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PubMed1985

4,4'-Bis[8-(phenylamino)naphthalene-1-sulfonate] binding to human thrombins: a sensitive exo site fluorescent affinity probe.

The binding of the fluorescent probe 4,4'-bis[8-(phenylamino)naphthalene-1-sulfonate] (bis-ANS) to human alpha- and gamma-thrombins was investigated. Bis-ANS binds in a 1:1 complex to both forms of the enzyme, with Kd = 14.8 +/- 2.2 microM and 5.8 +/- 1.0 microM for alpha- and gamma-thrombin, respectively, at pH 7.0 [25 mM tris(hydroxymethyl)aminomethane, 0.15 M NaC1]. Fluorescence changes upon complexation included a considerable (approximately 30-nm) blue shift in the fluorescence emission maximum as well as a dramatic increase in the fluorescence emission intensity: a 70-fold enhancement was observed with alpha-thrombin vs. a approximately 220-fold enhancement with gamma-thrombin. Proflavin was not displaced upon bis-ANS binding. The unknown thrombin effectors ATP, Ca(II)ATP, Co(III)ATP, phosphate, and pyrophosphate bound with enhancement of the fluorescence of the bis-ANS-alpha-thrombin complex. The two inhibitors benzamidine and p-chlorobenzylamine as well as heparin caused decreases in bis-ANS-thrombin fluorescence: valerylamidine had no effect on the fluorescence of the bis-ANS-thrombin complex. Kinetic measurements with two chromogenic substrates, S-2238 and S-2160, indicated that bis-ANS acts as a partial noncompetitive inhibitor of thrombin amidase activity. The kinetic evidence combined with the ligand binding results suggests that bis-ANS does not overlap the catalytic site. The fluorophore ANS complexed with equal affinity to both alpha- and gamma-thrombins (Kd = 24 +/- 4 microM); however, the gamma-thrombin-ANS complex emission at 470 nm was enhanced 26% more than that for the alpha form.

Adenosine Triphosphate↗
PubMed1989

Substitution of valine for glycine-558 in the congenital dysthrombin thrombin Quick II alters primary substrate specificity.

Thrombin Quick II is one of two dysfunctional forms of thrombin derived from the previously described congenital dysprothrombin prothrombin Quick. Thrombin Quick II does not clot fibrinogen, hydrolyze p-nitroanilide substrates of thrombin, or bind N2-[5-(dimethylamino)naphthalene-1-sulfonyl]arginine N,N-(3-ethyl-1,5-pentanediyl)amide, a high-affinity competitive inhibitor of thrombin. To determine the structural alteration in thrombin Quick II, the reduced, carboxymethylated protein was hydrolyzed by a lysyl endopeptidase. A peptide not present in a parallel thrombin hydrolysate was identified by reverse-phase chromatography. The peptide was purified by rechromatography and subjected to Edman degradation which showed that Gly-558 of human prothrombin had been replaced by Val. This corresponds to a point mutation of the Gly codon GGC to GUC. This Gly residue, which is highly conserved in the chymotrypsin family of serine proteases, forms part of the substrate binding pocket for bulky aromatic and basic side chains in chymotrypsin and trypsin, respectively. However, in porcine elastase 1, the corresponding residue is threonine. Consistent with the identified structural alteration, thrombin Quick II incorporates [3H]diisopropyl fluorophosphate stoichiometrically and hydrolyzes the elastase substrate succinyl-Ala-Ala-Pro-Leu-p-nitroanilide with a relative kcat/KM of 0.14 when compared to thrombin. This results from a 3-fold increase in KM and a 2.5-fold decrease in kcat for thrombin Quick II when compared to thrombin acting on the same substrate. These results and those of other investigators studying mutant trypsins support the conclusion that the catalytic activity of serine proteases is very sensitive to structural alterations in the primary substrate binding pocket.

Amino Acid Sequence↗
PubMed2006

Amide H/2H exchange reveals a mechanism of thrombin activation.

