| Thromboembolic diseases are life-threatening diseases, which could lead to ischemia and infarction of vital organs. To study the mechanism of thrombosis and develop novel antithrombotic therapy are the focuses in the field of medical science. Platelets play a crucial role in arterial thrombus formation through adhering to the exposed subendothelial collagen at sites of injury, which is mediated by several kinds of glycoproteins(GPs) located on the surface of platelets, such as GPIb-IX-V, GPIIb-IIIa, GPVI and GPIa-IIa. The adhered platelets subsequently recruit more platelets from blood stream, followed by the platelet plug formation as well as coagulation system activation at sites of injury. It has been shown that platelets play a pivotal role in the initial process of thrombosis, therefore it is necessary to treat and prevent thrombosis by means of anti-platelet adhesion. Recently, a monoclonal antibody(MoAb) against GPIIb-IIIa, Abciximab, which aborted the interaction between GPIIb-IIIa and fibrinogen, was used in treating thrombotic diseases. Despite its tremendous success, it could result in severe bleeding complication. Lately, inhibition of platelet adhesion to collagen, by blocking GPIb-vWF-collagen axis and GPVI/GPIa-IIa-collagen axis, has been documented as new kind antithrombotic method, which displayed ideal antithrombotic effects without significant bleeding side-effects.Under high shear stress, platelet adhesion to collagen is mediated by collagen receptors such as GPIb-IX-V, GPVI, and GPIa-IIa, among which, GPVI, the newly recognized collagen receptor, plays an important role. GPVI is present as a complex with signal-transducing Fc receptor- Y chain in the platelet membrane. There were growing evidences showed that GPVI-deletion and GPVI-deficiency platelets were unresponsive to collagen specifically without significant side-effect on hemostasis, which potentialized that GPVI might be an interesting target for safe anti-adhesion and anti-thrombotic therapy.In this study, the gene of extracellular domain of GPVI was cloned , the corresponding recombinant protein was expressed in prokaryotic cells. After the Balb/cmice were immunized with the recombinant protein, a MoAb against GPVI was developed by fusing mice spleen cells with myeloma cells and monoclonization. Meanwhile, the genes encoding the light- and heavy-chain variable regions(VL, VH) of the MoAb were amplified by RT-PCR, expression vector containing scFv was constructed and recombinant scFv was expressed in prokaryotic cells. The characterizations of MoAb and scFv were determined by platelet aggregation assay and parallel-plate flow chamber to evaluate their impacts on interaction of GPVI and collagen.1, The cDNA of extracellular domain of GPVI was amplified from the plasmid of pBluescript KS(-)-GPVI, and inserted into expression vector with 6X His tag, which was subsequently transferred into E.coli. Induced by IPTG, the recombinant protein (rGPVI) was expressed, which was about 32 kDa and mainly existed in inclusion body and accounted for 14% of total bacterial protein.The rGPVI was purified by chromatography on Ni-NTA resin and renatured by PBS containing GSH and GSSH. The purified rGPVI can be recognized by anti-Penta-His antibody and rabbit anti-GPVI polyclonal antibody by Western blot. In collagen binding assay the purified rGPVI exhibited binding activity to type I collagen. Furthermore, when incubated with fibnllar collagen, rGPVI could inhibited collagen-induced platelet aggregation with maximal inhibition rate of 77.8%. These results indicated that rGPVI could restore its biological activities similar to its native form.2, After immunizing mice with rGPVI and screening with HAT culture medium, nine clones of hybridoma were selected, among which one clonal hybridoma, named SZ118, which could secrete antibody against GPVI constantly, was intensively studied. It's confirmed by flow cytometry and Western blot that platelets and rGPVI, other than neutrophils and erythrocytes, could interact with MoAb SZ118 specifically with high affinity. In platelet aggregometry, we found that SZ118 could inhitbit fibnllar collagen(lug/ml) or convulxin(0.2nmol/L), a specific agonist to GPVI, -induced platelet aggregation in dose-dependent manner, resulting in maximal inhibition rate 85.9% and 43% respectively, though it had little effect on ADP-induced platelet aggregation. Simultaneously, the parallel-plate flow chamber was applied to detect itseffect on the inhibition of platelet adhesion to fibrillar collagen or immobilized vWF under high shear rate condition. After perfusing whole blood treated with SZl 18 ( final concentration 50ug/ml) at 1000s"1 for 5 minutes, platelet adhesion to the surface of collagen or vWF was markedly inhibited with maximal inhibition rate 95.5% to collagen , 35.7% to vWF. These results demonstrated that SZl 18 could impede the interaction between GPVI and collagen or vWF, and resulted in invalidating platelet adhesion and unstable platelet aggregates formation, therefore it might be an effective antithrombotic agent in preventing thrombosis.3, To reduce the human anti-mouse antibody response(HAMA) and the potential complement-mediated lysis of platelets caused by Fc region of the mouse-original antibody, we produced and purified the scFv of SZl 18. The genes encoding the VL and VH were cloned by RT-PCR from SZl 18 hybridoma. The DNA sequencing showed that VH gene was 333bp, encoding lllamino acid residues, and VL gene was 312bp, encoding 103 amino acid residues. The VH and VL genes were inserted into plasmid pSWl, which contained the oligonucleotide of the linker peptide ( (Gly4Ser) 3) by genetic engineering approaches and scFv of SZl 18 was obtained. Then the expression vector, pET20b(+)-SZ118scFv, was constructed by ligating scFv gene into pET20b(+) and sequenced again. The recombinent scFv protein (SZ118-scFv) was expressed in E. coli BL21(DE3) pLysS induced by IPTG. SZ118-scFv was mostly produced in the form of inclusion bodies with MW 32 kDa, the yield rate was about 20.5% of the total bacterial protein. After purification and renaturation mentioned above, SZ118-scFv retained platelet binding activity similar to that of parental SZl 18 by flow cytometry and ELISA, and was recognizes by anti-Penta-His antibody in Werstern blot. SZ118-scFv could also inhibit fibrillar collagen (lug/ml) or convulxin (0.2nmol/L)-induced platelet aggregation in dose-dependent manner with maximal inhibition rate 84.3% and 50.3% respectively, and with little effects on ADP-induced platelet aggregation. In perfusion test, the SZ118-scFv (50ug/ml) inhibited platelets adhesion to the surface of the fibrillar collagen at 1000s"1 shear rate with maximal inhibition rate 68.3%. These indicated that SZ118-scFv also could block the interaction of GPVI with collagen, similar to its parental antibody. That means that SZ118-scFvmight be a potential antithrombotic agent.4, It has been reported that there are 14 single nucleotide polymorphisms (SNPs) in GPVI gene up to now, six of them predict amino acid substitution, which have impacts on the GPVI density on the surface of platelets and platelet responses to collagen. In order to evaluate the association between T13254C polymorphism of GPVI gene and the thrombotic disease risk, the T13254C dimorphism of GPVI gene, which predict the substitution of serine219 by proline, was assessed in 274 patients with arterial thrombosis and 314 controls of Chinese Han population by means of PCR-RFLP and sequence analysis. The results showed that the frequencies of the T allele and C allele in the T13254C dimorphism were 0.9809 and 0.0191,respectively, which were significantly different from those reported in Caucasian (p<0.01). Compared with controls, there was no significant difference in T13254C genotype distribution in subjects with the arterial thrombotic diseases. Based on the results, we concluded that the GPVI T13254C polymorphism occurred in low frequency in Chinese Han population, and there is no association between T13254C polymorphism and the arterial thrombotic disease risk.
|
PDF Full Text Request