Substrate Specificity Of Thrombin Research Articles

Allosteric Modulation of Thrombin by Thrombomodulin: Insights from Logistic Regression and Statistical Analysis of Molecular Dynamics Simulations.

Thrombomodulin (TM), a transmembrane receptor integral to the anticoagulant pathway, governs thrombin's substrate specificity via interaction with thrombin's anion-binding exosite I. Despite its established role, the precise mechanisms underlying this regulatory function are yet to be fully unraveled. In this study, we deepen the understanding of these mechanisms through eight independent 1 μs all-atom simulations, analyzing thrombin both in its free form and when bound to TM fragments TM456 and TM56. Our investigations revealed distinct and significant conformational changes in thrombin mediated by the binding of TM56 and TM456. While TM56 predominantly influences motions within exosite I, TM456 orchestrates coordinated alterations across various loop regions, thereby unveiling a multifaceted modulatory role that extends beyond that of TM56. A highlight of our study is the identification of critical hydrogen bonds that undergo transformations during TM56 and TM456 binding, shedding light on the pivotal allosteric influence exerted by TM4 on thrombin's structural dynamics. This work offers a nuanced appreciation of TM's regulatory role in blood coagulation, paving the way for innovative approaches in the development of anticoagulant therapies and expanding the horizons in oncology therapeutics through a deeper understanding of molecular interactions in the coagulation pathway.

Targeting recombinant thrombomodulin fusion protein to red blood cells provides multifaceted thromboprophylaxis

AbstractThrombin generates fibrin and activates platelets and endothelium, causing thrombosis and inflammation. Endothelial thrombomodulin (TM) changes thrombin's substrate specificity toward cleavage of plasma protein C into activated protein C (APC), which opposes its thrombotic and inflammatory activities. Endogenous TM activity is suppressed in pathologic conditions, and antithrombotic interventions involving soluble TM are limited by rapid blood clearance. To overcome this problem, we fused TM with a single chain fragment (scFv) of an antibody targeted to red blood cells. scFv/TM catalyzes thrombin-mediated generation of activated protein C and binds to circulating RBCs without apparent damage, thereby prolonging its circulation time and bioavailability orders of magnitude compared with soluble TM. In animal models, a single dose of scFv/TM, but not soluble TM, prevents platelet activation and vascular occlusion by clots. Thus, scFv/TM serves as a prodrug and provides thromboprophylaxis at low doses (0.15 mg/kg) via multifaceted mechanisms inhibiting platelets and coagulation.

Thrombomodulin Links Coagulation to Inflammation and Immunity

Thrombomodulin (TM) is a type 1 membrane bound glycoprotein that has a C-type lectin domain at its Nterminus, 6 copies of the epidermal growth factor-like (EGF) motif and serine/threonine rich domain carrying a glycosoaminoglycan external to the membrane. TM binds thrombin changing thrombin's substrate specificity from procoagulant and pro-inflammatory to anti-coagulant and anti-inflammatory because of the activation of protein C (PC) and thrombin-activatable fibrinolysis inhibitor (TAFI). Thrombin's anion binding site 1 binds to TM's EGF domains 5 and 6. EGF4 is required for PC activation and EGF3 and 4 for TAFI activation in addition to EGF56. The X-ray structure of thrombin bound to TM has been solved and shows few major alterations in the active site of thrombin. The lectin domain can bind high mobility group box protein 1 (HMGB1) and a sugar, Lewis Y. TM's lectin domain behaves as an antagonist to HMGB1 endowing it with intrinsic anti-inflammatory activity. Treatment of dendritic cells with TM converts them from immunogenic to tolerogenic. TM is necessary for maintenance of pregnancy as well as prevention of coagulation throughout life. Soluble TM has been developed as an anticoagulant possessing favorable pharmacokinetics that has been approved for treatment of disseminated intravascular coagulation in Japan.

