GpIbα interacts exclusively with exosite II of thrombin.
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ABSTRACT:
SUBMITTER: Lechtenberg BC
PROVIDER: S-EPMC3919161 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Polyphosphate (a linear polymer of inorganic phosphate) is secreted from platelet dense granules, and we recently showed that it accelerates factor V activation by thrombin.To examine the interaction of polyphosphate with thrombin.Thrombin, but not prothrombin, altered the electrophoretic migration of polyphosphate in gel mobility assays. Thrombin binding to polyphosphate was influenced by ionic strength, and was evident even in plasma. Two positively charged exosites on thrombin mediate its interactions with other proteins and accessory molecules: exosite I (mainly with thrombin substrates), and exosite II (mainly with certain anionic polymers). Free thrombin, thrombin in complex with hirudin's C-terminal dodecapeptide and gamma-thrombin all bound polyphosphate similarly, excluding exosite I involvement. Mutations within exosite II, but not within exosite I, the Na(+)-binding site or hydrophobic pocket, weakened thrombin binding to polyphosphate as revealed by NaCl dependence. Surface plasmon resonance demonstrated tight interaction of polyphosphate with thrombin (K(d) approximately 5 nm) but reduced interaction with a thrombin exosite II mutant. Certain glycosaminoglycans, including heparin, only partially competed with polyphosphate for binding to thrombin, and polyphosphate did not reduce heparin-catalyzed inactivation of thrombin by antithrombin.Polyphosphate interacts with thrombin's exosite II at a site that partially overlaps with, but is not identical to, the heparin-binding site. Polyphosphate interactions with thrombin may be physiologically relevant, as the polyphosphate concentrations achievable following platelet activation are far above the approximately 5 nM K(d) for the polyphosphate-thrombin interaction.
| S-EPMC2856763 | biostudies-literature
Project description:Substrates and cofactors of the serine protease thrombin (IIa) employ two anion binding exosites (ABE-I and -II) to aid in binding. On the surface of platelets resides the GpIbalpha/beta-GpIX-GpV membrane-bound receptor complex. IIa's ABE-II is proposed to interact with an anionic portion of GpIbalpha which enhances IIa cleavage of PAR-1 and subsequent activation of platelets. In this work, one-dimensional (1D) and two-dimensional (2D) NMR, analytical ultracentrifugation (AUC), and hydrogen-deuterium exchange (HDX) coupled with MALDI-TOF MS were performed to further characterize the features of binding to IIa's ABEs. The described work builds upon investigations performed in a prior study with fibrin(ogen)'s gamma' peptide and IIa [Sabo, T. M., Farrell, D. H., and Maurer, M. C. (2006) Biochemistry 45, 7434-7445]. 1D line broadening NMR (1H and 31P) and 2D trNOESY NMR studies indicate that GpIbalpha residues D274-E285 interact extensively with the IIa surface in an extended conformation. AUC demonstrates that both GpIbalpha (269-286) and gamma' (410-427) peptides interact with IIa with a 1:1 stoichiometry. When the HDX results are compared to those for the ABE-I targeting peptide hirudin (54-65), the data imply that GpIbalpha (269-286), GpIbalpha (1-290), and gamma' (410-427) are indeed directed to ABE-II. The ABE-II binding fragments reduce HDX for sites distant from the interface, suggesting long-range conformational effects. These studies illustrate that GpIbalpha and gamma' target ABE-II with similar consequences on IIa dynamics, albeit with differing structural features.
| S-EPMC2842903 | biostudies-literature
Project description:Thrombin-induced platelet activation via PAR1 and PAR4 is an important event in haemostasis. Although the underlying mechanisms responsible for ensuring efficient PAR1 activation by thrombin have been extensively studied, the potential involvement of recognitions sites outside the active site of the protease in thrombin-induced PAR4 activation is largely unknown. In this study, we developed a new assay to assess the importance of exosite I and II for PAR4 activation with α - and γ-thrombin. Surprisingly, we found that exosite II is critical for activation of PAR4. We also show that this dependency on exosite II likely represents a new mechanism, as it is unaffected by blockage of the previously known interaction between thrombin and glycoprotein Ibα.
| S-EPMC6374991 | biostudies-literature
Project description:The serine proteinase alpha-thrombin plays a pivotal role in the regulation of blood fluidity, and therefore constitutes a primary target in the treatment of various haemostatic disorders. Haemadin is a slow tight- binding thrombin inhibitor from the land-living leech Haemadipsa sylvestris. Here we present the 3.1 A crystal structure of the human alpha-thrombin- haemadin complex. The N-terminal segment of haemadin binds to the active site of thrombin, forming a parallel beta-strand with residues Ser214-Gly216 of the proteinase. This mode of binding is similar to that observed in another leech-derived inhibitor, hirudin. In contrast to hirudin, however, the markedly acidic C-terminal peptide of haemadin does not bind the fibrinogen-recognition exosite, but interacts with the heparin-binding exosite of thrombin. Thus, haemadin binds to thrombin according to a novel mechanism, despite an overall structural similarity with hirudin. Haemadin inhibits both free and thrombomodulin-bound alpha-thrombin, but not intermediate activation forms such as meizothrombin. This specific anticoagulant ability of haemadin makes it an ideal candidate for an antithrombotic agent, as well as a starting point for the design of novel antithrombotics.
