Study On The Domains Of Von Willebrand Factor And The Generation Of Single Chain Fv To Its Domain A1

Thromboembolic diseases are life-threatening diseases that affect millions of people each year. Thrombosis research is a major subject in field of medical sciencs. von Willebrand factor (vWF) is produced in megakaryocytes and endothelial cells, and is present in plasma and vascular subendothelium. This huge protein with a unique multimeric structure plays a pivotal role in hemostasis and thrombosis. In this process vWF contributes to both platelet adhesion/aggregation and blood coagulation through its multiple interactions with the platelet membrane receptors, glycoprotein (GP) Ib-IX-V complex, GPIIb-IIIa complex, heparin, various types of collagen, and coagulation factor VIII. Platelet adhesion to the subendothelium of damaged blood vessels is the initial event in thrombosis. During this process, vWF form a bridge between collagen within the damaged vessel wall and the platelet receptor GPIb, which is especially important under high shear condition. vWF molecular is composed of several homologous domains with different functions; The vWF A1 domain interacts mainly with the GPIb-IX-V complex; Whereas its A3 domain is the binding site of fibrillar collagen. Moreover, vWF is now considered as a new target for the development of antithrombotic agents because the inhibition of vWF-mediated thrombosis may result in selective inhibition of platelet thrombosis at sites exposed to high shear stress, such as in atherosclerotic stenotic coronary arteries, without bleeding complications.The theme of my graduate study is to focus on the functions of different domains of vWF, using molecular biologic and immunologic approachs. My dissertation consists of two parts. Part I describes expression of vWF domains in E.coli and their function. Part II describes the construction of a phage display library of repertoire single chain antibody from mouse immunized with vWF-A1 and the identification of ScFv to rvWF-A1.In part I, we adopted reverse transcription-PCR to amplify cDNA of vWF domain A1 and A3 from human umbilical vein endothelial cells. After sequence analysis , the amplified DNA fragments were inserted into expression vectors with 6 ×his taq (pQE-31, pET-20b). The recombinant expression vectors were transformedinto E. coli, and induced by IPTG. In addition, chimeric molecule containing residues 449-728 of mature vWF subunit (vWF-A1 polypeptide) fused in frame to residues 918-1114 of the vWF (vWF-A3 polypetide) was also expressed. Each of the three recombinant proteins was purified by chromatography on Ni-NTA agarose column and renatured by Tris.HCl buffer containing GSH and GSSG, their purity were above 95%. FACS was adopted to analyse the rvWF-A1 function of binding to platelets and Chinese Hamaster Ovary cells transfected with pcDNA3.1-GPIb, meanwhile, platelet aggregometry was applied to detect its effect on the inhibition of ristocetin-induced platelet aggregation. The rvWF-Al was expressed successfully in E.coli, accounted for 30% of total bacterial protein. It was identified to have ability to bind GPIb, its rates of binding platelets and transfected CHO-K1 cells were 78.60% and 96.90%, respectively. The activity to inhibit ristocetin-induced platelet aggregation was dose-dependent, its maximal inhibiting rate was 84.70% at 1.4μ mol/L, IC50 was 0.56μmol/L. The results indicate that rvWF-Al might be an effective antithrombotic agent in preventing thrombosis.The rvWF-A3 was expressed successfully in Ecoli (strain DE3), accounting for 46.7% of total bacterial protein. It was identified to have the abilities to bind collagen and to inhibit vWF binding to collagen, its inhibiting rate was 54%.The rvWF-Al/A3 chimeric protein, accounted for 12.6% of total bacterial protein, can bind platelet glycoprotein Ib/IX regardless of modifier. It inhibited ristocetin-induced platelet aggregation with an IC50 of 0.76μmol/L, and inhibited vWF binding to collagen to the level of 76%. These results show that rvWF-Al/A3 chimera is bifunctional.One line of search for antiplatelet drugs in the prevention of thrombosi...