Thrombin is a dual action serine protease in the blood clotting cascade. Similar to other clotting factors, thrombin is mainly present in the blood in a zymogen form, prothrombin. Although the two cleavage events required to activate thrombin are well-known, little is known about why the thrombin precursors are inactive proteases. Although prothrombin is much larger than thrombin, prethrombin-2, which contains all of the same amino acids as thrombin, but has not yet been cleaved between Arg320 and Ile321, remains inactive. Crystal structures of both prethrombin-2 and thrombin are available and show almost no differences in the active site conformations. Slight differences were, however, seen in the loops surrounding the active site, which are larger in thrombin than in most other trypsin-like proteases, and have been shown to be important for substrate specificity. To explore whether the dynamics of the active site loops were different in the various zymogen forms of thrombin, we employed amide H/(2)H exchange experiments to compare the exchange rates of regions of thrombin with the same regions of prothrombin, prethrombin-2, and meizothrombin. Many of the surface loops showed less exchange in the zymogen forms, including the large loop corresponding to anion binding exosite 1. Conversely, the autolysis loop and sodium-binding site exchanged more readily in the zymogen forms. Prothrombin and prethrombin-2 gave nearly identical results while meizothrombin in some regions more closely resembled active thrombin. Thus, cleavage of the Arg320-Ile321 peptide bond is the key to formation of the active enzyme, which involves increased dynamics of the substrate-binding loops and decreased dynamics of the catalytic site.

Amides↗
PubMed2003

Inhibition of interleukin-12 expression by alpha-thrombin in human peripheral blood mononuclear cells: a potential mechanism for modulating Th1/Th2 responses.

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is a powerful regulator of inflammatory responses and is known to affect cell-mediated immunity. Interleukin (IL)-12 is a strong promoter of the development of Th1-type lymphocytes and its downregulation implies a positive feedback mechanism for development of Th2 responses. We have previously shown that thrombin enhances the release of IL-6, a Th2-related cytokine, in human peripheral blood mononuclear cells (PBMC). Here we show that thrombin downregulates IL-12 production at both protein and mRNA levels in human PBMC. The inhibition of IL-12 production was accompanied by an enhanced release of IL-10, which inhibits Th1-related processes and promotes Th2-type responses. The use of proteolytically inactive thrombin and of the specific thrombin receptor agonist peptide, SFLLRN, reveals that this downregulation is thrombin-specific and requires thrombin proteolytic activity. In addition, activation of coagulation inhibits IL-12 production in whole blood cultures, confirming the tight relationship between the coagulation pathway, where thrombin is a key enzyme, and inflammation. Decreased IL-12 production appears to be related also to IL-10 production, since the addition of an anti-IL-10 monoclonal antibody to thrombin-treated PBMC resulted in a partial restoration of IL-12 production. In conclusion, the observation that thrombin significantly affects the production of IL-12, as well as of IL-10, implies a concerted role orchestrated by thrombin in PBMC that could be crucial to effective immunity and inflammation.

Gene Expression Regulation↗
PubMed2005

Thrombin-induced glucose transport via Src-p38 MAPK pathway in vascular smooth muscle cells.

Thrombin is a mitogen for vascular smooth muscle cells (VSMC) and has been implicated in the development in atherosclerosis. However, little is known about the role of thrombin in glucose transport in VSMC. In this study, we examined the effect of thrombin on glucose uptake in rat A10 VSMC. We found that thrombin induced glucose uptake in a dose-dependent manner while hirudin, a potent thrombin inhibitor, prevented glucose uptake in the cells. PP2, a selective inhibitor of Src, prevented the thrombin-induced glucose uptake, but did not affect insulin-induced uptake. We also examined whether mitogen-activated protein kinase (MAPK) inhibitors influenced thrombin-induced glucose uptake. The p38 MAPK inhibitor (SB203580) inhibited thrombin-induced glucose uptake, but the MEK inhibitor (PD98059) did not. In contrast to thrombin, SB203580 did not affect insulin-induced glucose uptake. Furthermore, thrombin failed to translocate the insulin-sensitive glucose transporter GLUT4. These findings suggest that thrombin stimulates glucose transport via Src and subsequent p38 MAPK activation in VSMC.

Animals↗
PubMed1989

Effects of separate proteolytic and high-affinity binding activities of human thrombin on rapid platelet activation. A quenched-flow study.