Mutagenesis Studies toward Understanding Allostery in Thrombin

The binding of thrombomodulin (TM) to exosite-1 and the binding of Na(+) to 225-loop allosterically modulate the catalytic activity and substrate specificity of thrombin. To determine whether the conformation of these two cofactor-binding loops are energetically linked to each other and to the active site, we rationally designed two thrombin mutants in which either the 70-80 loop of exosite-1 or the 225-loop of the Na(+)-binding site was stabilized by an engineered disulfide bond. This was possible by replacing two residues, Arg-67 and Ile-82, in the first mutant and two residues, Glu-217 and Lys-224, in the second mutant with Cys residues. These mutants were expressed in mammalian cells as monomeric molecules, purified to homogeneity and characterized with respect to their ability to bind TM and Na(+) by kinetic and direct binding approaches. The Cys-67/Cys-82 mutant did not bind TM and exhibited a normal amidolytic activity, however, the activity of Cys-217/Cys-224 was dramatically impaired, though TM interacted with this mutant with >20-fold elevated K(D) to partially restore its activity. Both mutants exhibited approximately 2-3-fold higher K(D) for interaction with Na(+), and neither mutant clotted fibrinogen or activated protein C in the presence of TM. Both mutants interacted with heparin with a normal affinity. These results suggest that, while exosite-2 of thrombin is an independent cofactor binding-site, both Na(+)-binding and exosite-1 are energetically linked. Further studies with the fluorescein labeled Cys-195 mutant of thrombin revealed that the catalytic residue of thrombin is modulated by Na(+), but TM has no effect on the conformation of this residue.

The effects of exosite occupancy on the substrate specificity of thrombin

Thrombin (EC 3.4.4.13) has two exosites that mediate interactions between the enzyme and its substrates and cofactors. The binding of ligands to the exosites alters the functions of the protease, for example, when the cofactor thrombomodulin binds to both exosites I and II, it converts the enzyme from a procoagulant to an anticoagulant factor. It is unknown whether ligand binding to a thrombin exosite will alter the substrate specificity of the enzyme and thus contribute to the changed substrate repertoire of the enzyme upon engagement with cofactors. We first examined whether binding of ligands to exosites I and II altered the activity of the enzyme against fluorogenic peptide substrates. The efficiency of cleavage of substrates by thrombin did change when thrombomodulin or hirugen was present, indicating that exosite I occupancy changed the active site of the protease. The presence of heparin did not change the activity of the enzyme, indicating that exosite II occupancy had little effect on active site function. Investigation of the effects of exosite I occupancy by hirugen on thrombin specificity using phage display substrate libraries revealed that the ligand only changed the specificity of the enzyme to a small degree. Occupancy of both exosites by thrombomodulin induced greater changes to the specificity of the enzyme, with the prime side showing broader changes in amino acid frequencies. Thus, exosite I ligands do affect the activity and specificity of thrombin, but not greatly enough to explain the altered substrate profile of the enzyme when complexed with thrombomodulin.

Thrombomodulin Tightens the Thrombin Active Site Loops To Promote Protein C Activation

Thrombomodulin (TM) forms a 1:1 complex with thrombin. Whereas thrombin alone cleaves fibrinogen to make the fibrin clot, the thrombin-TM complex cleaves protein C to initiate the anticoagulant pathway. Crystallographic investigations of the complex between thrombin and TMEGF456 did not show any changes in the thrombin active site. Therefore, research has focused recently on how TM may provide a docking site for the protein C substrate. Previous work, however, showed that when the thrombin active site was occupied with substrate analogues labeled with fluorophores, the fluorophores responded differently to active (TMEGF1-6) versus inactive (TMEGF56) fragments of TM. To investigate this further, we have carried out amide H/(2)H exchange experiments on thrombin in the presence of active (TMEGF45) and inactive (TMEGF56) fragments of TM. Both on-exchange and off-exchange experiments show changes in the thrombin active site loops, some of which are observed only when the active TM fragment is bound. These results are consistent with the previously observed fluorescence changes and point to a mechanism by which TM changes the thrombin substrate specificity in favor of protein C cleavage.

Proteolysis of Non-phosphorylated and Phosphorylated Tau by Thrombin

The microtubule-associated protein tau aggregates intracellularly by unknown mechanisms in Alzheimer's disease and other tauopathies. A contributing factor may be a failure to break down free cytosolic tau, thus creating a surplus for aggregation, although the proteases that degrade tau in brain remain unknown. To address this issue, we prepared cytosolic fractions from five normal human brains and from perfused rat brains and incubated them with or without protease inhibitors. D-Phenylalanyl-L-prolylarginyl chloromethyl ketone, a thrombin-specific inhibitor, prevented tau breakdown in these fractions, suggesting that thrombin is a brain protease that processes tau. We next exposed human recombinant tau to purified human thrombin and analyzed the fragments by N-terminal sequencing. We found that thrombin proteolyzed tau at multiple arginine and lysine sites. These include Arg(155)-Gly(156), Arg(209)-Ser(210), Arg(230)-Thr(231), Lys(257)-Ser(258), and Lys(340)-Ser(341) (numbering according to the longest human tau isoform). Temporally, the initial cleavage occurred at the Arg(155)-Gly(156) bond. Proteolysis of the resultant C-terminal tau fragment then proceeded bidirectionally. When tau was phosphorylated by glycogen synthase kinase-3beta, most of these proteolytic processes were inhibited, except for the first cleavage at the Arg(155)-Gly(156) bond. Furthermore, paired helical filament tau prepared from Alzheimer's disease brain was more resistant to thrombin proteolysis than following dephosphorylation by alkaline phosphatase. The results suggest a possible role for thrombin in proteolysis of tau under physiological and/or pathological conditions in human brains. They are consistent with the hypothesis that phosphorylation of tau inhibits proteolysis by thrombin or other endogenous proteases, leading to aggregation of tau into insoluble fibrils.

