The Asp272–Glu282 Region of Platelet Glycoprotein Ibα Interacts with the Heparin-binding Site of α-Thrombin and Protects the Enzyme from the Heparin-catalyzed Inhibition by Antithrombin III

Cristofaro, Raimondo De; Candia, Erica De; Rutella, Sergio; Weitz, Jeffrey I.

doi:10.1074/jbc.275.6.3887

Cited by 66 publications

(81 citation statements)

References 51 publications

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“…Our findings confirm the role of exosite II (29)(30)(31)(32)(33)(34) and define that of exosite I (35), proving its necessary involvement in binding to immobilized but not soluble receptor. Although hirugen and HD1, exosite I ligands, lack inhibitory effect on FIIa interaction with GPIbα fragments linked to agarose (31) or plastic (30), we found that both inhibit binding to GPIbα fragments linked to immobilized antibodies and to membraneanchored platelet GPIb, the biologically relevant receptor.…”

Section: Discussionsupporting

confidence: 85%

“…For example, competition for exosite I that mediates binding to many substrates may limit proteolytic activity; in the case of fibrinogen, generation of the fibrin clot would be reduced and/or delayed with increasing FIIa binding to platelets. Blockade of exosite II, in turn, could affect factor VIII activation (39), with anticoagulant effects, but also protect from inhibition by serpins (30). It remains to be explored how these GPIb-dependent events may change the local balance of FIIa procoagulant and anticoagulant functions and influence hemostasis and thrombosis.…”

Section: Discussionmentioning

confidence: 99%

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Binding of α-thrombin to surface-anchored platelet glycoprotein Ibα sulfotyrosines through a two-site mechanism involving exosite I

Zarpellon

Celikel

Roberts

et al. 2011

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

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The involvement of exosite I in α-thrombin (FIIa) binding to platelet glycoprotein Ibα (GPIbα), which could influence interactions with other substrates, remains undefined. To address the problem, we generated the GPIbα amino terminal domain (GPIbα-N) fully sulfated on three tyrosine residues and solved the structure of its complex with FIIa. We found that sulfotyrosine (Tys) 278 enhances the interaction mainly by establishing contacts with exosite I. We then evaluated how substituting tyrosine with phenylalanine, which cannot be sulfated, affects FIIa binding to soluble or surface-immobilized GPIbα-N. Mutating Tyr 276 , which mostly contacts exosite II residues, markedly reduced FIIa interaction with both soluble and immobilized GPIbα-N; mutating Tyr 278 or Tyr 279 , which mostly contact exosite I residues, reduced FIIa complexing in solution by 0-20% but affinity for immobilized GPIbα-N 2 to 6-fold, respectively. Moreover, three exosite I ligands-aptamer HD1, hirugen, and lepirudin-did not interfere with soluble FIIa complexing to GPIbα-N, excluding that their binding caused allosteric effects influencing the interaction; nonetheless, all impaired FIIa binding to immobilized GPIbα-N and platelet GPIb nearly as much as aptamer HD22 and heparin, both exosite II ligands. Bound HD1 and hirugen alter Trp 148 orientation in a loop near exosite I preventing contacts with the sulfate oxygen atoms of Tys 279 . These results support a mechanism in which binding occurs when the two exosites of one FIIa molecule independently interact with two immobilized GPIbα molecules. Through exosite engagement, GPIbα may influence FIIa-dependent processes relevant to hemostasis and thrombosis.tyrosine sulfation | tyrosylprotein sulfotransferase-2 | protease-activated receptor | platelet activation | platelet aggregation

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Section: Discussionsupporting

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Binding of α-thrombin to surface-anchored platelet glycoprotein Ibα sulfotyrosines through a two-site mechanism involving exosite I

Zarpellon

Celikel

Roberts

et al. 2011

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

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“…Purification of platelet GpIb␣-(1-282) fragment was performed as detailed previously (20). Thrombin (20 nM to 1.28 M) was incubated in the presence of both 408 -427 ␥Ј peptide and fragment D* at fixed concentrations spanning from 10 to 320 M and from 0.2 and 3.2 M, respectively.…”

