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ADAMTS13 cleaves von Willebrand factor (VWF) between Tyr 1 Deficiency of plasma ADAMTS13 activity, due to either inherited mutations of the ADAMTS13 gene 2-9 or acquired autoantibodies against the ADAMTS13 protein, 10,11 results in thrombotic thrombocytopenic purpura (TTP).ADAMTS13 is primarily synthesized in hepatic stellate cells, 12-14 endothelial cells, 15,16 and megakaryocytes or platelets. 17,18 The plasma ADAMTS13 in healthy individuals ranges from 0.5 to 1 mg/L. 19,20 ADAMTS13 consists of metalloprotease, disintegrin, first thrombospondin type 1 (TSP1) repeat, and Cys-rich and spacer domains. 2,21 The C-terminus of ADAMTS13 has additional TSP1 repeats and 2 CUB (Cis/Cis/Urinary epidermal growth factor, Bone morphogenetic protein) domains. 2,21 Previous studies have shown that the N-terminus of ADAMTS13 is required and sufficient for recognition and cleavage of denatured multimeric VWF [22][23][24] or peptide substrate . 22 More recent studies have demonstrated that the spacer domain of ADAMTS13 binds the exosite (E 1660 APDLVLQR 1668 ) near the C-terminus of the VWF-A2 domain. 25,26 However, the role of the middle and distal C-terminal domains of ADAMTS13 in substrate recognition remains controversial. On the one hand, ADAMTS13 mutant lacking the CUB domains or truncated after the spacer domain cleaved multimeric VWF with similar efficiency as the full-length ADAMTS13 under static and denatured conditions 23,24 ; the mutant truncated after the spacer domain, when mixed with ADAMTS13-depleted plasma, was found to be "hyperactive" in cleaving a "stringlike" structure, which represents platelets attached to the newly released VWF on the endothelial cell surface in a parallelflow chamber-based assay. 27 These data suggest that the distal portion of ADAMTS13 molecule may be dispensable under static and denatured conditions but may play a role in modulating ADAMTS13-VWF interaction under flow. On the other hand, synthetic peptides or recombinant fragments derived from the CUB domains 28 appeared to block the cleavage of the stringlike structure on endothelial cells, suggesting that the CUB domains may directly participate in binding or recognition of VWF under flow. Although the parallel-flow chamber assay may mimic physiological condition, its complexity involving live endothelial cells, histamine stimulation, and platelets makes the quantitation less accurate and kinetic determination of ADAMTS13 and VWF interaction impossible.In the present study, we have developed a simple flow assay based on mechanical-induced shear stress on a mini vortexer or a laminar flow in a BIAcore (Uppsala, Sweden) system to determine the role of the C-terminal ADAMTS13 in recognition and cleavage of multimeric VWF. Our data demonstrate directly and quantitatively that the cooperative activity between the middle C-terminal TSP1 repeats and the distal C-terminal CUB domains of ADAMTS13 may be crucial for productive binding and cleavage of VWF under flow. Materials and methodsThe human study has been approved by the Institutional Re...

Dysregulated autophagy may lead to the development of disease. Role of autophagy and the diagnostic potential of microRNAs that regulate the autophagy in cardiac hypertrophy have not been evaluated. A rat model of cardiac hypertrophy was established using transverse abdominal aortic constriction (operation group). Cardiomyocyte autophagy was enhanced in rats from the operation group, compared with those in the sham operation group. Moreover, the operation group showed up-regulation of beclin-1 (an autophagy-related gene), and down-regulation of miR-30 in cardiac tissue. The effects of inhibition and over-expression of the beclin-1 gene on the expression of hypertrophy-related genes and on autophagy were assessed. Angiotensin II-induced myocardial hypertrophy was found to be mediated by over-expression of the beclin-1 gene. A dual luciferase reporter assay confirmed that beclin-1 was a target gene of miR-30a. miR-30a induced alterations in beclin-1 gene expression and autophagy in cardiomyocytes. Treatment of cardiomyocytes with miR-30a mimic attenuated the Angiotensin II-induced up-regulation of hypertrophy-related genes and decreased in the cardiomyocyte surface area. Conversely, treatment with miR-30a inhibitor enhanced the up-regulation of hypertrophy-related genes and increased the surface area of cardiomyocytes induced by Angiotensin II. In addition, circulating miR-30 was elevated in patients with left ventricular hypertrophy, and circulating miR-30 was positively associated with left ventricular wall thickness. Collectively, these above-mentioned results suggest that Angiotensin II induces down-regulation of miR-30 in cardiomyocytes, which in turn promotes myocardial hypertrophy through excessive autophagy. Circulating miR-30 may be an important marker for the diagnosis of left ventricular hypertrophy.

The soybean phytoestrogen, genistein (Gen), has anabolic effects on bone through mechanisms that remain to be elucidated. We examined the role of nitric oxide (NO) and its downstream effector guanylyl cyclase (GC) in mediating the effects of Gen on the proliferation and osteoblastic maturation of primary mouse bone marrow-derived mesenchymal stem cells (BMSCs). Gen (10(-8) approximately 10(-6) M) resulted in a dose-dependent increase in cell proliferation as measured by increased [3H]thymidine incorporation, and stimulated osteoblastic maturation as assessed by culture duration-dependent increments in alkaline phosphatase (ALP) activity, calcium deposition into extracellular matrix and Runx2/Cbfa1 gene expression in BMSCs cultures. Gen also resulted in a dose-dependent increase in NO synthase (NOS) activity, NO formation, and cGMP production in BMSCs cultures. The effects of Gen were mimicked by 17beta-estradiol (E2, 10(-8) M). Concurrent treatment with the estrogen receptor (ER) antagonist ICI182,780 (10(-7) M) or the NOS inhibitor L-NAME (3 x 10(-3) M) diminished the Gen (10(-6) M)-mediated increase in NOS activity, NO production, and cGMP content. In contrast, a soluble GC inhibitor 1H-[1,2,4]oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ, 10(-6) M) selectively blocked the Gen (10(-6) M)-mediated increase in cGMP content but not in NO production and NOS activity. Moreover, inhibition of ER, NOS activity or cGMP blocked Gen-induced proliferation and osteoblastic differentiation of BMSCs and Runx2/Cbfa1 gene expression in culture. Gen has estrogen-like activity and stimulates the proliferation and osteoblastic differentiation of mouse BMSCs at least in part through NO/cGMP pathway.