We have used a general quenched-flow approach to study platelet function as early as 0.3 s after stimulation with three types of human thrombin: alpha-thrombin, gamma-thrombin, which is proteolytically active but does not bind to the high-affinity sites, and di-isopropyl fluorophosphate-derivatized (DIP)-alpha-thrombin, an active site-inhibited analogue that does bind to the high-affinity site. Large doses of gamma-thrombin evoked moderate aggregation and serotonin release, but minimal phosphorylation of the 20 and 47 kDa proteins. The initial (1.5-3.0 s) increase in cystolic free calcium concentration ([Ca2+]i) indicated by Indo-1 was also diminished, but by 5 s was nearly as high (1.0 microM) as with alpha-thrombin. A large dose of DIP-alpha-thrombin, on the other hand, induced minimal aggregation, serotonin secretion and [Ca2+]i response within 6 s. There was, however, a transient dephosphorylation of the 20 kDa protein. When combined, gamma- and DIP-alpha-thrombin were approximately additive in their ability to induce aggregation and serotonin secretion, but strongly synergistic in phosphorylating the 20 and 47 kDa proteins. The [Ca2+]i increase was not, however, enhanced over that induced by gamma-thrombin alone. These results demonstrate that phosphorylation of either the 20 or 47 kDa proteins is not correlated with [Ca2+]i dynamics and is neither required nor directly involved in platelet aggregation and secretion induced by thrombin. The high-affinity binding activity of thrombin is not necessary for rapid platelet Ca2+ influx, aggregation and serotonin release within the first critical seconds of activation.

Blood Platelets↗
PubMed1989

Thrombin causes increased monocytic-cell adhesion to endothelial cells through a protein kinase C-dependent pathway.

The coagulation protein thrombin has been shown to stimulate multiple endothelial-cell (EC) functions, including production of platelet-derived growth factor and of platelet-activating factor (PAF), and neutrophil adhesion. We have found that thrombin causes increased binding of monocytic cells (U937 cells and normal human monocytes) to cultured EC of various species. Maximum adhesion of monocytes to pig aortic EC occurred 6 h after thrombin treatment and remained elevated through 24 h. Stimulation of adherence by bovine alpha-thrombin was half-maximal at 15 units/ml, and reached a plateau at 50 units/ml. Catalytically inactive thrombin (phenylmethanesulphonyl fluoride-treated) had no effect on monocyte adhesion to EC. Heparin, but not the endotoxin antagonist polymyxin B, suppressed the stimulation of adhesion by thrombin without altering basal adhesion. Two lines of evidence suggested that protein kinase C (PKC) was involved in the intracellular signalling to increase monocyte adhesion to EC. First the PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated monocytic-cell adhesion to EC at a dose consistent with stimulation of PKC (half-maximal response at 1-3 nM) and with a time course similar to that for thrombin stimulation (maximal by 4 h). Diacylglycerol, a physiological activator of PKC, also stimulated U937-cell adhesion to EC. Secondly, H7, a PKC inhibitor, completely blocked stimulation of monocyte adhesion to EC by thrombin or PMA. The structural analogue of H7, HA1004, which preferentially inhibits cyclic-AMP- and cyclic-GMP-dependent protein kinases, had no effect on stimulated monocyte adhesion. The PKC inhibitor also blocked the stimulation of monocyte adhesion to EC by interleukin-1 and endotoxin, but did not alter the basal level of monocyte binding to unstimulated EC. Thrombin stimulation of monocyte adhesion differed from the reported stimulation of neutrophil adhesion by thrombin in that the latter process reached a maximum in minutes rather than hours. In addition, neither PAF itself nor agents known to stimulate PAF production by EC, such as arachidonate and the Ca2+ ionophore A23187, had any effect on monocyte adhesion. These results demonstrate a PKC-dependent cytokine-like action of the coagulation protein thrombin in modulating monocytic-cell adhesion to EC, a phenomenon of potential importance in many pathological and physiological processes.

Animals↗
PubMed1995

Regulation of neurite outgrowth from differentiated human neuroepithelial cells: a comparison of the activities of prothrombin and thrombin.