Binding of Exosite Ligands to Human Thrombin: RE-EVALUATION OF ALLOSTERIC LINKAGE BETWEEN THROMBIN EXOSITES I AND II

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. An array of fluorescent thrombin derivatives and fluorescein-labeled hirudin(54-65) ([5F]Hir(54-65)(SO(3)(-))) were used as probes in quantitative equilibrium binding studies to resolve whether the affinities of the exosite I-specific ligands, Hir(54-65)(SO(3)(-)) and fibrinogen, and of the exosite II-specific ligands, prothrombin fragment 2 and a monoclonal antibody, were affected by alternate exosite occupation. Hir(54-65)(SO(3)(-)) and fibrinogen bound to exosite I with dissociation constants of 16-28 nm and 5-7 microm, respectively, which were changed < or =2-fold by fragment 2 binding. Native thrombin and four thrombin derivatives labeled with different probes bound fragment 2 and the antibody with dissociation constants of 3-12 microm and 1.8 nm, respectively, unaffected by Hir(54-65)(SO(3)(-)). The results support a ternary complex binding model in which exosites I and II can be occupied simultaneously. The thrombin catalytic site senses individual and simultaneous binding of exosite I and II ligands differently, resulting in unique active site environments for each thrombin complex. The results indicate significant, ligand-specific allosteric coupling between thrombin exosites I and II and catalytic site perturbations but insignificant inter-exosite thermodynamic linkage.

Importance of the Arg-Gly-Asp Triplet in Human Thrombin for Maintenance of Structure and Function

Site-directed mutagenesis was employed to assess the importance of the Arg-Gly-Asp triplet that comprises residues 197 to 199 in the B-chain of thrombin. Properties of the R197E and the D199E variants were compared with those of ζ-thrombin and the inactive S205A variant wherein the active site Ser is replaced by Ala. Relative to ζ-thrombin, the R197E thrombin variant under the assay conditions used exhibits 26% activity toward a small chromogenic substrate, 13% activity in the activation of protein C in the presence of thrombomodulin, <3% activity in processing fibrinogen, and 1% activity in inducing platelet activation. Thus, the substrate specificity of thrombin was altered by the R197 → E replacement. The D199E variant was essentially inactive. It exhibited only 0.02% of the activity of thrombin toward the chromogenic substrate and its reactivity toward the active site-directed alkylating agent D-Phe-Pro-Arg-CH 2Cl was 10,000-fold lower than that of thrombin. Like the inactive S205A thrombin variant, the D199E variant antagonized the interactions of thrombin with hirudin and thrombomodulin, but was a less effective antagonist. The dependence of the antagonism of the thrombin-thrombomodulin interaction on the concentration of D199E thrombin variant provided evidence suggesting the presence of two or more domains in thrombin that independently interact with their counterparts in thrombomodulin. Although the S205A thrombin variant antagonized the action of thrombin on platelets no such activity could be demonstrated for the D199E variant in the concentration range studied (<800 nM). Comparison of the circular dichroism spectra of ζ-thrombin, the D199E, R197E, and S205A variants indicated that subtle differences in conformation exist between the Dl 99E variant and the other thrombins. These differences in conformation might well account for the altered behavior of the D199E variant with respect to its interactions toward thrombomodulin, hirudin, and platelets.

Formation of a ternary complex between thrombin, fibrin monomer, and heparin influences the action of thrombin on its substrates.