Section: Synthesis Of Fibrinogenmentioning

confidence: 99%

“…The D domains contain a ␥ chain variant, termed ␥Ј, arising from an alternative mRNA splicing (4), resulting in an elongated chain composed of 427 instead of 411 residues. The inserted region at the C terminus is composed of 20 (5). The elongated ␥ chain, termed ␥Ј, mainly heterodimerizes in the fibrinogen molecule with the more abundant ␥A chain, thus generating the ␥A/␥Ј dimers (6).…”

mentioning

confidence: 99%

Fibrinogen-elongated γ Chain Inhibits Thrombin-induced Platelet Response, Hindering the Interaction with Different Receptors

Lancellotti

Rutella

Filippis

et al. 2008

Journal of Biological Chemistry

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The expression of the elongated fibrinogen ␥ chain, termed ␥, derives from alternative splicing of mRNA and causes an insertion sequence of 20 amino acids. This insertion domain interacts with the anion-binding exosite (ABE)-II of thrombin. This study investigated whether and how ␥ chain binding to ABE-II affects thrombin interaction with its platelet receptors, i.e. glycoprotein Ib␣ (GpIb␣), protease-activated receptor (PAR) 1, and PAR4. Both synthetic ␥ peptide and fibrinogen fragment D*, containing the elongated ␥ chain, inhibited thrombin-induced platelet aggregation up to 70%, with IC 50 values of 42 ؎ 3.5 and 0.47 ؎ 0.03 M, respectively. Solid-phase binding and spectrofluorimetric assays showed that both fragment D* and the synthetic ␥ peptide specifically bind to thrombin ABE-II and competitively inhibit the thrombin binding to GpIb␣ with a mean K i ≈ 0.5 and ≈35 M, respectively. Both these ␥ chain-containing ligands allosterically inhibited thrombin cleavage of a synthetic PAR1 peptide, of native PAR1 molecules on intact platelets, and of the synthetic chromogenic peptide D-Phe-pipecolyl-Arg-p-nitroanilide. PAR4 cleavage was unaffected. In summary, fibrinogen ␥ chain binds with high affinity to thrombin and inhibits with combined mechanisms the platelet response to thrombin. Thus, its variations in vivo may affect the hemostatic balance in arterial circulation.Fibrinogen is a key molecule in both primary and secondary hemostasis, because of its role in forming the platelet plug by connecting activated platelets and in forming plasma fibrin clot upon thrombin cleavage. Fibrinogen consists of two symmetric half-molecules, each containing a set of three different polypeptide chains termed A␣, B␤, and ␥. The latter contains several sites that interact with different ligands such as other fibrin(ogen) molecules, coagulation enzymes, growth factors, and integrins (1). The product of thrombin digestion of fibrinogen, i.e. fibrin, binds with a considerable specificity thrombin, so that in the early studies fibrin was termed antithrombin I (2). Thrombin has a divalent interaction with two classes of binding sites on fibrin, one of low affinity in the E domain and the other of high affinity in the D domain of fibrin(ogen) molecules (3). Binding of thrombin to fibrinogen involves sequences of both A␣ and B␤ chain, which contain recognition sites in the fibrinogen E domain. These recognition sites are still able to interact with thrombin after cleavage of fibrinopeptide A and B and form the low affinity binding site for the enzyme. The D domains contain a ␥ chain variant, termed ␥Ј, arising from an alternative mRNA splicing (4), resulting in an elongated chain composed of 427 instead of 411 residues. The inserted region at the C terminus is composed of 20 amino acids ( 408 VRPEH-PAETEYDSLYPEDDL 427 ), rich of acidic side chains and two sulfate anions linked to Tyr 418 and Tyr 422 (5). The elongated ␥ chain, termed ␥Ј, mainly heterodimerizes in the fibrinogen molecule with the more abundant ␥A chain, thus generating ...

