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PubMed1993

A player of many parts: the spotlight falls on thrombin's structure.

The wealth of structural information now available for thrombin, its precursors, its substrates, and its inhibitors allows a rationalization of its many roles. alpha-thrombin is a rather rigid molecule, binding to its target molecules with little conformational change. Comparison of alpha-thrombin with related trypsin-like serine proteinases reveals an unusually deep and narrow active site cleft, formed by loop insertions characteristic of thrombin. This canyon structure is one of the prime causes for the narrow specificity of thrombin. The observed modularity of thrombin allows a diversity in this specificity; its "mix-and-match" nature is exemplified by its interactions with macromolecules (Fig. 20). The apposition of the active site to a hydrophobic pocket (the apolar binding site) on one side and a basic patch (the fibrinogen recognition exosite) on the other allows for a fine tuning of enzymatic activity, as seen for fibrinogen. Thrombin receptor appears to use the same sites, but in a different way. Protein C seems only able to interact with thrombin if the recognition exosite is occupied by thrombomodulin. These two sites are also optimally used by hirudin, allowing the very tight binding observed; thrombin inhibition is effected by blocking access to the active site. On the other hand, antithrombin III makes little use of the recognition exosite; instead, its interactions are tightened with the help of heparin, which binds to a second basic site (the heparin binding site). Thrombin's modularity is a result of the conjunction of amino acid residues of like properties, such as charge or hydrophobicity. The charge distribution plays a role, not only in the binding of oppositely charged moieties of interacting molecules, but also in selection and preorientation of them. Nonproteolytic cellular properties are attributed to 1) the rigid insertion loop at Tyr60A, and 2) a partially inaccessible RGD sequence. The former can interact with cells in the native form; the latter would appear to be presented only in an (at least partially) unfolded state. The membrane binding properties of prothrombin can be understood from the ordered arrangement of calcium ions on binding to the Gla domain. Kringle F2 binds to thrombin at the heparin binding site through charge complementarity; a conformational change appears to occur on binding. The observed rigidity of the thrombin molecule in its complexes makes thrombin ideal for structure based drug design. Thrombin can be inhibited either at the active site or at the fibrinogen recognition exosite, or both.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2001

Activation of p44/42 mitogen activated protein kinases in thrombin-induced brain tolerance.

BACKGROUND: Our recent studies have shown that prior intracerebral injection of a low dose of thrombin attenuates the brain edema formation that results from either an intracerebral hematoma, an intracerebral injection of a large dose of thrombin or cerebral ischemia. The aim of the current study is to investigate whether thrombin-induced tolerance (thrombin preconditioning; TPC) is associated with activation of p44/42 mitogen activated protein (MAP) kinases. METHODS: This study contained three parts. In the first, rats received an intracerebral infusion of either saline or one unit thrombin (the TPC dose) into the right caudate nucleus. After 1, 3 and 7 days, the rats will be killed and brains used to detect p44/42 MAP kinases activation using Western blot analysis and immunohistochemistry. In the second and third parts, rats received intracerebral infusions of either vehicle, one unit thrombin (TPC) or one unit thrombin and 5 nmol PD 098059. These rats were either killed to detect kinases activation after 24 h or received a second intracerebral infusion of five-unit thrombin 7 days later with brain edema being assessed after a further 24 h. RESULTS: Western blot analysis demonstrated that p44/42 MAP kinases were activated in the ipsilateral basal ganglia after the intracerebral infusion of thrombin one unit. Cells immunoreactive for activated p44/42 MAP kinases were found in the ipsilateral basal ganglia and ipsilateral cortex. PD 098059, a MAP kinase kinase inhibitor, abolished thrombin-induced activation of p44/42 MAP kinases. TPC suppressed thrombin-induced brain edema while PD 098059 blocked this protective effect. The water contents in the ipsilateral basal ganglia 24 h after infusion of thrombin five units were 82.6+/-0.8%, 79.2+/-0.4% and 81.8+/-1.9% in the control, TPC alone and TPC plus PD 098059 groups, respectively. CONCLUSION: Thrombin can activate p44/42 MAP kinases within the brain and the protective effects of thrombin preconditioning on brain edema formation are related to this activation.

Animals↗
PubMed1997

Transfected adenosine A1 receptor-mediated modulation of thrombin-stimulated phospholipase C and phospholipase A2 activity in CHO cells.