| S-EPMC305786 | biostudies-literature
Project description:Thrombin, derived from zymogen prothrombin (ProT), is a serine protease involved in procoagulation, anticoagulation, and platelet activation. Thrombin's actions are regulated through anion-binding exosites I and II (ABE I and ABE II) that undergo maturation during activation. Mature ABEs can utilize exosite-based communication to fulfill thrombin functions. However, the conformational basis behind such long-range communication and the resultant ligand binding affinities are not well understood. Protease activated receptors (PARs), involved in platelet activation and aggregation, are known to target thrombin ABE I. Unexpectedly, PAR3 (44-56) can already bind to pro-ABE I of ProT. Nuclear magnetic resonance (NMR) ligand-enzyme titrations were used to characterize how individual PAR1 (49-62) residues interact with pro-ABE I and mature ABE I. 1D proton line broadening studies demonstrated that binding affinities for native PAR1P (49-62, P54) and for the weak binding variant PAR1G (49-62, P54G) increased as ProT was converted to mature thrombin. 1H,15N-HSQC titrations revealed that PAR1G residues K51, E53, F55, D58, and E60 exhibited less affinity to pro-ABE I than comparable residues in PAR3G (44-56, P51G). Individual PAR1G residues then displayed tighter binding upon exosite maturation. Long-range communication between thrombin exosites was examined by saturating ABE II with phosphorylated GpIbα (269-282, 3Yp) and monitoring the binding of PAR1 and PAR3 peptides to ABE I. Individual PAR residues exhibited increased affinities in this dual-ligand environment supporting the presence of interexosite allostery. Exosite maturation and beneficial long-range allostery are proposed to help stabilize an ABE I conformation that can effectively bind PAR ligands.
| S-EPMC6716794 | biostudies-literature
Project description:Thrombin is a serine protease of the chymotrypsin family that acts both as a procoagulant and as an anticoagulant by cleaving either factor VIII, factor V and fibrinogen or protein C, respectively. Numerous previous studies have shown that electropositive regions at a distance from the active site, so called exosites, are of major importance for the cleavage by human thrombin. Upstream of all the known major cleavage sites for thrombin in factor VIII, factor V and fibrinogen are clusters of negatively charged amino acids. To study the importance of these sites for the interaction with the exosites and thereby the cleavage by thrombin, we have developed a new type of recombinant substrate. We have compared the cleavage rate of the minimal cleavage site, involving only 8-9 amino acids (typically the P4-P4' positions) surrounding the cleavage site, with the substrates also containing the negatively charged regions upstream of the cleavage sites. The results showed that addition of these regions enhanced the cleavage rate by more than fifty fold. However, the enhancement was highly dependent on the sequence of the actual cleavage site. A minimal site that showed poor activity by itself could be cleaved as efficiently as an optimal cleavage site when presented together with these negatively charged regions. Whereas sites conforming closely to the optimal site were only minimally enhanced by the addition of these regions. The possibility to mimic this interaction for the sites in factor V and factor VIII by recombinant substrates, which do not have the same folding as the full size target, indicates that the enhancement was primarily dependent on a relatively simple electrostatic interaction. However, the situation was very different for fibrinogen and protein C where other factors than only charge is of major importance.
