Purification and functional characterization of two thrombosis-related molecules: von Willebrand Factor and ADAMTS-13
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Abstract:
von Willebrand Factor (vWF) is secreted as the ultra-large (UL) multimeric glycoprotein by endothelial cells and megakaryocytes. It can be cleaved into small multimers by metalloprotease ADAMTS-13 following its secretion. As a member of a disintegrin and metalloproteinase with thrombospondin family, ADAMTS-13 plays a crucial role in thrombosis regulation. Many studies have revealed that unsuccessful cleavage of UL-vWF by ADAMTS-13 induced thrombosis. Here, we have purified A domain, which is the most important activity unit of vWF, and the full-length ADAMTS-13. Results of SDS-PAGE and Western blot confirmed two purified proteins have correct molecular weights. Flow cytometry analysis showed that the microspheres were functionalized via coating purified ADAMTS-13 on their surfaces. Using binding assay, microspheres coated with ADAMTS-13 can adhere on petri dishes coated with immobilized vWF A1A2A3 under static conditions, which were resulted from the binding between ADAMTS-13 and vWF A1A2A3. These results demonstrated purified ADAMTS-13 and vWF A1A2A3 have functional activities and can be used as an important tool for exploring the mechanism of interaction between vWF A1A2A3 and ADAMTS-13 in further studies for thrombosis regulations.Keywords:
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A disintegrin-like and metalloproteinase with thrombospondin type 1 motifs(ADAMTS),which is a new family of secreted zinc-dependent metaloproteases.The resent studies of ADAMTS's structure showed that the hypermethylation of TSP impacted the stabilization of oncogenes or tumor suppressor genes,or mRNA and that domain was related with the proteolysis of aggrecan in human osteoarthritic.CUB domain is associated with vWF-cleaving activity which played an important part in thrombotic thrombocytopenia purpura.These advances provide a new way of thought in diagnosis and treatment in the future.
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ADAMTS13
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ADAMTS
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Aggrecan
ADAMTS
Motif (music)
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The ADAMs (a disintegrin and metalloproteinase) and the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin-like motifs) are extracellular proteases that mediate cellular interactions and cell signaling via the modulation of adhesion and the cleavage of cell surface protein ectodomains and extracellular matrix molecules. Gene expression profiling was undertaken to better understand the role of the ADAM and ADAMTS families in the clear native human lenses and following surgical injury with particular relevance to posterior capsule opacification.To carry out profile analysis, the lens (t=0d) was dissected into three regions; anterior epithelia, equatorial region, and fiber cells. Capsular bag culture was undertaken as a means of assessing short-term changes (t=6d) and post-cataractous lens capsular bags (ex vivo) were used to predict long-term changes in ADAM/ADAMTS gene expression. RNA was isolated and quantitative real-time (TaqMan) reverse transcription-PCR (RT-PCR) performed. Data were analyzed in terms of cycle threshold number (C(T)) and also normalized relative to endogenous 18S rRNA.High expression of ADAM-9, -10, -15, and -17 was detected in all native lens regions. ADAM-15 expression was a characteristic of the native lens epithelia more than the fibers. Post-surgical injury, lens capsular bags showed a positive shift in ADAM/ADAMTS expression that was significant for ADAM-9, -15, and ADAMTS-3. Ex vivo capsular bags, with a long-term post surgical injury period, maintained high expression of ADAM-9 and -10 genes.The native human lens expresses ADAM and ADAMTS genes that are differentially regulated following surgical injury. Roles in maintaining cellular adhesion may be of particular importance to native lens tissue integrity and may be lost in the lens wound healing response following cataract surgery.
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ADAMTS
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Disintegrin-like and Metalloproteinase with Thrombospondin Motifs (ADAMTS) proteins that are fundamentally located in the extracellular matrix (ECM) have critical roles on different cellular processes by altering the ECM architecture. It has been known that expression of some members of these proteinases increases in aneurismal and dissectional aortic tissue. The purpose of this study is to investigate ADAMTS1, 5, 16 and tissue inhibitors of metalloproteinases-1, -2 (TIMP-1, -2) levels in aortic tissue obtained from patients with thoracic aortic aneurysms and dissections and to achieve new insights about the function of ADAMTS family members.We investigated ADAMTS1, 5, and 16 expression in human thoracic aortic aneurysms (TAA) (n = 22), thoracic aortic dissections (TAD) (n = 12), and thoracic aortas from age-matched control organ donors (n = 6) (a total number of 34 cases and 6 controls). The expression levels of ADAMTS proteins were determined by Western blot technique using anti-ADAMTS1, ADAMTS5, ADAMTS16, TIMP-1 and TIMP-2 antibodies.ADAMTS1, 5, and 16 protein expressions were significantly higher in thoracic aortic aneurysm and dissection tissues compared to control aortic tissues. Furthermore, TIMP-1 protein levels decreased in TAA and TAD tissues, TIMP-2 did not change.Under the light of our findings, increased expression of ADAMTS1, 5, and 16 proteins may promote deceleration in thoracic aortic aneurysm progression. This is the first study that demonstrates ADAMTS5 and ADAMTS16 proteolytic activity in aneurysm and dissection.
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Thoracic aortic aneurysm
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