, and subseq...