Thrombin receptor activation in Chinese hamster ovary (CHO) cells stimulates the hydrolysis of inositol phospholipids and the release of arachidonic acid. Our previous studies have shown that activation of the human transfected adenosine A1 receptor in CHO cells (CHO-A1) potentiates the accumulation of inositol phosphates elicited by endogenous P2U purinoceptors and CCKA receptors. In this study we have investigated whether adenosine A1 receptor activation can modulate thrombin-stimulated arachidonic acid release and/or inositol phospholipid hydrolysis in CHO-A1 cells. Thrombin stimulated [3H]arachidonic acid release and total [3H]inositol phosphate accumulation in CHO-A1 cells. Both these responses to thrombin were were insensitive to pertussis toxin. The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), potentiated thrombin-stimulated [3H]arachidonic acid. In marked contrast, PMA inhibited thrombin-stimulated [3H]inositol phosphate accumulation. The selective protein kinase C inhibitor Ro 31-8220 (3-¿1-[3-(2-isothioureido)propyl] indol-3-yl¿-4-(1-methylindol-3-yl)-3-pyrrolin-2,5-dione) had no effect on thrombin-stimulated [3H]arachidonic acid release but reversed the potentiation of thrombin-stimulated [3H]arachidonic acid release elicited by PMA. The selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) augmented the release of [3H]arachidonic acid produced by thrombin. Co-activation of the adenosine A1 receptor also potentiated thrombin-stimulated [3H]inositol phosphate accumulation. The synergistic interactions between the adenosine A1 receptor and thrombin were abolished in pertussis-toxin-treated cells. The potentiation of [3H]arachidonic acid release by CPA was blocked by the protein kinase C inhibitors Ro 31-8220 and GF 109203X (3-[1-[3-(dimethylamino)propyl]-1 H-indol-3-yl]-4-(1 H-indol-3-yl)- 1H-pyrrole-2,5-dione). In conclusion, thrombin receptor activation in CHO-A1 cells stimulates the accumulation of [3H]inositol phosphates and the release of [3H]arachidonic acid through pertussis-toxin-insensitive G-proteins. Experiments using PMA suggest that protein kinase C differentially regulates thrombin receptor activation of phospholipase C and phospholipase A2. Co-activation of the transfected human adenosine A1 receptor augments thrombin-stimulated phospholipase C and phospholipase A2 activity. Finally, the augmentation of phospholipase A2 activity by the adenosine A1 receptor is inhibited by selective protein kinase C inhibitors, suggesting the involvement of protein kinase C.

Animals↗
PubMed1999

Synergy between thrombin and serotonin in inducing vascular smooth muscle cell proliferation.

Previous studies have indicated that apart from playing an important role in hemostasis and thrombosis, thrombin may also contribute to the development of postangioplasty restenosis caused by the stimulation of vascular smooth muscle cell (VSMC) proliferation. Because thrombin generation in vivo is accompanied by platelet activation and release of smooth muscle cell (SMC) growth factors such as serotonin, we examined the possible interaction between these two compounds on VSMC proliferation. Thrombin (0.01 to 100 nmol/L), thrombin receptor-activating peptide (0.1 to 1000 micromol/L), and serotonin (5HT; 0.1 to 1000 micromol/L) increased tritiated thymidine incorporation into the DNA of canine aortic VSMCs in a dose-dependent manner. When thrombin and 5HT were added together at sub-threshold concentrations, they acted synergistically in inducing tritiated thymidine incorporation. These findings were paralleled by a 90%+/-5% increase in the cell number at 48 hours, as compared with a 37%+/-2% increase with 50 micromol/L serotonin and a 13%+/-3% increase with 0.1 nmol/L thrombin. We also demonstrated that a brief exposure to thrombin (1 hour) is sufficient to show its potentiating effect on serotonin. The mitogenic effect of serotonin and its synergistic interaction with thrombin on VSMC proliferation was abolished by serotonin type 2 receptor antagonist LY281067. Similarly, gamma-hirudin--a direct thrombin inhibitor--blocked the mitogenic effect of thrombin and its synergistic interaction with serotonin. When LY281067 and gamma-hirudin were used together, they abolished the mitogenic effects of both the agonists. Because clot-bound active thrombin can escape inactivation by anti-thrombin, this thrombin may potentiate the mitogenic effect of serotonin and keep the SMCs in a proliferative state for a long period of time. These findings support the use of 5HT2 receptor antagonists in combination with thrombin inhibitors in the prevention of SMC proliferation after coronary angioplasty.

Animals↗
PubMed2002

Inhibition of a thrombin anion-binding exosite-2 mutant by the glycosaminoglycan-dependent serpins protein C inhibitor and heparin cofactor II.