| S-EPMC4482499 | biostudies-literature
Project description:BACKGROUND: Heparin cofactor II (HCII) is a circulating protease inhibitor, one which contains an N-terminal acidic extension (HCII 1-75) unique within the serpin superfamily. Deletion of HCII 1-75 greatly reduces the ability of glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, and abrogates HCII binding to thrombin exosite 1. While a minor portion of HCII 1-75 can be visualized in a crystallized HCII-thrombin S195A complex, the role of the rest of the extension is not well understood and the affinity of the HCII 1-75 interaction has not been quantitatively characterized. To address these issues, we expressed HCII 1-75 as a small, N-terminally hexahistidine-tagged polypeptide in E. coli. RESULTS: Immobilized purified HCII 1-75 bound active ?-thrombin and active-site inhibited FPR-ck- or S195A-thrombin, but not exosite-1-disrupted ?T-thrombin, in microtiter plate assays. Biotinylated HCII 1-75 immobilized on streptavidin chips bound ?-thrombin and FPR-ck-thrombin with similar KD values of 330-340 nM. HCII 1-75 competed thrombin binding to chip-immobilized HCII 1-75 more effectively than HCII 54-75 but less effectively than the C-terminal dodecapeptide of hirudin (mean Ki values of 2.6, 8.5, and 0.29 ?M, respectively). This superiority over HCII 54-75 was also demonstrated in plasma clotting assays and in competing the heparin-catalysed inhibition of thrombin by plasma-derived HCII; HCII 1-53 had no effect in either assay. Molecular modelling of HCII 1-75 correctly predicted those portions of the acidic extension that had been previously visualized in crystal structures, and suggested that an ?-helix found between residues 26 and 36 stabilizes one found between residues 61-67. The latter region has been previously shown by deletion mutagenesis and crystallography to play a crucial role in the binding of HCII to thrombin exosite 1. CONCLUSIONS: Assuming that the KD value for HCII 1-75 of 330-340 nM faithfully predicts that of this region in intact HCII, and that 1-75 binding to exosite 1 is GAG-dependent, our results support a model in which thrombin first binds to GAGs, followed by HCII addition to the ternary complex and release of HCII 1-75 for exosite 1 binding and serpin mechanism inhibition. They further suggest that, in isolated or transferred form, the entire HCII 1-75 region is required to ensure maximal binding of thrombin exosite 1.
| S-EPMC3601010 | biostudies-literature
Project description:Thrombin participates in procoagulation, anticoagulation, and platelet activation. This enzyme contains anion binding exosites, ABE I and ABE II, which attract regulatory biomolecules. As prothrombin is activated to thrombin, pro-ABE I is converted into mature ABE I. Unexpectedly, certain ligands can bind to pro-ABE I specifically. Moreover, knowledge of changes in conformation and affinity that occur at the individual residue level as pro-ABE I is converted to ABE I is lacking. Such changes are transient and were not captured by crystallography. Therefore, we employed nuclear magnetic resonance (NMR) titrations to monitor development of ABE I using peptides based on protease-activated receptor 3 (PAR3). Proton line broadening NMR revealed that PAR3 (44-56) and more weakly binding PAR3G (44-56) could already interact with pro-ABE I on prothrombin. 1H-15N heteronuclear single-quantum coherence NMR titrations were then used to probe binding of individual 15N-labeled PAR3G residues (F47, E48, L52, and D54). PAR3G E48 and D54 could interact electrostatically with prothrombin and tightened upon thrombin maturation. The higher affinity for PAR3G D54 suggests the region surrounding thrombin R77a is better oriented to bind D54 than the interaction between PAR3G E48 and thrombin R75. Aromatic PAR3G F47 and aliphatic L52 both reported on significant changes in the chemical environment upon conversion of prothrombin to thrombin. The ABE I region surrounding the 30s loop was more affected than the hydrophobic pocket (F34, L65, and I82). Our NMR titrations demonstrate that PAR3 residues document structural rearrangements occurring during exosite maturation that are missed by reported X-ray crystal structures.
| S-EPMC6076386 | biostudies-literature
Project description:Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.
| S-EPMC3107735 | biostudies-literature
Project description:INTRODUCTION:Thrombin is a primary target of most anticoagulants. Yet, thrombin's dual and opposing role in pro- as well as anti- coagulant processes imposes considerable challenges in discovering finely tuned regulators that maintain homeostasis, rather than disproportionately changing the equilibrium to one side. In this connection, we have been studying exosite 2-mediated allosteric modulation of thrombin activity using synthetic agents called low molecular weight lignins (LMWLs). Although the aromatic scaffold of LMWLs is completely different from the polysaccharidic scaffold of heparin, the presence of multiple negatively charged groups on both ligands induces binding to exosite 2 of thrombin. This work characterizes the nature of interactions between LMWLs and thrombin to understand the energetic cooperativity between exosite 2 and active site of thrombin. MATERIALS AND METHODS:The thermodynamics of thrombin-LMWL complexes was studied using spectrofluorimetric titrations as a function of ionic strength and temperature of the buffer. The contributions of enthalpy and entropy to binding were evaluated using classic thermodynamic equations. Label-free surface plasmon resonance was used to assess the role of sodium ion in LMWL binding to thrombin at a fixed ionic strength. RESULTS AND CONCLUSIONS:Exosite 2-induced conformational change in thrombin's active site is strongly dependent on the structure of the ligand, which has consequences with respect to regulation of thrombin. The ionic and non-ionic contributions to binding affinity and the thermodynamic signature were highly ligand specific. Interestingly, LMWLs display preference for the sodium-bound form of thrombin, which supports the existence of an energetic coupling between exosite 2 and sodium-binding site of thrombin.
| S-EPMC5943173 | biostudies-literature