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Kenji Kitazato - One of the best experts on this subject based on the ideXlab platform.
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Prolonged bleeding time induced by anticoagulant glycosaminoglycans in dogs is associated with the Inhibition of Thrombin-induced platelet aggregation
Thrombosis Research, 2003Co-Authors: Kenji Kitazato, Eiji Sasaki, Kazuhisa Minamiguchi, Hideki NagaseAbstract:Abstract Introduction: The clinical use of unfractionated heparin (UFH) is complicated by hemorrhage. This has led to a search for safer alternatives, one of which, the recently identified depolymerized holothurian glycosaminoglycan (DHG), causes less bleeding and exhibits a better antithrombotic–hemorrhagic ratio in rats and dogs than UFH and low-molecular-weight heparin (LMWH). In contrast to UFH and LMWH, which exert their anticoagulant effects by inhibiting Thrombin in the presence of antiThrombin III (AT), DHG exerts its anticoagulant effect by inhibiting the intrinsic factor Xase complex and Thrombin in the presence of heparin cofactor II (HCII). Materials and Methods: The hemorrhagic effect of DHG was compared with those of UFH and LMWH in healthy dogs, and the mechanism responsible for prolonging bleeding time was examined both in dogs and with human platelets. Results: DHG prolonged template-bleeding time in dogs less than UFH and LMWH do. Although the maximum noneffective concentrations of each glycosaminoglycan (GAG) that prolong the bleeding time are almost the same as the concentrations that inhibit Thrombin-induced platelet aggregation, they are not related to those that inhibit ADP-induced platelet aggregation. Results of experiments on gel-filtered platelets from humans indicate that the Inhibition of Thrombin-induced platelet aggregation caused by UFH and LMWH in the presence of AT is more prominent than that caused by DHG with HCII. Conclusions: These results suggest that the prolongation of bleeding time caused by GAGs are associated with the Inhibition of Thrombin-induced platelet aggregation, and DHG may cause less bleeding than UFH and LMWH because of its different Thrombin Inhibition mechanism in platelet-rich plasma (PRP).
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Prolonged bleeding time induced by anticoagulant glycosaminoglycans in dogs is associated with the Inhibition of Thrombin-induced platelet aggregation.
Thrombosis research, 2003Co-Authors: Kenji Kitazato, Eiji Sasaki, Kazuhisa Minamiguchi, Hideki NagaseAbstract:The clinical use of unfractionated heparin (UFH) is complicated by hemorrhage. This has led to a search for safer alternatives, one of which, the recently identified depolymerized holothurian glycosaminoglycan (DHG), causes less bleeding and exhibits a better antithrombotic-hemorrhagic ratio in rats and dogs than UFH and low-molecular-weight heparin (LMWH). In contrast to UFH and LMWH, which exert their anticoagulant effects by inhibiting Thrombin in the presence of antiThrombin III (AT), DHG exerts its anticoagulant effect by inhibiting the intrinsic factor Xase complex and Thrombin in the presence of heparin cofactor II (HCII). The hemorrhagic effect of DHG was compared with those of UFH and LMWH in healthy dogs, and the mechanism responsible for prolonging bleeding time was examined both in dogs and with human platelets. DHG prolonged template-bleeding time in dogs less than UFH and LMWH do. Although the maximum noneffective concentrations of each glycosaminoglycan (GAG) that prolong the bleeding time are almost the same as the concentrations that inhibit Thrombin-induced platelet aggregation, they are not related to those that inhibit ADP-induced platelet aggregation. Results of experiments on gel-filtered platelets from humans indicate that the Inhibition of Thrombin-induced platelet aggregation caused by UFH and LMWH in the presence of AT is more prominent than that caused by DHG with HCII. These results suggest that the prolongation of bleeding time caused by GAGs are associated with the Inhibition of Thrombin-induced platelet aggregation, and DHG may cause less bleeding than UFH and LMWH because of its different Thrombin Inhibition mechanism in platelet-rich plasma (PRP).
Hideki Nagase - One of the best experts on this subject based on the ideXlab platform.