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“…Exosite II binds heparin and other glycosaminoglycans (2,12,13), prothrombin activation fragment 2 (F2) (14), the chondroitin sulfate moiety of thrombomodulin (15,16), the leech peptide hemadin (17), and an exosite II-specific human monoclonal antibody (18). Factors V (19 -22), Va (21,22), and VIII (19), platelet glycoprotein Ib␣ (23)(24)(25), and the snake venom protein bothrojaracin (26) have been reported to interact with both exosites I and II.…”

mentioning

confidence: 99%

Binding of Exosite Ligands to Human Thrombin

Verhamme¹,

Olson²,

Tollefsen³

et al. 2002

Journal of Biological Chemistry

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The substrate specificity of thrombin is regulated by binding of macromolecular substrates and effectors to exosites I and II. Exosites I and II have been reported to be extremely linked allosterically, such that binding of a ligand to one exosite results in near-total loss of affinity for ligands at the alternative exosite, whereas other studies support the independence of the interactions. The specificity of thrombin toward its procoagulant and anticoagulant physiological substrates is allosterically regulated by interactions of macromolecular substrates, inhibitors, and effectors with either of two electropositive sites, exosites I and II, in near-opposition on the enzyme surface (1, 2). Exosite I binds fibrinogen (Fbg) 1 (3), fibrin I and II (3, 4), the 12-residue carboxyl-terminal hirudin 54 -65 sequence (5, 6), thrombomodulin (7), the thrombin receptor (8, 9), and an acidic sequence on the serpin, heparin cofactor II (10, 11). Exosite II binds heparin and other glycosaminoglycans (2, 12, 13), prothrombin activation fragment 2 (F2) (14), the chondroitin sulfate moiety of thrombomodulin (15, 16), the leech peptide hemadin (17), and an exosite II-specific human monoclonal antibody (18). Factors V (19 -22), Va (21,22), and VIII (19), platelet glycoprotein Ib␣ (23-25), and the snake venom protein bothrojaracin (26) have been reported to interact with both exosites I and II.Binding of exosite ligands to thrombin is correlated with significant changes in the kinetics of hydrolysis of peptide ester and peptide p-nitroanilide substrates (3,7,9,18,(27)(28)(29)(30)(31) in addition to profound effects on specificity and reactivity toward its natural macromolecular substrates and inhibitors (10,11,15,(32)(33)(34)(35)(36). These studies indicate that exosite binding of allosteric effectors is coupled to conformational changes affecting the S1-S3 substrate specificity subsites in the thrombin catalytic site (37-39). Binding studies of F2, thrombomodulin, fibrin, and heparin with various active site-labeled thrombin derivatives in which the S1-S4 subsites were occupied (16,34,40), and studies of the effect of exosite ligand binding on the hydrolysis of tripeptide p-nitroanilide substrates suggest that structurally different ligands produce ligand-specific changes in the catalytic site. Extreme allosteric linkage between exosites I and II (30,31,41) has been reported to prevent simultaneous occupation of exosites I and II (30,41), whereas other studies provide contrasting evidence for binary and ternary complex formation with similar affinities among thrombin and exosite I and II ligands (18). In favor of inter-exosite linkage, the dissociation constant for fluorescently labeled, Tyr 63 -sulfated hirudin [53][54][55][56][57][58][59][60][61][62][63][64] and bovine thrombin was weakened 10-fold by F2 binding, although ternary complex formation was demonstrated (31). Extremely negative interexosite interactions were reported for Tyr 63 -sulfated hirudin 54 -65 (Hir 54 -65 (SO 3 Ϫ )) and human F2 or a synthetic peptide...

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The Asp272–Glu282 Region of Platelet Glycoprotein Ibα Interacts with the Heparin-binding Site of α-Thrombin and Protects the Enzyme from the Heparin-catalyzed Inhibition by Antithrombin III

Cited by 66 publications

References 51 publications

Binding of α-thrombin to surface-anchored platelet glycoprotein Ibα sulfotyrosines through a two-site mechanism involving exosite I

Binding of α-thrombin to surface-anchored platelet glycoprotein Ibα sulfotyrosines through a two-site mechanism involving exosite I

Fibrinogen-elongated γ Chain Inhibits Thrombin-induced Platelet Response, Hindering the Interaction with Different Receptors

Binding of Exosite Ligands to Human Thrombin

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