Comparison of the inhibition of thrombin by three plasma protease inhibitors

Downing, Michael R.; Bloom, James W.; Mann, Kenneth G.

doi:10.1021/bi00606a030

Cited by 154 publications

(51 citation statements)

References 27 publications

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“…␣ 2 M inhibits both ADAMTS-4 and ADAMTS-5 with secondorder rate constants on the order of 10 6 (37,46). ␣ 2 M is found at very high concentrations in the synovial fluid higher than 2 mg/ml, suggesting that the half-lives of free ADAMTS-4 and ADAMTS-5 under these conditions would be on the order of 0.1 and 1 s, respectively.…”

Section: Discussionmentioning

confidence: 98%

“…The densitometric response was found to be linear over the density ranges required for the blots as assessed by loading varying amounts of AGEG product and quantifying the immunoreactive bands by scanning densitometry using the software OneDscan. Active site concentrations were determined by fitting these data to the equation (37), where y is the measured 1772 AGEG product, y max is the amount of product in the absence of ␣ 2 M, k i is the second-order rate constant, t is time, and E o and I o are the initial concentrations of enzyme and ␣ 2 M, respectively. For this purpose the nonlinear curve-fitting program "the nonlinear regression software package GraFit" (Erithacus Software Ltd., Staines, UK) was used, and the second-order rate constants were fixed at 10 5 and 10…”

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confidence: 99%

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α2-Macroglobulin Is a Novel Substrate for ADAMTS-4 and ADAMTS-5 and Represents an Endogenous Inhibitor of These Enzymes

Tortorella

Arner

Hills

et al. 2004

Journal of Biological Chemistry

134

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Osteoarthritis is characterized by the loss of aggrecan and collagen from the cartilage extracellular matrix. The proteinases responsible for the breakdown of cartilage aggrecan include ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). Post-translational inhibition of ADAMTS-4/-5 activity may be important for maintaining normal homeostasis of aggrecan metabolism, and thus, any disruption to this inhibition could lead to accelerated aggrecan breakdown. To date TIMP-3 (tissue inhibitor of matrix metalloproteinases-3) is the only endogenous inhibitor of ADAMTS-4/-5 that has been identified. In the present studies we identify Loss of aggrecan from the cartilage extracellular matrix is an early and sustained feature of osteoarthritis (OA).1 Aggrecan, the major proteoglycan in cartilage, consists of a protein backbone of 210 -250 kDa containing 3 globular domains referred to as G1 (located at the N terminus), followed by G2 and G3 (located at the C terminus) (1). Attached to the core protein between G2 and G3 are the glycosaminoglycans, chondroitin sulfate, and keratan sulfate. The chondroitin sulfate chains (100 -125 per monomer) are located in the C-terminal portion of the core protein, whereas the keratan sulfate chains (25-50 per monomer) are preferentially located toward the N terminus (2). Multiple aggrecan monomers interact with hyaluronic acid via their G1 domain to form aggregates of very high molecular weight. The negatively charged glycosaminoglycan chains are responsible for the extremely high osmotic swelling pressure of cartilage, which is counteracted by the resistance of type II collagen fibers (the other major macromolecule of cartilage). Thus, aggrecan provides cartilage with the ability to resist compressive forces, and its loss will have a severe effect upon the functionality of the cartilage.The cleavage of cartilage aggrecan in OA has been attributed primarily to aggrecanase 1 and 2 (3-9). These enzymes are metalloproteinases that belong to the a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family and have been designated ADAMTS-4 and ADAMTS-5, respectively. Both enzymes cleave the core protein of aggrecan after the amino acids Glu 373 , Glu 1545 , Glu 1714 , Glu 1819 , and Glu 1919(human sequence) (10, 11). Because of their preference for cleaving at the C terminus of glutamic acid, these enzymes have been referred to as glutamyl endopeptidases (12). In fact, it has been shown that ADAMTS-4 can cleave other chondroitin sulfate proteoglycans, including brevican and versican after Glu 393 and Glu 441 , respectively (13-15). Tight regulation of aggrecanase activity is critical for maintaining a fine balance between aggrecan anabolism and catabolism. In diseases such as OA the balance is disturbed in favor of catabolism, and this could be attributed to de novo synthesis of ADAMTS-4 (9, 16) and/or post-translational activation of 18). Another control mechanism for aggrecan catabolism may involve endogenous inhibitors of the aggrecanases. Recently it has been shown that t...

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Section: Discussionmentioning

confidence: 98%

mentioning

confidence: 99%

α2-Macroglobulin Is a Novel Substrate for ADAMTS-4 and ADAMTS-5 and Represents an Endogenous Inhibitor of These Enzymes

Tortorella

Arner

Hills

et al. 2004

Journal of Biological Chemistry

134

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“…ATIII appears to be the most important plasma proteinase inhibitor of thrombin (19). In vivo studies demonstrate that thrombin binds to high affinity sites on endothelial cells.…”

Section: Introductionmentioning

confidence: 99%

Regulation of factor Xa in vitro in human and mouse plasma and in vivo in mouse. Role of the endothelium and plasma proteinase inhibitors.