Antithrombin (ATIII), heparin cofactor II (HCII) and protein C inhibitor (PCI; also named plasminogen activator inhibitor-3) are serine protease inhibitors (serpins) whose thrombin inhibition activity is accelerated in the presence of glycosaminoglycans. We compared the inhibition properties of PCI and HCII to ATIII using R93A/R97A/R101A thrombin, an anion-binding exosite-2 (exosite-2) mutant that has greatly reduced heparin-binding properties. Heparin-enhanced PCI inhibition of R93A/R97A/R101A thrombin was only approximately 2-fold compared to 40-fold enhancement with wild-type recombinant thrombin. Thrombomodulin (TM) (with or without the chondroitin sulfate moiety) accelerated PCI inhibition of both wild-type and R93A/R97A/R101A thrombins. HCII achieved the same maximum activity in the presence of heparin with both wild-type and R93A/R97A/R101A thrombins; however, the optimum heparin concentration was 20 times greater than the reaction with wild-type thrombin, indicative of a decrease in heparin affinity. Dermatan sulfate (DSO4)-catalyzed HCII thrombin inhibition was unchanged in R93A/R97A/R101A thrombin compared to wild-type recombinant thrombin. These results suggest that PCI is similar to ATIII and depends upon ternary complex formation with heparin and these specific thrombin exosite-2 residues to accelerate thrombin inhibition. In contrast, HCII does not require Arg(93), Arg(97) and Arg(101) of thrombin exosite-2 and further supports the hypothesis that HCII uses an allosteric process following glycosaminoglycan binding to inhibit thrombin.

Allosteric Regulation↗
PubMed1982

Progressive development of a thrombin inhibitor binding site.

The studies reported here were undertaken to determine whether the thrombin precursors prothrombin, prethrombin 1, prethrombin 2, and Meizo thrombin interact with the fluorescent, reversible thrombin inhibitor dansylarginine N,N-(3-ethyl-1,5-pentanediyl)amide (DAPA) [Nesheim, M. E., Prendergast, F. G., & Mann, K. G. (1979) Biochemistry 18, 996--1003]. The results indicate that prothrombin and prethrombin 1, in which the cleavage sites at Arg274-Thr275 and Arg323-Ile324 both remain intact, do not bind DAPA, while prethrombin 2 or Meizo thrombin, which results respectively from a single cleavage of prothrombin at Arg274-Thr275 or Arg323-Ile324, do bind the inhibitor. Since prethrombin 2 is a precursor of thrombin without measurable enzymatic activity, a thorough characterization of its interaction with DAPA was undertaken. The interaction of DAPA with bovine thrombin similarly was studied for comparative purposes. The binding of DAPA to either protein is accompanied by changes in the fluorescence properties of the dansyl moiety including increases in emission intensity, excited-state lifetime, polarization, and a slight blue shift in the wavelength of maximum emission intensity. Corrected excitation spectra indicate energy transfer to DAPA from one or more aromatic side chains of both proteins. Values of P0 for both complexes were extrapolated from Perrin plots of polarization vs. temperature and suggest that the dansyl moiety is held more rigidly in thrombin than in prethrombin 2. With excitation at either 280 or 335 nm the emission intensity of DAPA-prethrombin 2 is substantially less than that of the DAPA-thrombin complex. In contrast, the intensity of the Meizo thrombin-DAPA complex is greater than that of the DAPA-thrombin complex. From measurements of intensity changes the dissociation constants and stoichiometry of DAPA binding to thrombin and prethrombin 2 were measured. Prethrombin 2 binds to DAPA with a Kd = 5.9 x 10(-7) M (n = 1) while thrombin binds about 30 times more tightly with a Kd = 2.0 x 10(-8) M (n = 1). The active site directed irreversible thrombin inhibitors diisopropyl phosphorofluoridate and D-phenylalanylprolylarginyl chloromethyl ketone displace DAPA from thrombin but not from prethrombin 2. The results of these studies indicate the binding of a presumed substrate analogue (DAPA) to an inactive zymogen, prethrombin 2. In addition, the lack of DAPA binding to prothrombin and prethrombin 1, under conditions in which it binds to prethrombin 2, implicates events that accompany cleavage at Arg274-Arg275 in the "progressive" formation of an active site, even though further cleavage at Arg323-Ile324 is required for expression of enzymatic activity.

Animals↗
PubMed1998

Binding of human alpha-thrombin to platelet GpIb: energetics and functional effects.

