The Research Of ADAMTS13 Protein C-terminal Domain And TSP1 On The Regulation Of ADAMTS13 Activity

ADAMTS13 as von willebrand factor-cleaving protease, its main function is specific cleavage of von Willebrand factor, which regulate VWF-mediated platelet thrombus formation. In 2001, Human ADAMTS13 gene was first cloned and reported. It was located at the end of the long arm of chromosome 9 (9q34), and the gene contained 37kb and 29 exons. The gene expression of open reading frame contain 4284bp, encoding 1427 amino acids. The structure of ADAMTS13 protein is mainly composed by nine domains: including signal peptide, propeptide, metalloproteinase domain, disintegrin domain and thrombospondin-1 (TSP1) motif, cysteine-rich region, spacer region, thrombospondin 1 (TSP1) repeat motifs 2-8, complement binding domain (CUB), the signal peptide and propeptide of ADAMTS13 will be cut off before secretion. Present study confirmed that ADAMTS13 activity severe deficiency can lead to thrombotic thrombocytopenic purpura. Not only in TTP, ADAMTS13 activity deficiency also detected in most of the other diseases accompaned with the thrombotic tendency in recent years. ADAMTS13 gene defects or the presence of autoantibodies is known as the reasons for serious deficiencies, then, what other factors can result in reduced ADAMTS13 activity, leading to a damage degradation of plasma VWF levels? The research of the regulation of ADAMTS13 activity has been warm spot in the field of thrombosis and hemostasis. On the basis of our previous work related, we made a further research and analysis of some regulatory factors of ADAMTS13 activity, and the relationship between ADAMTS13 and microvascular endothelial cells. We also established a new type detection method of ADAMTS13 enzymatic activity.1, the regulation of ADAMTS13 protein C-terminal domain to its activity Present study shows that, there is a wide range of binding sites between ADAMTS13 and VWF. Metalloproteinase domain is the pivotal center of ADAMTS13 protease activity, while disintegrin domain, thrombospondin-1 motif, cysteine-rich region, spacer region, these domains play an important effect for ADAMTS13 activity under static conditions, especially spacer region, has been confirmed as an mainly recognized site by autoantibodies in most patients with acquired TTP. ADAMTS13 protein C-terminal domain is considered to have small contribution to its activity, but C-terminal CUB region is a unique structure for ADAMTS13 in its family. In recent years, studies have suggested that the CUB domain peptide could competitive inhibit ADAMTS13 activity on the high shear conditions, and the C-terminal TSP1 motifs and CUB domain may play an cooperated role for ADAMTS13 activity. Thus, we perform a further research to the function of ADAMTS13 protein C-terminal domain.First, using plasmid pSecTag-ADAMTS13-His, transfection to Hela cells by liposome-mediation, and selection by hygromycin B, we obtained two cell lines of stablely expressed rADAMTS13. Serum-free culture supernatant was collected and concentrated, then purified by Ni-NTA Agarose column, and then calculated protein concentration by the spectrophotometer OD280 and identified by Westerblot. The results showed that: We got a full-length wild-type and C-terminal truncated rADAMTS13 protein, initial OD280 calculated protein concentration (wild-type and truncated rADAMTS13 proteins were 116ug/ml, 126ug/ml), Westerblot results also confirmed the specificity of the recombinant protein. The molecular size was consistent with the expected.Second, by the R-CBA and Reduced-SDS-PAGE assay, we used plasma purified VWF protein as substrate to detect the two rADAMTS13 protein activity on static or vortex dynamic conditions respectively. While by using ELISA method, we also detect the binding capacity of the two rADAMTS13 protein to VWF substrate. The results showed that: by Reduced-SDS-PAGE assay on the static urea denaturing conditions, we used VWF176KD digested fragments to reflect the specific cleavage activity of rADAMTS13 protein, found that the C-terminal truncated rADAMTS13 protease activity was significant higher than that of wild type, R-CBA assay for quantification of enzyme activity suggested that the average activity of wild-type and truncated protein were 61.23±7.67%, 92.84±0.38 % respectively, there was a significant difference(p <0.01). ELISA analysis detected the binding capacity of rADAMTS13 to coated VWF under static conditions and suggested that both wild-type and truncated rADAMTS13 protein had an increasing binding capacity to VWF as the protein concentration enhanced, but the binding capacity of truncated rADAMTS13 protein to VWF was significantly higher than that of wild type. However, on the high shear conditions, only full-length wild-type rADAMTS13 protein could be specific cleavage of VWF, weaker than static conditions. The average activity of wild-type was 41.34±18.06%, while truncated rADAMTS13 had an undetectable activity, less than 5%.2, the regulation of TSP1 to ADAMTS13 activityPlatelet-sensitive protein-1 (Thrombospondin-1, TSP1), like the VWF, also stored in platelet a-particles. Study found that TSP1 could protect VWF-mediated platelet thrombosis on the surface of endothelial cells, and suggested that TSP1 might be competitive inhibition of ADAMTS13 activity through effective integration with VWFA3 domain. There was a widespread interaction