Study On The Reaction Of VWF And ADAMTS13

Part I Stable expression of the von Willebrand factor cleaving protease and the research of its biological activity Objective: This study was to acquire recombinant protein of von Willebrand factor cleaving protease(ADAMTS13, a disintegrin and metalloprotease with a thrombo Spondin type 1 motifs 13), for further studies on its biological function in thrombosis and hemostasis. Methods: We transfected the Hela cells with the plasmid p Sec Tag-ADAMTS13 by lipofectamine. A positive cell cloning was selected by hygromycin-B. The recombinant protein was purified with Ni-NTA agarose column by gradient imidazole. The purity and immune activity of purified products were identified with SDS-PAGE and Western blotting respectively. We also measured the enzymatic activity of recombinant protein(r ADAMTS13) by GST-His two-site ELISA assay. Results: The results showed that we successfully constructed Hela cells ADAMTS2-4 which expressed high level of r ADAMTS13. We received about 5.8 mg recombinant protein in culture supernantants per liter purified with Ni-NTA column. The protein formed a main lane at the position of 190 k Da with SDS-PAGE and reacted with polyclonal antibody against ADAMTS13 by Western blotting. The amount of r ADAMTS13 activity was 6.4 U/m L, according to the normal plasma defined as 1 U/m L. Conclusions: In conclusion, r ADAMTS13 protein had high purity, immune activity and good enzymatic activity, which could establish the experimental foundation for further research on biological function and mechanism of this unique metalloprotease.Part II The co-influence of VWD type 2B/2M mutations in A1 domain and platelet GPIbα on the rate of cleavage to VWF by ADAMTS13 Objectives: In plasma, the size of von Willebrand factor(VWF) multimer is down regulated by ADAMTS13(A disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13). The binding of platelets or glycoprotein(GP) Ibα recombinant fragment to VWF domain A1 may increase the cleavage by ADAMTS13 to VWF. Both type 2B and type 2M von Willebrand disease(VWD) result in bleeding disorders with the diathesis of increased and decreased binding affinity between GPIbα and VWF, respectively. However, the influence of 2B/2M VWD mutations in A1 domain and GPIbα on cleavage by ADAMTS13 to VWF needs further study. Methods: Different types of full-length human recombinant VWF(r VWF) were expressed including three type 2B mutations(P1337L, H1268 D and R1308C), one type 2M mutation(D1302G) and wide type(WT). The three types of r VWF were characterized by multimeric analysis, ristocetin-induced platelet aggregation(RIPA) and VWF: RCo–ELISA. Five characterized r VWF were digested by ADAMTS13 in the absence or presence of GPIbα(H1-V289) under static conditions or high shear stress. The interaction of r VWF and ADAMTS13 was also tested by plate binding assays. Results: Under static(nature) conditions or high shear stress, type 2B mutants exhibited a higher susceptibility to ADAMTS13 than r VWF-WT, whereas type 2M mutant normalized. While under static(denatured) conditions or high shear stress(with GPIbα fragment) r VWF-WT showed even higher susceptibility to ADAMTS13 with two type 2B mutants studied. Conclusion: Type 2B mutations localized in the VWF A1 domain could enhance the affinity to ADAMTS13 and the sensitivity to ADAMTS13-mediated proteolysis by their own conformational change. When GPIbα participates, dramatically increased proteolytic cleavage of VWF by ADAMTS13 would happen to r VWF-WT excluding some type 2B mutants. Part III Characterization of monoclonal antibodies against ADAMTS13 and study on their function Objectives: ADAMTS13 is a plasma metalloprotease that proteolytically regulates von Willebrand factor(VWF), a critical mediator of platelet tethering and primary haemostasis. The physiological importance of the ADAMTS13-VWF axis is highlighted by cases of either inherited or acquired ADAMTS13 deficiency, which is associated with life-threatening thrombotic thrombocytopenic purpura(TTP). Preparation of murine monoclonal antibody against human ADAMTS13 is crucial for further study for the function of ADAMTS13. Methods: Balb/c mice were immunized by purified recombinant ADAMTS13 truncated eukaryotic protein(ADAMTS13-T7). Murine anti-human ADAMTS13 monoclonal antibodies(Mc Abs) were developed by standard hybridoma technology and identified by ELISA. The recognition of Mc Abs with full-length recombinant ADAMTS13 protein was identified by Western blotting. In function assay, the influence of Mc Abs on the proteolysis of VWF by ADAMTS13 was observed. Results: A group of 6 murine anti-ADAMTS13 Mc Abs was obtained with the clone number 1G11, 2F11, 6G3, 9E1, 10A2 and 10B4. In ELISA, the highest titers of 1G11 and 2F11 were observed, which both showed the high affinity to ADAMTS13-T7 compared with full-length ADAMTS13. The results of Western blotting demonstrated that 6 Mc Abs could all recognize ADAMTS13, among which 1G11 and 2F11 showed stronger reaction with ADAMTS13. In addition, under static(nature) conditions, 1G11 and 2F11 could inhibit cleavage of VWF by ADAMTS13 dramatically in increasing concentrations of Mc Abs. Conclusions: Our results further indicated that ADAMTS13 distal domains are adjacent to its proximal domains. Mc Abs against ADAMTS13 could be provided as the useful tool for further study of mechanism of ADAMTS13.