Thrombin interaction with platelet glycocalicin (GC), the 140 kDa extracytoplasmic fragment of the membrane glycoprotein Ib, was investigated by using a solid-phase assay. Thrombin bound to GC-coated polystyrene wells was detected by measuring the hydrolysis of a chromogenic substrate. The monoclonal antibody LJ-Ib10, which specifically binds to the thrombin-binding site of GC, could displace thrombin from immobilized GC, whereas the monoclonal antibody LJ-Ib1, which interacts with the von Willebrand factor-binding domain of GC, did not affect thrombin binding to GC. Competitive inhibition of thrombin binding to immobilized GC was also observed using GC in solution or ligands that bind to the thrombin heparin-binding site, such as heparin and prothrombin fragment 2. Furthermore functional experiments demonstrated that GC binding to thrombin competes with heparin for thrombin inactivation by the antithrombin III-heparin complex as well. Thrombin-GC interaction was also studied as a function of temperature over the range 4-37 degreesC. A large negative heat capacity change (DeltaCp), of -4.14+/-0.8 kJ.mol-1.K-1, was demonstrated to dominate the thermodynamics of thrombin-GC complex-formation. Finally it was demonstrated that GC binding to thrombin can allosterically decrease the enzyme affinity for hirudin via a simultaneous decrease in association rate and increase in the dissociation velocity of the enzyme-inhibitor adduct. Together these observations indicate the GC binding to the heparin-binding domain of thrombin is largely driven by a hydrophobic effect and that such interaction can protect the enzyme from inhibition by the heparin-anti-thrombin III complex.

Antithrombin III↗
PubMed1999

Allosteric modulation of BPTI interaction with human alpha- and zeta-thrombin.

In this study, thrombin interaction with the basic pancreatic trypsin inhibitor (BPTI) was investigated in the presence of different allosteric modulators of thrombin, that is the C-terminal hirudin peptide 54-65 (Hir54-65), a recombinant thrombomodulin form (TMEGF4-6) and Na+. BPTI binding to alpha-thrombin is positively linked to Na+. Under low sodium concentration (5 mM Na+) the BPTI affinity for alpha-thrombin was roughly threefold lower than in the presence of 150 mM sodium (Ki = 320 microM vs. 100 microM). The hirudin fragment, which binds to the fibrinogen recognition site (FRS) of thrombin, induced a progressive and saturable decrease (3.6-fold) of alpha-thrombin affinity for BPTI, whereas the thrombomodulin peptide, which binds to a more extended region of FRS, caused a 5.5-fold increase of the enzyme affinity for the inhibitor. The opposite effect exerted by Hir54-65 and TMEGF4-6 was also observed for BPTI interaction with zeta-thrombin, in which the amidic bond between W148 and T149 is cleaved. However, in this case the effect by Hir54-65 and TMEGF4-6, although qualitatively similar to that observed with alpha-thrombin, had a smaller magnitude. Thrombin hydrolysis of Protein C was also differently affected by Hir54-65 and TMEGF4-6 peptides. While the latter enhanced the Protein C activation, the former caused a reduction of both alpha- and zeta-thrombin kcat/K(m)' for Protein C cleavage. These results showed that (a) Na+ facilitates BPTI interaction with thrombin; (b) Hir54-65 and TMEGF4-6, though sharing in part the same binding site at the thrombin FRS, can affect in opposite way thrombin's interaction with BPTI and Protein C; (c) such findings along with the results obtained with zeta-thrombin might be explained by admitting that the thermodynamic linkage between FRS and the critical W60-loop is also controlled by ligation and/or conformational state of the W148 insertion loop.

Allosteric Regulation↗
PubMed1996

Identification and characterization of the thrombin binding sites on fibrin.

Thrombin binds to fibrin at two classes of non-substrate sites, one of high affinity and the other of low affinity. We investigated the location of these thrombin binding sites by assessing the binding of thrombin to fibrin lacking or containing gamma' chains, which are fibrinogen gamma chain variants that contain a highly anionic carboxyl-terminal sequence. We found the high affinity thrombin binding site to be located exclusively in D domains on gamma' chains (Ka, 4.9 x 10(6) M-1; n, 1.05 per gamma' chain), whereas the low affinity thrombin binding site was in the fibrin E domain (Ka, 0.29 x 10(6) M-1; n, 1.69 per molecule). The amino-terminal beta15-42 fibrin sequence is an important constituent of low affinity binding, since thrombin binding at this site is greatly diminished in fibrin molecules lacking this sequence. The tyrosine-sulfated, thrombin exosite-binding hirudin peptide, S-Hir53-64 (hirugen), inhibited both low and high affinity thrombin binding to fibrin (IC50 1.4 and 3.0 microM respectively). The presence of the high affinity gamma' chain site on fibrinogen molecules did not inhibit fibrinogen conversion to fibrin as assessed by thrombin time measurements, and thrombin exosite binding to fibrin at either site did not inhibit its catalytic activity toward a small thrombin substrate, S-2238. We infer from these findings that there are two low affinity non-substrate thrombin binding sites, one in each half of the dimeric fibrin E domain, and that they may represent a residual aspect of thrombin binding and cleavage of its substrate fibrinogen. The high affinity thrombin binding site on gamma' chains is a constitutive feature of fibrin as well as fibrinogen.