The mechanism by which thrombin and prothrombin control neurite retraction was studied in Ad12E1HER10 human neuroepithelial cells. Morphological changes in differentiated cells were apparent within minutes of the addition of very low concentrations of thrombin (3 pM). Higher concentrations (2 nM) of prothrombin were required to elicit a similar response. Doses of thrombin and prothrombin sufficient to cause neurite retraction stimulated protein tyrosine kinase activity. Protein tyrosine kinase activation also correlated positively with thrombin- and prothrombin-induced phosphoinositide 3-kinase activation and InsP6 dephosphorylation. However, thrombin-stimulated Ins(1,4,5)P3 generation and intracellular Ca2+ mobilization only occurred at concentrations in excess of those needed to induce retraction. No fluctuations in Ins(1,4,5)P3 were detected after stimulation with prothrombin, and no rapid synchronized release of Ca2+ was observed, even at very high concentrations. Prothrombin did, however, cause small oscillations in the intracellular Ca2+ concentration, similar to those produced by low concentrations of thrombin, after approximately 30 min. We conclude that prothrombin- and thrombin-induced neurite retractions are not dependent on PtdIns(4,5)P2 and Ca2+ mobilization, but are more probably mediated through an effector mechanism involving protein tyrosine kinase activation. No intracellular Ca2+ mobilization, protein tyrosine kinase activity or neurite retraction was observed after treatment of cells with proteolytically inactive mutant thrombin (S205-->A). Prothrombin-mediated intracellular Ca2+ mobilization and neurite retraction were inhibited by hirudin, which was shown to interact with thrombin but not prothrombin. It is concluded that cleavage of prothrombin to thrombin is a necessary prerequisite for biological activity on differentiated Ad12E1HER10 cells and that differences in agonist concentration are capable of coupling the thrombin receptor to different pathways within the cell.

Blotting, Western↗
PubMed1996

Thrombin causes cell spreading and redistribution of beta-amyloid immunoreactivity in cultured hippocampal neurons.

Culture of rat embryonic hippocampal neurons in serum-free B27/Neurobasal for 4 days enabled tests of the effect of added thrombin on differentiated cell morphology and processing of the amyloid precursor protein (APP). By fluorescence microscopy of neurons labeled with dil and by scanning electron microscopy, an increase in spreading of the neuron soma was clearly seen in cells treated with 1 microg/ml (27 nM) of thrombin for 24 h. This treatment also caused a dose-dependent increase in immunoreactive area/cell, detected with antibody 4G8 binding to the beta-amyloid region of APP. Thrombin treatment also produced a dose-dependent increase in immunoreactive brightness detected with the Alz-50 antibody. Thrombin did not affect viability or cause neurite retraction. The thrombin effect on 4G8 immunoreactivity required 24 h for full effect and could be blocked by the thrombin inhibitor antithrombin III or hirudin. A thrombin receptor appeared to be activated because a full immunoreactive response was observed by treatment of neurons with the thrombin receptor-activating peptide SFL-LRNPNNKYEPF. When cytoplasmic extracts were analyzed by western immunoblots or by pulse-chase radiolabeling, no thrombin-dependent changes in processing of 127- and 120-kDa bands were seen. Material migrating in the region of synthetic betaA4 was not found. Together, these results suggest that thrombin acts on neurons through a thrombin receptor to stimulate cell spreading and redistribution of APP without amyloidogenic changes. The adhesion responsible for this spreading could be important in altering synaptic connections in the brain.

Amyloid beta-Peptides↗
PubMed1992

Thrombin regulation in mother and fetus during pregnancy.

Pregnancy is characterized by an increase in plasma concentration of several coagulants and with little or no change in plasma concentrations of inhibitors of thrombin or thrombin generation with the exception of a decrease in protein S. The net effect of these physiologic differences from nonpregnant adults is that, in vitro, thrombin generation is slightly increased in pregnancy plasma. Thrombin inhibition may be altered by the presence of circulating dermatan sulfate proteoglycan. Based on information on placental structure and function and ex vivo markers of thrombin generation, it is clear that there must be local generation of thrombin with fibrin deposition in the placenta. The roles of the alterations in the hemostatic system in regulating this increased thrombin activity remain to be elucidated. In addition, regulation of thrombin at the vessel wall of the placenta needs to be further explored, since it is likely important in preventing locally increased thrombin generation from producing systemic effects, that is, thrombosis. The fetal coagulation system is an immature, developing system, as is evident from low and gradually increasing plasma concentrations of both coagulants and inhibitors. Some of these low levels result from decreased gene expression in the immature liver. However, others (Factors II, VII and VIII) are related to other processes that remain to be determined. There is a progressive increase in the levels of these factors with increasing gestational age but most remain below adult values at term. As a result, thrombin generation and inhibition in vitro are decreased. Since bleeding and thrombotic complications are not a feature of normal fetal life, it is likely that the hemostatic system is balanced. In vivo data on thrombin generation and inhibition are lacking and the interaction of the physiologically different plasma of the fetus with the vessel wall is only now being explored.

Blood Coagulation Factors↗
PubMed1988

Thrombin modulates and reverses neuroblastoma neurite outgrowth.

