The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) within endovascular platelet aggregates, and ADAMTS13 deficiency causes fatal microvascular thrombosis. The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains of ADAMTS13 recognize a cryptic site in VWF that is exposed by tensile force. Another seven T and two complement C1r/C1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains of uncertain function are C-terminal to the MDTCS domains. We find that the distal T8-CUB2 domains markedly inhibit substrate cleavage, and binding of VWF or monoclonal antibodies to distal ADAMTS13 domains relieves this autoinhibition. Small angle X-ray scattering data indicate that distal T-CUB domains interact with proximal MDTCS domains. Thus, ADAMTS13 is regulated by substrate-induced allosteric activation, which may optimize VWF cleavage under fluid shear stress in vivo. Distal domains of other ADAMTS proteases may have similar allosteric properties. 1A) (1-5), a metalloprotease that severs VWF and releases adherent platelets. Deficiency of ADAMTS13 disrupts this feedback regulatory mechanism and causes thrombotic thrombocytopenic purpura (TTP), which is characterized by life-threatening microvascular thrombosis (3, 6, 7).The recognition and cleavage of VWF is a formidable challenge. VWF and ADAMTS13 occur at ∼10 μg/mL and ∼1 μg/mL, respectively, compared with total plasma protein of ∼80,000 μg/mL. ADAMTS13 is constitutively active and has no known inhibitors in vivo. Nevertheless, VWF is the only identified ADAMTS13 substrate, and VWF is resistant to cleavage until subjected to fluid shear stress (8), adsorbed on a surface (9), or treated with denaturants (8, 10). This specificity depends on structural features of both ADAMTS13 and VWF that have not been characterized fully.The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains domains of ADAMTS13 bind to cryptic sites that are uncovered by unfolding VWF domain A2 (11-15) (Fig. 1B), and these interactions are required for efficient cleavage of VWF or peptide substrates. More distal ADAMTS13 domains bind to sites in or near VWF domain D4 that are always available (16-18). Deletion of distal ADAMTS13 domains impairs the cleavage of VWF multimers in vitro (16,19) and increases VWF-dependent microvascular thrombosis in vivo (20) but accelerates the cleavage of peptide substrates (12, 13). In addition, ADAMTS13 cleaves guanidine hydrochloride-treated VWF multimers with an apparent K m of ∼15 nM (21), which is 100-fold lower than the K m of ∼1.6-1.7 μM for peptide substrates that are based on the sequence of VWF domain A2 (12,14). These striking differences suggest that distal T or complement c1r/c1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains regulate ADAMTS13 activity. We have now shown that these distal domains inhibit ADAMTS13, and binding to VWF relieves this autoinhibition. Result...

A green, simple, and cost effective electrochemical method to synthesize pure graphene oxide (GO) and graphene nanosheets (GNs) using pencil in ionic liquid medium is reported. The morphology and microstructure of prepared GNs and GO are examined using scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), X‐ray diffraction (XRD), and Raman spectroscopy; the experiments confirm the formation of high quality graphene. The synthesized GO is used for the real‐time and label‐free surface plasmon resonance (SPR) sensing of the biological warfare agent Salmonella typhi.

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The metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats member 13) prevents microvascular thrombosis by cleaving von Willebrand factor (VWF) within platelet-rich thrombi, and cleavage depends on allosteric activation of ADAMTS13 by the substrate VWF. Human ADAMTS13 has a short propeptide, metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains (proximal domains), followed by 7 T and 2 CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains (distal domains). Distal domains inhibit the catalytic proximal domains; binding of distal T8-CUB domains to the VWF D4 domain relieves autoinhibition and promotes cleavage of the nearby VWF A2 domain. However, the role of specific ADAMTS13 distal domains in this allosteric mechanism is not established. Assays of plasma ADAMTS13 from 20 placental mammals, birds, and amphibians show that allosteric regulation is broadly conserved, and phylogenetic analysis of 264 vertebrates shows the long propeptide, T3, T4, T6, and T6a domains have been deleted several times in placental mammals, birds, and fish. Notably, pigeon ADAMTS13 has only 3 distal T domains but was activated normally by human VWF D4 and cleaved VWF multimers, preferentially under fluid shear stress. Human ADAMTS13 constructed to resemble pigeon ADAMTS13 retained normal allosteric regulation and shear-dependent cleavage of VWF. Thus, the T3-T6 domains of human ADAMTS13 are dispensable. Conversely, deletion of T7 or T8 abolished allosteric activation. For most species, some sequence changes in the VWF substrate can markedly increase the rate of cleavage, suggesting that ADAMTS13 and VWF have not evolved to be optimal enzyme-substrate pairs. These properties may reflect evolutionary pressure to balance the risk for VWF-dependent bleeding and thrombosis.

An efficient natural product inspired diversity oriented syn thesis of tetrahydroquinoline analogues has been developed using the natural carbohydrate derived solid acid catalyst via multicomponent aza-Diels-Alder reaction of imine (generated in situ from aromatic amine and aldehyde) with dienophile in acetonitrile in a diastereoselective manner. The use of water as solvent reverses the diastereoselectivity toward the cis isomer. Interestingly, tricyclic pyrano/furano benzopyran with cis diastereoselectivity is obtained when salicylaldehyde is used as an alternative of aromatic aldehyde under the same condition. These synthesized quinolines and benzopyrans analogues have been evaluated for their Antitubercular activity against M. tuberculosis H₃₇Ra, and M. tuberculosis H₃₇Rv, and some of the analogues shows better activity profile than their natural product analogues. The protocol is not only mild, efficient, ecofriendly, but also involves reusable and biodegradable catalyst and provides route for both the diastereoisomer.