Amino Acid Chloromethyl Ketones↗
PubMed1996

Thrombin primes responsiveness of selective chemoattractant receptors at a site distal to G protein activation.

To define the molecular basis of human chemoattractant receptor regulation, rat basophilic leukemia RBL-2H3 cells, which are thrombin-responsive, were transfected to stably express epitope-tagged receptors for C5a, interleukin-8 (IL-8), formylpeptides (e.g. N-formyl-methionyl-leucyl-phenylalanine (fMLP)), and platelet-activating factor (PAF). Here we demonstrate that both thrombin and a synthetic peptide ligand for the thrombin receptor (sequence SFLLRN) caused phosphorylation and heterologous desensitization of the receptors for C5a, IL-8, and PAF but not that for formylpeptides as measured by agonist-stimulated [35S]guanosine 5'-3-O-(thio)triphosphate binding to membranes. Consistent with the PAF receptor phosphorylation, both thrombin and thrombin receptor peptide inhibited phosphoinositide hydrolysis, Ca2+ mobilization, and degranulation stimulated by PAF. Unexpectedly, despite heterologous desensitization at the level of receptor/G protein activation, there was enhancement ("priming") by thrombin of subsequent activities stimulated by C5a and IL-8 as well as fMLP. The priming effect of thrombin was blocked by its inhibitor, hirudin. However, two other activators of the thrombin receptor, the peptide SFLLRN and trypsin, stimulated Ca2+ mobilization in RBL-2H3 cells but did not cause priming. In addition, SFLLRN and the thrombin receptor antagonist peptide FLLRN both inhibited thrombin-induced Ca2+ mobilization but not priming. Furthermore, the proteolytically active gamma-thrombin, which does not stimulate the tethered ligand thrombin receptor and caused little or no Ca2+ mobilization in RBL-2H3 cells, effectively primed the response to fMLP. These data demonstrate that heterologous receptor phosphorylation and attenuation of G protein activation are not, by themselves, sufficient for the inhibition of biological responses mediated by C5a and IL-8. Moreover, thrombin appears to utilize mechanism(s) independent of its tethered ligand receptor to selectively prime phospholipase C-mediated biological responses of the C5a, IL-8, and formylpeptide receptors but not PAF. Because C5a, IL-8, and formylpeptide activate phospholipase Cbeta2, whereas PAF stimulates a different phospholipase C, the striking selectivity of thrombin's priming may be mediated via its ability to enhance receptor-mediated activation of phospholipase Cbeta2.

Amino Acid Sequence↗
PubMed1997

Heparin promotes proteolytic inactivation by thrombin of a reactive site mutant (L444R) of recombinant heparin cofactor II.

A heparin cofactor II (HCII) mutant with an Arg substituted for Leu444 at the P1 position (L444R-rHCII) was previously found to have altered proteinase specificity (Derechin, V. M., Blinder, M. A., and Tollefsen, D. M. (1990) J. Biol. Chem. 265, 5623-5628). The present study characterizes the effect of glycosaminoglycans on the substrate versus inhibitor activity of L444R-rHCII. Heparin increased the stoichiometry of inhibition of L444R-rHCII with alpha-thrombin (compared with minus glycosaminoglycan) but decreased it with R93A,R97A,R101A-thrombin, a mutant thrombin that does not bind glycosaminoglycans. Dermatan sulfate decreased the stoichiometry of inhibition of L444R-rHCII with both proteinases. SDS-polyacrylamide gel electrophoresis showed no proteolysis of L444R-rHCII when incubated with R93A,R97A,R101A-thrombin in the absence or the presence of glycosaminoglycan or with alpha-thrombin and dermatan sulfate. In contrast, greater than 75% of the L444R-rHCII was converted to a lower molecular weight form when incubated with alpha-thrombin/heparin. A time course of alpha-thrombin inhibition by L444R-rHCII/heparin showed a rapid but transient inhibition with approximately 80% of the alpha-thrombin activity being regained after 6 h of incubation. In contrast, all other combinations of inhibitor, proteinase, and glycosaminoglycan resulted in complete and sustained inhibition of the proteinase. Heparin fragments of 8-20 polysaccharides in length rapidly accelerated L444R-rHCII inhibition of both alpha-thrombin and R93A,R97A,R101A-thrombin. After extended incubations, R93A,R97A,R101A-thrombin was completely inhibited by L444R-rHCII with all the heparin fragments, but approximately 30-50% of alpha-thrombin activity remained with fragments long enough to bridge HCII-thrombin. These results collectively indicate that ternary complex formation, mediated by heparin, increases L444R-rHCII inactivation by alpha-thrombin.

