HD1, a thrombin- and prothrombin-binding DNA aptamer, inhibits thrombin generation by attenuating prothrombin activation and thrombin feedback reactions

Kretz, Colin A.; Cuddy, Karl; Stafford, Alan R.; Fredenburgh, James C.; Roberts, Robin S.; Weitz, Jeffrey I.

doi:10.1160/th09-04-0237

Cited by 26 publications

(24 citation statements)

References 42 publications

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“…Phosphatidylcholine-phosphatidylserine (3:1) vesicles (PCPS) were prepared as described. 29 Nucleic acid preparations. RNA and DNA were isolated from cultured A549 non-small cell lung cancer cells, a generous gift from Dr. P. Liaw, using RNeasy Plus mini and DNeasy mini isolation kits, respectively (Qiagen, Mississauga, ON, Canada), according to the manufacturer's protocol.…”

Section: Methodsmentioning

confidence: 99%

Arterial thrombosis is accelerated in mice deficient in histidine-rich glycoprotein

Zhou

Leslie

et al. 2015

Blood

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Section: Methodsmentioning

confidence: 99%

Arterial thrombosis is accelerated in mice deficient in histidine-rich glycoprotein

Zhou

Leslie

et al. 2015

Blood

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“…Fulllength hirudin was from Dade-Behring (Marburg, Germany). Citrated human plasma was prepared as described (31). Unless otherwise specified, other reagents were from Sigma.…”

Section: Methodsmentioning

confidence: 99%

Batroxobin Binds Fibrin with Higher Affinity and Promotes Clot Expansion to a Greater Extent than Thrombin

Stafford

Leslie

et al. 2013

Journal of Biological Chemistry

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Batroxobin is a thrombin-like serine protease from the venom of Bothrops atrox moojeni that clots fibrinogen. In contrast to thrombin, which releases fibrinopeptide A and B from the NH2-terminal domains of the Aα- and Bβ-chains of fibrinogen, respectively, batroxobin only releases fibrinopeptide A. Because the mechanism responsible for these differences is unknown, we compared the interactions of batroxobin and thrombin with the predominant γA/γA isoform of fibrin(ogen) and the γA/γ′ variant with an extended γ-chain. Thrombin binds to the γ′-chain and forms a higher affinity interaction with γA/γ′-fibrin(ogen) than γA/γA-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (Kd values of about 0.5 μm) even though it does not interact with the γ′-chain. The batroxobin-binding sites on fibrin(ogen) only partially overlap with those of thrombin because thrombin attenuates, but does not abrogate, the interaction of γA/γA-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a Kd value of 2–5 μm, the α(17–51) and Bβ(1–42) regions bind thrombin but not batroxobin. Once bound to fibrin, the capacity of batroxobin to promote fibrin accretion is 18-fold greater than that of thrombin, a finding that may explain the microvascular thrombosis that complicates envenomation by B. atrox moojeni. Therefore, batroxobin binds fibrin(ogen) in a manner distinct from thrombin, which may contribute to its higher affinity interaction, selective fibrinopeptide A release, and prothrombotic properties.

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“…Such methods include engineering deletion mutants (3), use of competitive ligands (4,5), and site-directed mutagenesis (6,7). In contrast to these techniques, substrate phage display is a highthroughput, unbiased approach to studying protease substrate specificity (8)(9)(10).…”

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confidence: 99%

Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

Kretz

Dai

Söylemez

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Proteases play important roles in many biologic processes and are key mediators of cancer, inflammation, and thrombosis. However, comprehensive and quantitative techniques to define the substrate specificity profile of proteases are lacking. The metalloprotease ADAMTS13 regulates blood coagulation by cleaving von Willebrand factor (VWF), reducing its procoagulant activity. A mutagenized substrate phage display library based on a 73-amino acid fragment of VWF was constructed, and the ADAMTS13-dependent change in library complexity was evaluated over reaction time points, using high-throughput sequencing. Reaction rate constants (k cat /K M ) were calculated for nearly every possible single amino acid substitution within this fragment. This massively parallel enzyme kinetics analysis detailed the specificity of ADAMTS13 and demonstrated the critical importance of the P1-P1′ substrate residues while defining exosite binding domains. These data provided empirical evidence for the propensity for epistasis within VWF and showed strong correlation to conservation across orthologs, highlighting evolutionary selective pressures for VWF.phage display | protease | high-throughput sequencing | ADAMTS13 | von Willebrand factor P rotease specificity is critical for maintaining diversity and compartmentalization of function, and is tightly controlled. For many proteases, a substrate initially docks to an exosite, which captures and orients the substrate scissile bond toward the active site of the enzyme. At the active site, the Px-Px′ (1) substrate amino acid side chains align with the complementary Sx-Sx′ pockets of the enzyme to optimize recognition by the active site residues that execute the proteolytic reaction (2).Conventional techniques for probing the substrate recognition requirements of a protease are cumbersome and time-consuming and require intimate knowledge of the enzyme/substrate pair. Such methods include engineering deletion mutants (3), use of competitive ligands (4, 5), and site-directed mutagenesis (6, 7). In contrast to these techniques, substrate phage display is a highthroughput, unbiased approach to studying protease substrate specificity (8-10). In this method, a library consisting of 10 6 -10 9 independent phage clones, each expressing a unique potential substrate on its surface, is panned for multiple rounds with a protease, and the cleaved or uncleaved phages after each reaction are removed and amplified for subsequent rounds of selection. In this manner, the library complexity is iteratively reduced and becomes populated by peptide sequences that are most informative. This methodology, although useful, is limited by the number of clones selected for individual Sanger sequencing after the last round of selection, and the selection of phages based on competitive growth advantages unrelated to enzyme specificity. The availability of high-throughput DNA sequencing technology (11) has facilitated detailed analysis of the changing complexity within a phage display library (12-16) without requiring multip...

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HD1, a thrombin- and prothrombin-binding DNA aptamer, inhibits thrombin generation by attenuating prothrombin activation and thrombin feedback reactions

Cited by 26 publications

References 42 publications

Arterial thrombosis is accelerated in mice deficient in histidine-rich glycoprotein

Arterial thrombosis is accelerated in mice deficient in histidine-rich glycoprotein

Batroxobin Binds Fibrin with Higher Affinity and Promotes Clot Expansion to a Greater Extent than Thrombin

Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

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