Previous studies have shown that neuroblastoma cells and several types of primary neuronal cells in culture rapidly extend neurites when switched from serum-containing to serum-free medium. The present studies on cloned neuroblastoma cells show that thrombin blocked this spontaneous differentiation at 2 nM with a half-maximal potency of 50 pM. This required the catalytic activity of thrombin and was reversed upon thrombin removal. Thrombin also caused cells in serum-free medium to retract their neurites at equally low concentrations. Two other serine proteases, urokinase and plasmin, did not block or reverse neurite extension even at 100-fold higher concentrations. A specific assay for thrombin indicated that thrombin detected in serum-containing medium from neuroblastoma cultures was derived from serum and that it was likely responsible for much of the known capacity of serum to maintain neuroblastoma cells in a nondifferentiated state. This was supported by the finding that heparin addition reduced the thrombin concentration in serum-containing medium and stimulated neurite outgrowth from neuroblastoma cells in serum-containing medium. Studies on the ability of thrombin to modulate neurite outgrowth by other agents showed that it blocked and reversed the neurite outgrowth activity of two thrombin inhibitors: protease nexin-1 (which is identical to glial-derived neurite-promoting factor) and hirudin. Thrombin, however, did not block the neurite-promoting activity of dibutyryl cAMP or prostaglandin E1. These results suggest a specific role for thrombin in control of neurite outgrowth.

Alprostadil↗
PubMed1997

Thrombin cleaves recombinant human thrombopoietin: one of the proteolytic events that generates truncated forms of thrombopoietin.

A heterogeneity in the molecular weight (Mr) of thrombopoietin (TPO) has been reported. We found several thrombin cleavage sites in human, rat, murine, and canine TPOs, and also found that human TPO undergoes selective proteolysis by thrombin. Recombinant human TPO (rhTPO) was incubated with human platelets in the presence of calcium ions to allow the generation of thrombin, and was cleaved into low Mr peptide fragments. The cleavage was completely inhibited by hirudin, indicating that the proteolysis was mediated by thrombin. In a platelet-free system, analyses of thrombin cleavage by immunoblotting using anti-human TPO peptide antibodies revealed that the four major thrombin-cleaved peptide fragments were selectively generated depending on the digestion time. The amino acid sequences of the thrombin-polypeptides were further analyzed, and two major thrombin cleavage sites were determined. One of them was at AR191-T192 in the C-terminal domain of TPO, and thrombin cleaved first at this site. The other site at GR117-T118 in the N-terminal domain was subsequently cleaved by prolonged thrombin digestion. As a result, the biological activity of TPO was modulated. The generation of truncated forms of TPO by thrombin may be a notable event in view of the platelet-related metabolism of TPO.

Amino Acid Sequence↗
PubMed1995

Thrombin stimulates phosphorylation of insulin-like growth factor-1 receptor, insulin receptor substrate-1, and phospholipase C-gamma 1 in rat aortic smooth muscle cells.

It has recently been reported that protein-tyrosine kinase activity is required for thrombin-induced growth in vascular smooth muscle cells (VSMC). In the present study, we have identified several phosphoproteins that are tyrosine-phosphorylated in response to thrombin in quiescent VSMC. These proteins are insulin-like growth factor-1 receptor beta-subunit (IGF-IR beta), insulin receptor substrate-1 (IRS-1), and phospholipase C-gamma 1 (PLC-gamma 1). Thrombin-stimulated phosphorylation of these proteins was rapid; it was maximal at 1 min and reduced thereafter. Thrombin also activated mitogen-activated protein kinases (MAPK) in quiescent VSMC in a biphasic manner with a rapid and larger peak at 10 min (6-fold) followed by a sustained smaller second peak at 2 h (2-fold). Inhibition of protein-tyrosine kinase activity by the use of two structurally different protein-tyrosine kinase inhibitors, genistein and herbimycin A, significantly blocked the thrombin-induced tyrosine phosphorylation of IGF-1R beta, IRS-1, and PLC-gamma 1 and decreased thrombin-stimulated DNA synthesis. In contrast, however, inhibition of protein-tyrosine kinase activity had no effect on thrombin activation of MAPK. Collectively, these findings suggest a role for tyrosine phosphorylation of IGF-IR beta, IRS-1, and PLC-gamma 1 in thrombin-induced mitogenic signaling events in VSMC. Furthermore, while protein tyrosine phosphorylation is essential for thrombin-induced DNA synthesis, it is not required for thrombin-stimulated MAPK activation. Since thrombin rapidly activated Src in VSMC, Src may be involved in the cross-talk between the G-protein-coupled receptor agonist and a tyrosine kinase receptor such as IGF-1R.