Heparin↗
PubMed1984

Ni-mediated inhibition of human platelet adenylate cyclase by thrombin.

The influence of the proteolytic enzyme, thrombin, was studied on adenylate cyclase activity in human platelet membranes. Thrombin had a biphasic inhibitory effect on the enzyme. Up to a concentration of about 1 U/ml, thrombin inhibited the enzyme in a strictly GTP-dependent manner by maximal 60-70%, with half-maximal inhibition occurring at about 0.005 U/ml thrombin. At higher concentrations, thrombin-induced inhibition of platelet adenylate cyclase was independent of GTP. The inhibitory effect of thrombin observed at low concentrations was further evaluated. The inhibition was half-maximal and maximal at about 0.1 and 3 microM GTP, respectively, occurred without major lag phase and was observed with the unstimulated and the forskolin or prostaglandin E1-stimulated platelet adenylate cyclase. Additionally, thrombin accelerated and potentiated the enzyme inhibition caused by the stable GTP analog, guanosine 5'-(gamma-thio)triphosphate. Furthermore, at identical concentrations causing adenylate cyclase inhibition, thrombin effectively stimulated GTP hydrolysis in platelet membranes. Finally, the thrombin inhibition of human platelet adenylate cyclase was impaired or abolished by Mn2+ and by treatment of the platelet membranes with N-ethylmaleimide or pertussis toxin. All of these data indicate that thrombin at low concentrations inhibits platelet adenylate cyclase by a process involving the inhibitory guanine nucleotide-binding regulatory component, Ni, in a manner similar to hormonal factors. However, in contrast to typical hormonal inhibition, thrombin inhibition of the platelet enzyme was not reversed by washing of the platelet membranes or by subsequent addition of the thrombin inactivator, hirudin, which prevented the inhibition when added together with thrombin. These data suggest that thrombin by its proteolytic capacity causes a persistent activation of its receptor site which leads to a persistent activation of Ni with subsequent persistent adenylate cyclase inhibition.

Adenylyl Cyclase Inhibitors↗
PubMed1999

Thrombin activates factor XI on activated platelets in the absence of factor XII.

Thrombin can activate factor XI in the presence of dextran sulfate or sulfatides. However, a physiological cofactor for thrombin activation of factor XI has not been identified. We examined this question in a cell-based, tissue factor-initiated model system. In the absence of factor XII, factor XI enhanced thrombin generation in this model. The effect on thrombin generation was reproduced by 2 to 5 pmol/L factor XIa. A specific inhibitor of factor XIIa did not diminish the effect of factor XI. Thus, factor XI can be activated in a model system that does not contain factor XIIa or nonphysiological cofactors. Preincubation of factor XI with activated platelets and thrombin or factor Xa enhanced subsequent thrombin generation in the model system. Preincubation of factor XI with thrombin or factor Xa, but without platelets, did not enhance thrombin generation, suggesting that these proteases might activate factor XI on platelet surfaces. Thrombin and factor Xa were then directly tested for their ability to activate factor XI. In the presence of dextran sulfate, thrombin or factor Xa activated factor XI. Thrombin, but not factor Xa, also cleaved detectable amounts of factor XI in the presence of activated platelets. Thus, thrombin activates enough factor XI to enhance subsequent thrombin generation in a model system. Platelet surfaces might provide the site for thrombin activation of functionally significant amounts of factor XI in vivo.

Anticoagulants↗
PubMed1998

Thrombin binds to soluble fibrin degradation products where it is protected from inhibition by heparin-antithrombin but susceptible to inactivation by antithrombin-independent inhibitors.