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Prolonged bleeding time induced by anticoagulant glycosaminoglycans in dogs is associated with the Inhibition of Thrombin-induced platelet aggregation
Thrombosis Research, 2003Co-Authors: Kenji Kitazato, Eiji Sasaki, Kazuhisa Minamiguchi, Hideki NagaseAbstract:Abstract Introduction: The clinical use of unfractionated heparin (UFH) is complicated by hemorrhage. This has led to a search for safer alternatives, one of which, the recently identified depolymerized holothurian glycosaminoglycan (DHG), causes less bleeding and exhibits a better antithrombotic–hemorrhagic ratio in rats and dogs than UFH and low-molecular-weight heparin (LMWH). In contrast to UFH and LMWH, which exert their anticoagulant effects by inhibiting Thrombin in the presence of antiThrombin III (AT), DHG exerts its anticoagulant effect by inhibiting the intrinsic factor Xase complex and Thrombin in the presence of heparin cofactor II (HCII). Materials and Methods: The hemorrhagic effect of DHG was compared with those of UFH and LMWH in healthy dogs, and the mechanism responsible for prolonging bleeding time was examined both in dogs and with human platelets. Results: DHG prolonged template-bleeding time in dogs less than UFH and LMWH do. Although the maximum noneffective concentrations of each glycosaminoglycan (GAG) that prolong the bleeding time are almost the same as the concentrations that inhibit Thrombin-induced platelet aggregation, they are not related to those that inhibit ADP-induced platelet aggregation. Results of experiments on gel-filtered platelets from humans indicate that the Inhibition of Thrombin-induced platelet aggregation caused by UFH and LMWH in the presence of AT is more prominent than that caused by DHG with HCII. Conclusions: These results suggest that the prolongation of bleeding time caused by GAGs are associated with the Inhibition of Thrombin-induced platelet aggregation, and DHG may cause less bleeding than UFH and LMWH because of its different Thrombin Inhibition mechanism in platelet-rich plasma (PRP).
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Prolonged bleeding time induced by anticoagulant glycosaminoglycans in dogs is associated with the Inhibition of Thrombin-induced platelet aggregation.
Thrombosis research, 2003Co-Authors: Kenji Kitazato, Eiji Sasaki, Kazuhisa Minamiguchi, Hideki NagaseAbstract:The clinical use of unfractionated heparin (UFH) is complicated by hemorrhage. This has led to a search for safer alternatives, one of which, the recently identified depolymerized holothurian glycosaminoglycan (DHG), causes less bleeding and exhibits a better antithrombotic-hemorrhagic ratio in rats and dogs than UFH and low-molecular-weight heparin (LMWH). In contrast to UFH and LMWH, which exert their anticoagulant effects by inhibiting Thrombin in the presence of antiThrombin III (AT), DHG exerts its anticoagulant effect by inhibiting the intrinsic factor Xase complex and Thrombin in the presence of heparin cofactor II (HCII). The hemorrhagic effect of DHG was compared with those of UFH and LMWH in healthy dogs, and the mechanism responsible for prolonging bleeding time was examined both in dogs and with human platelets. DHG prolonged template-bleeding time in dogs less than UFH and LMWH do. Although the maximum noneffective concentrations of each glycosaminoglycan (GAG) that prolong the bleeding time are almost the same as the concentrations that inhibit Thrombin-induced platelet aggregation, they are not related to those that inhibit ADP-induced platelet aggregation. Results of experiments on gel-filtered platelets from humans indicate that the Inhibition of Thrombin-induced platelet aggregation caused by UFH and LMWH in the presence of AT is more prominent than that caused by DHG with HCII. These results suggest that the prolongation of bleeding time caused by GAGs are associated with the Inhibition of Thrombin-induced platelet aggregation, and DHG may cause less bleeding than UFH and LMWH because of its different Thrombin Inhibition mechanism in platelet-rich plasma (PRP).
Umesh R. Desai - One of the best experts on this subject based on the ideXlab platform.
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A small group of sulfated benzofurans induces steady-state submaximal Inhibition of Thrombin.
Bioorganic & medicinal chemistry letters, 2018Co-Authors: Daniel K. Afosah, Stephen Verespy, Rami A. Al-horani, Rio S. Boothello, Rajesh Karuturi, Umesh R. DesaiAbstract:Abstract Despite the development of promising direct oral anticoagulants, which are all orthosteric inhibitors, a sizable number of patients suffer from bleeding complications. We have hypothesized that allosterism based on the heparin-binding exosites presents a major opportunity to induce sub-maximal Inhibition of coagulation proteases, thereby avoiding/reducing bleeding risk. We present the design of a group of sulfated benzofuran dimers that display heparin-binding site-dependent partial allosteric Inhibition of Thrombin against fibrinogen (ΔY = 55–75%), the first time that a small molecule (MW
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A novel allosteric pathway of Thrombin Inhibition: Exosite II mediated potent Inhibition of Thrombin by chemo-enzymatic, sulfated dehydropolymers of 4-hydroxycinnamic acids.