The consequences of the combined effects of fibrin II monomer (FnIIm) and heparin (H) on the hydrolysis of peptidyl p-nitroanilide substrates by thrombin (IIa), the cleavage of prothrombin by thrombin and the thrombin-catalyzed release of fibrinopeptides from fibrinogen have been studied at pH 7.4 and I 0.15. The effects of fibrin II monomer and heparin on chromogenic substrate hydrolysis can be described by a hyperbolic mixed inhibition model in which substrate can interact with four possible enzyme species (IIa, IIa.H, IIa.FnIIm, and IIa.FnIIm.H) that arise as a result of random formation of a ternary complex among thrombin, fibrin II monomer, and heparin (Hogg, P. J. and Jackson, C. M. (1990) J. Biol. Chem. 265, 241-247). The formation of the ternary IIa.FnIIm.H complex results in an increase in the Km values of 7.03 +/- 1.17-fold (1.37-9.65 microM) and 1.94 +/- 0.60-fold (38.1-73.9 microM) for H-D-Ile-Pro-Arg-pNA and Cbz-Gly-Pro-Arg-pNA hydrolysis, respectively, and a decrease in the kc values of 0.45 +/- 0.08-fold (49.5-22.3 s-1) and 0.52 +/- 0.05-fold (93.1-48.4 s-1). Fibrin II monomer and heparin in combination also decrease the efficiency (kc/Km) with which thrombin cleaves prothrombin to produce Fragment 1 and Prethrombin 1 by 2.3-fold from 607 +/- 30 to 264 +/- 13 M-1 s-1. In contrast to the effects of fibrin II monomer and heparin on thrombin hydrolysis of chromogenic substrates, its proteolysis of prothrombin and its inactivation by antithrombin III (Hogg, P. J., and Jackson, C. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3619-3623), these components have no discernible influence on the ability of thrombin to cleave fibrinogen. These observations indicate that the substrate specificity of thrombin is altered when it is bound in a complex with fibrin II monomer and heparin and suggest that the catalytic efficiency of thrombin for its physiological substrates will be affected differentially by these interactions. Such ternary complex formation involving thrombin, fibrin II monomer, and heparin may provide a mechanism for selectively regulating thrombin action.

Microthrombomodulin: Residues 310–486 from the epidermal growth factor precursor homology domain of thrombomodulin will accelerate protein C activation

Thrombomodulin is the endothelial cell cofactor for thrombin-catalyzed activation of protein C. Recently, we isolated a 10-kDa thrombin binding fragment, CB3, from the epidermal growth factor precursor homology domain (epidermal growth factor (EGF)-like regions) of thrombomodulin (Kurasawa, S., Stearns, D. J., Jackson, K.W., and Esmon, C.T. (1988) J. Biol. Chem. 263, 5993-5996). The CB3 fragment did not, however, support protein C activation. A 29-kDa fragment, called CB23, has now been isolated and corresponds to residues 310-486 in the EGF-like region of thrombomodulin. The CB23 fragment bound thrombin and accelerated thrombin-catalyzed protein C activation. With two separate preparations of CB23, the Km for protein C was 1.6 and 1.9 microM and the Kd for thrombin was 8.9 and 13.2 nM. The carboxyl terminus of CB23 and CB3 was identified by isolation and sequence analysis of a tryptic peptide from CB3. The sequence of this peptide corresponded to Asn457-Ser486, indicating that the carboxyl terminus of these fragments is 6 residues beyond the sixth EGF-like region of thrombomodulin. In addition, although CB3 cannot accelerate protein C activation, CB3 did inhibit the rate of thrombin-catalyzed fibrinopeptide release from fibrinogen. Thus, like native thrombomodulin, CB3 will alter thrombin's substrate specificity, but protein C activation requires additional information all of which can be provided by other regions of the EGF-like domain.

Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation.

Protein C is activated rapidly when thrombin binds to a specific cell surface cofactor protein, thrombomodulin. Studies were initiated to determine the influence of thrombin-thrombomodulin complex formation on the substrate specificity of thrombin. When thrombin binds to thrombomodulin, the resultant complex retains less than 1% of the fibrinogen clotting activity of free thrombin. Permanent alteration of the thrombin molecule is not involved since full clotting activity is regenerated by incubation of the complex with excess diisopropyl phosphothrombin. Unlike the activation of protein C by the thrombin-thrombomodulin complex which is dependent on Ca2+, inhibition of fibrinogen clotting activity is not dependent on the presence of divalent metal ions. Formation of the thrombin-thrombomodulin complex also inhibits thrombin activation of factor V. Despite these changes in macromolecular substate specificity, no significant change in the hydrolysis of the synthetic substrates p-tosyl-L-arginine methyl ester and N alpha-benzoyl-L-arginine ethyl ester is detected upon formation of the thrombin-thrombomodulin complex. Formation of this complex results in a slight increase in the Km (from 9.0 +/- 0.4 to 10.2 +/- 0.6 microM) and Vmax (from 230 +/- 10 to 270 +/- 10 mol/s/mol of thrombin) for the specific thrombin substrate H-D-Phe-Pip-Arg-p-nitroanilide. These studies suggest that thrombomodulin has two distinct anticoagulant functions: 1) to inhibit the ability of thrombin to clot fibrinogen and to activate factor V; and 2) to accelerate the formation of the anticoagulant, activated protein C.