BACKGROUND: Thrombolytic therapy induces a procoagulant state characterized by elevated plasma levels of fibrinopeptide A (FPA), but the responsible mechanism is uncertain. METHODS AND RESULTS: Washed plasma clots were incubated in citrated plasma in the presence or absence of tissue plasminogen activator (t-PA), and FPA generation was monitored as an index of unopposed thrombin activity. FPA levels are almost twofold higher in the presence of t-PA than in its absence. This primarily reflects the action of thrombin bound to soluble fibrin degradation products because (a) there is progressive FPA generation even after clots are removed from t-PA-containing plasma, and (b) clot lysates produce concentration-dependent FPA generation when incubated in citrated plasma. Using thrombin-agarose affinity chromatography, (DD)E and fragment E but not D-dimer were identified as the thrombin-binding fibrin fragments, indicating that the thrombin-binding site is located within the E domain. Heparin inhibits thrombin bound to fibrin degradation products less effectively than free thrombin. In contrast, D-Phe-Pro-ArgCH2Cl, hirudin and hirugen inhibit free thrombin and thrombin bound to fibrin degradation products equally well. CONCLUSIONS: Thrombin bound to soluble fibrin degradation products is primarily responsible for the increase in FPA levels that occurs when a clot undergoes t-PA-induced lysis. Like clot-bound thrombin, thrombin bound to fibrin derivatives is protected from inhibition by heparin but susceptible to inactivation by direct thrombin inhibitors. These findings help to explain the superiority of direct thrombin inhibitors over heparin as adjuncts to thrombolytic therapy.

Analysis of Variance↗
PubMed1996

Thrombin receptor actions in neonatal rat ventricular myocytes.

Previous studies established that thrombin stimulates phosphoinositide hydrolysis and modulates contractile function in neonatal rat ventricular myocytes. The present study further defines the signaling pathways activated by the thrombin receptor and their role in thrombin's actions in cardiac myocytes. The thrombin receptor-derived agonist peptide (TRAP, a portion of the tethered ligand created by thrombin's proteolytic activity) stimulates the rapid and transient accumulation of inositol bis- and tris-phosphates (IP2 and IP3, respectively), which is followed by the more gradual and sustained accumulation of inositol monophosphate (IP1). TRAP elicits a larger and more sustained accumulation of IP1 than does thrombin. Thrombin and TRAP also activate mitogen-activated protein kinase (MAPK) in cultured neonatal rat ventricular myocytes. Differences in the kinetics and magnitude of thrombin- and TRAP-dependent inositol phosphate (IP) accumulation are paralleled by differences in the kinetics and magnitude of thrombin- and TRAP-dependent activation of MAPK. Pretreatment with phorbol 12-myristate 13-acetate (PMA) to downregulate protein kinase C (PKC) attenuates thrombin- and TRAP-dependent activation of MAPK, although small and equivalent effects of thrombin and TRAP to stimulate MAPK persist in PMA-pretreated cells. These results support the notion that the thrombin receptor activates MAPK through PKC-dependent pathways and that the incremental activation of MAPK by TRAP over that induced by thrombin is the consequence of enhanced activation through the PKC limb of the phosphoinositide lipid pathway. TRAP also increases the beating rate of spontaneously contracting ventricular myocytes and elevates cytosolic calcium in myocytes electrically driven at a constant basic cycle length. The effects of TRAP to modulate contractile function and elevate intracellular calcium are not inhibited by tricyclodecan-9-yl-xanthogenate (D609, to block TRAP-dependent IP accumulation) or pretreatment with PMA (to downregulate PKC). The TRAP-dependent rise in intracellular calcium also is not inhibited by verapamil or removal of extracellular calcium but is markedly attenuated by depletion of sarcoplasmic reticular calcium stores by caffeine. Patch-clamp experiments demonstrate that TRAP elevates intracellular calcium in cells held at a membrane potential of -70 mV. Taken together, these results support the conclusion that the thrombin receptor modulates contractile function by mobilizing intracellular calcium through an IP3-independent mechanism and that this response does not require activation of voltage-gated ion channels.

Animals↗
PubMed1972

The effects of thrombin on adenyl cyclase activity and a membrane protein from human platelets.