The Journal of biological chemistry, 2007Co-Authors: Brian L. Henry, Bernhard H. Monien, Paul E. Bock, Umesh R. DesaiAbstract:Thrombin and factor Xa, two important pro-coagulant proteinases, can be regulated through direct and indirect Inhibition mechanisms. Recently, we designed sulfated dehydropolymers (DHPs) of 4-hydroxycinnamic acids that displayed interesting anticoagulant properties (Monien, B. H., Henry, B. L., Raghuraman, A., Hindle, M., and Desai, U. R. (2006) Bioorg. Med. Chem. 14, 7988-7998). To better understand their mechanism of action, we studied the direct Inhibition of Thrombin, factor Xa, factor IXa, and factor VIIa by CDSO3, FDSO3, and SDSO3, three analogs of sulfated DHPs. All three sulfated DHPs displayed a 2-3-fold preference for direct Inhibition of Thrombin over factor Xa, whereas this preference for inhibiting Thrombin over factor IXa and factor VIIa increased to 17-300-fold, suggesting a high level of selectivity. Competitive binding studies with a Thrombin-specific chromogenic substrate, a fluorescein-labeled hirudin peptide, bovine heparin, enoxaparin, and a heparin octasaccharide suggest that CDSO3 preferentially binds in or near anion-binding exosite II of Thrombin. Studies of the hydrolysis of H-D-hexahydrotyrosol-Ala-Arg-p-nitroanilide indicate that CDSO3 inhibits Thrombin through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. Overall, designed sulfated DHPs appear to be the first molecules that bind primarily in the region defined by exosite II and allosterically induce Thrombin Inhibition. The molecules are radically different in structure from all the current clinically used anticoagulants and thus represent a novel class of potent dual Thrombin and factor Xa inhibitors.
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Mechanism of poly(acrylic acid) acceleration of antiThrombin Inhibition of Thrombin: implications for the design of novel heparin mimics.
Journal of medicinal chemistry, 2005Co-Authors: Bernhard H. Monien, Kai I. Cheang, Umesh R. DesaiAbstract:The bridging mechanism of antiThrombin Inhibition of Thrombin is a dominant mechanism contributing a massive ∼2500-fold acceleration in the reaction rate and is also a key reason for the clinical usage of heparin. Our recent study of the antiThrombin-activating properties of a carboxylic acid-based polymer, poly(acrylic acid) (PAA), demonstrated a surprisingly high acceleration in Thrombin Inhibition (Monien, B. H.; Desai, U. R. J. Med. Chem. 2005, 48, 1269). To better understand this interesting phenomenon, we have studied the mechanism of PAA-dependent acceleration in antiThrombin Inhibition of Thrombin. Competitive binding studies with low-affinity heparin and a heparin tetrasaccharide suggest that PAA binds antiThrombin in both the pentasaccharide- and the extended heparin-binding sites, and these results are corroborated by molecular modeling. The salt-dependence of the KD of the PAA−antiThrombin interaction shows the formation of five ionic interactions. In contrast, the contribution of nonionic for...
Deshun Lu - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of Thrombin activatable fibrinolysis inhibitor by cysteine derivatives.
Thrombosis Research, 2005Co-Authors: Yee H., Donetta S. Gifford-moore, Douglas W. Beight, Radhakrishnan Rathnachalam, Valentine J. Klimkowski, Alan M. Warshawsky, Deshun LuAbstract:Abstract Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a basic carboxypeptidase that functions as a fibrinolysis inhibitor through the cleavage of C-terminal lysine on partially degraded fibrin. Modulation of TAFI activity provides a potential therapy for thrombosis complications by potentiating fibrinolysis. In our study, we identified three novel TAFI inhibitors containing a cysteine backbone. Three cysteine derivatives, guanidinyl- l -cysteine, glycyl- l -cysteine, and glycyl-glycyl- l -cysteine were tested in TAFI substrate assays and showed Kappi=0.08, 0.14, and 0.99 μM, respectively. Subsequent fibrinolysis assays confirmed their TAFI inhibitory activities. Guanidinyl- l -cysteine showed inhibitory activity in a human plasma clot lysis assay (IC50=9.4 μM). Identification of these cysteine derivatives represents an opportunity to develop potent and specific TAFI inhibitors.
Douglas M Tollefsen - One of the best experts on this subject based on the ideXlab platform.