between ADAMTS13 and VWFA domains, especially between ADAMTS13 and VWFA2 domain. Consideration that our laboratory had constructed expression of the VWFA1, A3 domain protein, we further constructed expression of the VWFA2 domain protein, and researched the specific regulation mechanisms of TSP1 to ADAMTS13 activity.First, we used pSVvWF plasmid as a template, amplified VWFA2 gene sequence with designed specific primers to A2 by PCR, and constructed pUCm-T -VWFA2 recombinant plasmid with T4 DNA ligase, followed by restriction enzyme digestion and DNA sequencing analysis, the results showed that the cloned VWFA2 gene was completely correct. Then we once again successfully constructed and connected PQE30-VWFA2 recombinant plasmid, used the expression vector to express in the M15, and purified the expression protein by Ni-NTA Agarose column, after electrophoresis and identified by Coomassie blue staining, showing that a single protein of approximately 25 kD band, the molecular weight was consistent with the expected VWFA2 domain. The results proved that we obtained high purity VWFA2 prokaryotic expression protein, which provided us a basis to further study of the biological function of ADAMTS13 enzyme protein.Secondly, by ELISA and Westerblot analysis, we used plasma purified VWF protein or VWFA1, A2, A3 domain as substrate to observe the regulation of TSP1 to ADAMTS13 activity. While by using ELISA method, we also detected the influence of TSP1 to the binding capacity of ADAMTS13 to the substrates. The results suggested that: TSP1 could significantly inhibit the binding of ADAMTS13 to VWF and directly inhibits ADAMTS13 cleavage of VWF on the conditions of existence of the largest physical concentrations, the maximum inhibition rate could be up to 70%. Between ADAMTS13 and TSP1 exist competitive binding sites in VWF A2 and A3 domains, TSP1 could directly inhibit ADAMST13 cleavage of VWFA2 domain.3, the research of inter-relationship between ADAMTS13 and microvascular endothelial cellsLike hepatic stellate cells, vascular endothelial cells are also a major source of plasma ADAMTS13, which has been confirmed in the study abroad. However, these studies focus on some of the major vascular endothelial cells, and as to the research about ADAMTS13 expression in microvascular cells is less. Considering that ADAMTS13 regulates VWF-mediated platelet thrombus formation mainly on the surface of microvascular cells, so whether the microvascular cells participate the regulation of the local ADAMTS13 activity around the blood vessels? In addition, reports suggest ATRA can down-regulate the expression of tissue factor, and the up-regulate the expression of TM, t-PA to improve vascular endothelial cells hypercoagulable state. Whether ATRA also affected the balance between ADAMTS13 and VWF of microvascular endothelial cells? Therefore, we take these issues to perform a further research about the relationship between ADAMTS13 and microvascular endothelial cells.First, we used flow cytometry and immunofluorescence microscopy to directly observe the expression of ADAMTS13 in microvascular endothelial cells. We also observed the expression and distribution of VWF in microvascular endothelial cells by immunofluorescence microscopy. We found that a large number of ADAMTS13 expression did exist in microvascular endothelial cells, and there was some same distribution of stacking area between ADAMTS13 and VWF, which was consistent with some study by other researchers in large vascular endothelial cell lines. We also used flow cytometry to detect interactions between recombinant ADAMTS13 protein and HMEC-1 membrane surface, our results suggested that ADAMTS13 could bind to microvascular endothelial cells membrane surface, and this binding was not dependent on the C-terminal domain of ADAMTS13, and could be blocked by TSP1 protein.Second, we used real-time quantitative PCR and Westerblot technology to confirm that ATRA can increase ADAMTS13 expression of microvascular endothelial cells from gene and protein levels respectively. We also confirmed that some of the inflammatory cytokines such as TNF-αcould inhibit the expression of ADAMTS13 in endothelial cells, which was consistent with other reports. Our results also showed that ATRA could also reverse the inhibitory effect of TNF-αto ADAMTS13 expression, and did not affect the secretion of VWF. Therefore, we believe that ATRA can effectively improve the balance secretion of ADAMTS13 and VWF in vascular endothelial cells, thereby improving the body’s coagulation status.4, establish and evaluate a new type method of ADAMTS13 activity assayADAMTS13 activity detection has a significant value not only in clinical diagnosis of TTP, but also for the prognosis evaluation in TTP patients. However, the present detection methods exist some different defects, and can not be promoted. In view of this, study and development of a new ADAMTS13 activity detection method is still necessary. We used two new VWF monoclonal antibodies SZ129, SZ125——specific to VWFA1 and the A3 domain, to establish a new type method of ADAMTS13 activity assay, and make a preliminary evaluation and clinical application. Our results show that we have created a new type method of ADAMTS13 activity assay, and the test results are accurate. Our method is simple and easy to carry out, reproducible, suitable for application in ordinary clinical units and laboratory labs.