Washed human platelets were incubated with 0.1-1.0 U/ml human thrombin and the effects on adenyl cyclase activity and on a platelet membrane protein (designated thrombin-sensitive protein) were studied. Adenyl cyclase activity was decreased 70-90% when intact platelets were incubated with thrombin. The T(1/2) for loss of adenyl cyclase activity was less than 15 sec at 1 U/ml thrombin. There was no decrease of adenyl cyclase activity when sonicated platelets or isolated membranes were incubated with these concentrations of thrombin. Loss of adenyl cyclase activity was relatively specific since the activities of other platelet membrane enzymes were unaffected by thrombin. Prior incubation of platelets with dibutyryl cyclic adenosine monophosphate (AMP), prostaglandin E(1), or theophylline protected adenyl cyclase from inhibition by thrombin. Incubation of intact but not disrupted platelets with thrombin resulted in the release of thrombin-sensitive protein from the platelet membrane. The rapid release of this protein (T(1/2) < 15 sec) at low concentrations of thrombin suggested that removal of thrombin-sensitive protein from the platelet membrane is an integral part of the platelet release reaction. This hypothesis is supported by the parallel effects of thrombin on adenyl cyclase activity and thrombin-sensitive protein release in the presence of dibutyryl cyclic AMP, prostaglandin E(1), and theophylline at varying concentrations of thrombin.

Adenylyl Cyclases↗
PubMed1989

Binding of thrombin to subendothelial extracellular matrix. Protection and expression of functional properties.

We have analyzed the binding of thrombin, a serine protease with central roles in hemostasis, to the subendothelial extracellular matrix (ECM) produced by cultured endothelial cells. This substrate provides a thrombogenic surface where hemostasis is initiated. Binding was saturable and equilibrium was achieved after 3 h incubation with 125I-alpha-thrombin. Scatchard analysis of thrombin binding revealed the presence of 5.1 X 10(9) binding sites per squared millimeter ECM, with an apparent Kd of 13 nM. The catalytically blocked enzyme, diisofluorophosphate (DIP)-alpha-thrombin competed efficiently with 125I-alpha-thrombin, indicating that the binding was independent of its catalytic site. Moreover, high concentrations of the synthetic tetradecapeptide, representing residues 367-380 of thrombin B chain (the macrophage mitogenic domain of thrombin), competed with thrombin binding to ECM, indicating that the binding site may reside in the vicinity of "loop B" region. Thrombin binds to dermatan sulfate in the ECM, as demonstrated by the inhibition of 125I-alpha-thrombin binding to ECM pretreated with chondroitinase ABC, but not with heparitinase or chondroitinase AC. This stands in contrast to 125I-FGF (fibroblast growth factor) binding to ECM, which was inhibited by heparitinase but not by chondroitinase ABC, ECM-bound thrombin exhibits an exposed proteolytic site as monitored by the Chromozyme TH assay and by its ability to convert fibrinogen to a fibrin clot and to induce platelet activation as indicated by 14C-serotonin release. ECM-bound thrombin failed to form a complex with its major circulating inhibitor-antithrombin III (AT III), compared with rapid complex formation with soluble thrombin. We propose that thrombin binds to subendothelial ECM where it remains functionally active, localized, and protected from inactivation by circulating inhibitors.

Amides↗
PubMed1990

Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors.

Propagation of venous thrombi or rethrombosis after coronary thrombolytic therapy can occur despite heparin administration. To explore potential mechanisms, we set out to determine whether clot-bound thrombin is relatively protected from inhibition by heparin-antithrombin III but susceptible to inactivation by antithrombin III-independent inhibitors. Using plasma fibrinopeptide A (FPA) levels as an index of thrombin activity, we compared the ability of thrombin inhibitors to block FPA release mediated by fluid-phase thrombin with their activity against the clot-bound enzyme. Incubation of thrombin with citrated plasma results in concentration-dependent FPA generation, which reaches a plateau within minutes. In contrast, there is progressive FPA generation when fibrin clots are incubated with citrated plasma. Heparin, hirudin, hirudin dodecapeptide (hirugen), and D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK) produce concentration-dependent inhibition of FPA release mediated by fluid-phase thrombin. However, heparin is much less effective at inhibiting thrombin bound to fibrin because a 20-fold higher concentration is necessary to block 70% of the activity of the clot-bound enzyme than is required for equivalent inhibition of fluid-phase thrombin (2.0 and 0.1 U/ml, respectively). In contrast, hirugen and PPACK are equally effective inhibitors of fluid- and solid-phase thrombin, while hirudin is only 50% as effective against the clot-bound enzyme. None of the inhibitors displace bound 125I-labeled thrombin from the clot. These studies indicate that (a) clot-bound thrombin is relatively protected from inhibition by heparin, possibly because the heparin binding site on thrombin is inaccessible when the enzyme is bound to fibrin, and (b) clot-bound thrombin is susceptible to inactivation by antithrombin III-independent inhibitors because the sites of their interaction are not masked by thrombin binding to fibrin. For these reasons, antithrombin III-independent inhibitors may be more effective than heparin in certain clinical settings.

Amino Acid Chloromethyl Ketones↗