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heparin cofactor ii
Advances in Experimental Medicine and Biology, 1997Co-Authors: Douglas M TollefsenAbstract:Heparin cofactor II (HCII) is a serpin that inhibits Thrombin rapidly in the presence of dermatan sulfate or heparin. Both of these glycosaminoglycans bind to HCII and increase the rate of Inhibition of Thrombin >1000-fold. This review will focus on the biochemistry of HCII and the mechanism by which glycosaminoglycans stimulate its activity.
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Specific glycosaminoglycans support the Inhibition of Thrombin by plasminogen activator inhibitor 1
Biochemistry, 1993Co-Authors: Raymond Klein Gebbink, Douglas M Tollefsen, Craig H. Reynolds, Koen Mertens, Hans PannekoekAbstract:In the absence of accessory components, plasminogen activator inhibitor 1 (PAI-1) rapidly forms equimolar, inactive complexes both with tissue-type (t-PA) and with urokinase-type (u-PA) plamsinogen activator. In the presence of either the glycoprotein vitronectin or the glycosaminoglycan heparin, PAI-1 is endowed with additional, efficient Thrombin-inhibitory properties (Ehrlich et al., 1990, 1991a). Here, we have investigated the interaction between PAI-1, Thrombin, and glycosaminoglycans in more detail. Inhibition of Thrombin by PAI-1 was quantitatively analyzed in the presence of a wide range of concentrations of heparin, heparan sulfate, dermatan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, keratan sulfate, and hyaluronic acid by measuring residual amidolytic activity. In addition, a qualitative analysis was performed by determining the formation of SDS-stable, equimolar complexes between Thrombin and PAI-1 in the presence of various glycosaminoglycans. Heparin, at concentrations between 0.1 and 1 microgram/mL, significantly promoted Thrombin Inhibition by PAI-1 as well as SDS-stable complex formation. Suboptimal Inhibition was observed with dermatan sulfate, chondroitin 4-sulfate, and heparan sulfate at concentrations that are at least 1 order of magnitude higher than that required for optimal Inhibition in the presence of heparin. Virtually no Inhibition of Thrombin and SDS-stable complex formation was detected with any of the other glycosaminoglycans at concentrations between 0.1 and 1 microgram/mL.(ABSTRACT TRUNCATED AT 250 WORDS)
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site directed mutagenesis of arginine 103 and lysine 185 in the proposed glycosaminoglycan binding site of heparin cofactor ii
Journal of Biological Chemistry, 1990Co-Authors: Morey A Blinder, Douglas M TollefsenAbstract:Abstract Inhibition of Thrombin by heparin cofactor (HCII) is accelerated approximately 1000-fold by heparin or dermatan sulfate. We found recently that the mutation Arg189----His decreases the affinity of HCII for dermatan sulfate but not for heparin (Blinder, M. A., Andersson, T. R., Abildgaard, U., and Tollefsen, D. M. (1989) J. Biol. Chem. 264, 5128-5133). Other investigators have implicated Arg47 and Lys125 of anti-Thrombin (homologous to Arg103 and Lys185 of HCII) in heparin binding. To investigate the corresponding residues in HCII, we have constructed amino acid substitutions (Arg103----Leu, Gln, or Trp; Lys185----Met, Asn, or Thr) by oligonucleotide-directed mutagenesis of the cDNA and expressed the products in Escherichia coli. The recombinant HCII variants were assayed for binding to heparin-Sepharose and for Inhibition of Thrombin in the presence of various concentrations of heparin or dermatan sulfate. All of the Arg103 variants bound to heparin with normal affinity. Furthermore, Inhibition of Thrombin by the Arg103----Leu variant occurred at a normal rate in the absence of a glycosaminoglycan and was accelerated by normal concentrations of heparin and dermatan sulfate. These results indicate that HCII, unlike anti-Thrombin, does not require a positive charge at this position for the interaction with heparin or dermatan sulfate. The Arg103----Gln and Arg103----Trp variants inhibited Thrombin at about one-third of the normal rate in the absence of a glycosaminoglycan, suggesting that these mutations exert an effect on the reactive site (Leu444-Ser445) of HCII. All of the Lys185 variants bound to heparin with decreased affinity but inhibited Thrombin at approximately the normal rate in the absence of a glycosaminoglycan. These variants required greater than 10-fold higher concentrations of heparin to accelerate Inhibition of Thrombin and were not stimulated significantly by dermatan sulfate, suggesting that heparin and dermatan sulfate interact with Lys185 of HCII. These results provide evidence that the glycosaminoglycan-binding site in HCII includes Lys185 but not Arg103, both of which were predicted to be involved by homology to anti-Thrombin.