Fuchs¹,

Pizzo²

1983

J. Clin. Invest.

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A B S T R A C T The regulation of human Factor Xa was studied in vitro in human and mouse plasma, and in vivo in mouse. In human plasma, '251-Factor Xa bound to a1-proteinase inhibitor, antithrombin III, and a2-macroglobulin in a ratio of 4.9:1.9:1 as determined by gel electrophoresis and by adsorption to IgG-(antiproteinase inhibitor)-Sepharose beads. The distribution of Factor Xa in mouse plasma was similar. The clearance of Factor Xa in mice was rapid (50% clearance in 3 min) and biphasic. a1-Proteinase inhibitor-trypsin, even at a 2,000-fold molar excess, failed to inhibit the clearance of Factor Xa, while a2-macroglobulin-trypsin inhibited only the later phase of clearance. The plasma clearance of diisopropylphosphoryl-Factor Xa was more rapid than native Factor X. (50% clearance in 2.5 min), and the clearance was blocked by diisopropylphosphoryl-thrombin. Electrophoresis experiments confirmed that by 2 min after injection into the murine circulation, 90% of the bound Factor Xa was on a2-macroglobulin, in marked contrast to the in vitro results. Organ distribution studies at 3 and 15 min with 125I_ Factor X. demonstrated that the majority of radioactivity was in the liver, with significant radioactivity also present in lung and kidney. Autopsies performed 30 s after injection of 125I-Factor Xa also demonstrated significant binding to the aorta and vena cava. These studies indicate that Factor Xa binds to specific thrombin-binding sites on endothelial cells, and that this binding alters its proteinase inhibitor specificity. Factor Xa binds to a2-macroglobulin in vivo, whereas the predominant in vitro inhibitor of Factor X. is a,-proteinase inhibitor.

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“…The laboratory of Dr Hemker 83 identified that a significant fraction of thrombin is bound to ␣ 2 -macroglobulin, and our studies suggest accumulation of the thrombin ␣ 1 -antitrypsin complex. 84 Furthermore, significant destructive prothrombin cleavage by thrombin leads to production of prothrombin fragment 1 and prethrombin 1.…”

Section: Discussionmentioning

confidence: 99%

Cellular regulation of blood coagulation: a model for venous stasis

Campbell

Brummel‐Ziedins

Butenas

et al. 2010

Blood

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We have adapted the corn-trypsin inhibitor whole-blood model to include EA.hy926 as an endothelium surrogate to evaluate the vascular modulation of blood coagulation initiated by relipidated recombinant tissue factor (rTf) and a cellular Tf surrogate, lipopolysaccharide (LPS)-stimulated THP1 cells (LPS-THP-1). Compared with bare tubes, EA.hy926 with rTf decreased the rate of thrombin formation, ITS accumulation, and the production of fibrinopeptide A. These phenomena occurred with increased rates of factor Va (fVa) inactivation by cleavages at R 506 and R 306 . Thus, EA.hy926 provides thrombin-dependent protein C activation and APC fVa inactivation. Comparisons of rTf with LPS-THP-1 showed that the latter gave reduced rates for TAT formation but equivalent fibrinopeptide A, and fV activation/inactivation. In the presence of EA.hy926, the reverse was obtained; with the surrogate endothelium and LPS-THP-1 the rates of TAT generation, fibrinopeptide release, and fV activation were almost doubled, whereas cleavage at R 306 was equivalent. These observations suggest cooperativity between the 2 cell surrogates. These data suggest that the use of these 2 cell lines provides a reproducible quasiendothelial quasi-inflammatory cytokinestimulated monocyte system that provides a method to evaluate the variations in blood phenotype against the background of stable inflammatory cell activator and a stable vascular endothelial surrogate.(Blood. 2010; 116(26):6082-6091) IntroductionVirchow postulated 3 phenomena that lead to venous thrombosis: (1) the components contained within the blood itself lead to systemic hypercoagulability; (2) alteration of the endothelium from injury or dysfunction; and (3) alterations of blood flow. 1 These postulates are the foundation for the physiologic understanding of blood coagulation and the maintenance of the hemostatic balance in the venous circulation.The maintenance of blood fluidity and vascular integrity is actively maintained by collaborations between the vascular wall and the procoagulant and anticoagulant functions in blood. Cellbound and soluble constituents cooperate to maintain this quasiequilibrium fluid state. Upon vascular perforation, multiple interactions initiate a local coagulant response, promoting platelet adhesion/ aggregation, the generation of membrane-bound catalysts, thrombin generation, and fibrin deposition that overcome local anticoagulants. These processes arrest blood loss with dimensions relevant to the injury. Subsequently, fibrinolysis and cellular proliferation repair 2 the injured tissue.The coagulation processes have been studied in vitro using anticoagulated phlebotomy blood, 3 derived plasma, 4,5 isolated component systems, [6][7][8] and numerical simulations. 9,10 We have used corn-trypsin inhibitor (CTI) stabilized whole blood to study the tissue factor (Tf)-initiated process in the absence of chelating anticoagulants. 11,12 Several hundred of these experiments have been performed to characterize the temporal dynamics of various blood clotting reactions...

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Comparison of the inhibition of thrombin by three plasma protease inhibitors

Cited by 154 publications

References 27 publications

α2-Macroglobulin Is a Novel Substrate for ADAMTS-4 and ADAMTS-5 and Represents an Endogenous Inhibitor of These Enzymes

α2-Macroglobulin Is a Novel Substrate for ADAMTS-4 and ADAMTS-5 and Represents an Endogenous Inhibitor of These Enzymes

Regulation of factor Xa in vitro in human and mouse plasma and in vivo in mouse. Role of the endothelium and plasma proteinase inhibitors.

Cellular regulation of blood coagulation: a model for venous stasis

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