Animals↗
PubMed1996

Cyclic AMP inhibition of thrombin-induced growth in vascular smooth muscle cells correlates with decreased JNK1 activity and c-Jun expression.

Thrombin is a potent modulator of vascular tone and vascular smooth muscle cell (VSMC) mitogenesis. Early studies from other laboratories demonstrated that cyclic AMP (cAMP) antagonizes the mitogenic effects of platelet-derived growth factor and epidermal growth factor by inhibiting the extracellular signal-regulated protein kinases (ERKs; p42, p44) group of mitogen-activated protein kinases (MAPKs) in several cell types. This report examines the role of ERKs and Jun N-terminal kinase 1 (JNK1) groups of mitogen-activated protein kinases in thrombin-induced DNA synthesis in VSMCs using agents such as forskolin and dibutyrylcyclic AMP that increase intracellular cAMP levels. Both agents significantly inhibited thrombin-stimulated DNA synthesis in VSMCs. These agents, however, had no effect on thrombin induction of ERKs activation and c-Fos expression, suggesting divergence of the latter two events from the growth-signaling events of thrombin that are sensitive to inhibition by cAMP. Thrombin activated JNK1 and induced c-Jun expression in VSMCs in a time-dependent manner. In contrast to ERKs and c-Fos, thrombin-induced JNK1 activation and c-Jun expression were sensitive to inhibition by forskolin, suggesting an association of these events with thrombin-stimulated growth in these cells. Thrombin also increased AP-1 activity, and this response was significantly blunted by forskolin. Together, these results demonstrate a correlation between JNK1 activation and c-Jun expression by thrombin and their association with the mitogenic signaling events of thrombin in VSMCs.

Animals↗
PubMed1997

The thrombin receptor second cytoplasmic loop confers coupling to Gq-like G proteins in chimeric receptors. Additional evidence for a common transmembrane signaling and G protein coupling mechanism in G protein-coupled receptors.

Thrombin activates human platelets and other cells in part by cleaving an unusual G protein-coupled receptor. Thrombin cleavage of this receptor's amino-terminal exodomain unmasks a new amino terminus. This then binds intramolecularly to the body of the receptor to trigger transmembrane signaling and activation of Gi- and Gq-like G proteins. Toward identifying the domains responsible for thrombin receptor-G protein interactions, we examined the signaling properties of chimeric receptors in which thrombin receptor cytoplasmic sequences replaced the cognate sequences in the Gs-coupled beta2-adrenergic receptor (beta2AR) or the Gi-coupled dopamine D2 receptor (D2R). In Xenopus oocytes, a chimeric beta2AR bearing the thrombin receptor second cytoplasmic (C2) loop gained the ability to trigger intracellular Ca2+ release in response to adrenergic agonist, whereas a beta2AR bearing the cognate C2 loop from the D2R did not. Similarly, in COS-7 cells, a chimeric D2R bearing the thrombin receptor C2 loop gained the ability to trigger phosphoinositide hydrolysis in response to dopaminergic agonist, apparently by coupling to a Gq-like G protein. No detectable Gs coupling was seen. Thus, the thrombin receptor C2 loop was able to confer Gq-like coupling in several different receptor contexts. These observations suggest that the thrombin receptor C2 loop specifies Gq coupling by directly contacting Gq or by contributing to a structure required for Gq coupling. The ability of the thrombin receptor C2 loop to function in the context of the D2R and beta2AR strongly suggests that the transmembrane switching and G protein activation strategies used by the thrombin receptor must be very similar to those used by the D2R and beta2AR despite the thrombin receptor's strikingly different liganding mechanism.

Animals↗
PubMed1997

Inhibitory mechanism of the protein C pathway on tissue factor-induced thrombin generation. Synergistic effect in combination with tissue factor pathway inhibitor.

The effects of the components of the protein C pathway on thrombin generation were studied in a reconstituted model in which thrombin is generated by factor VIIa and relipidated tissue factor (TF) via the activation of the purified coagulation factors X, IX, VIII, V, and prothrombin. The influence of protein C and soluble thrombomodulin on thrombin generation was correlated with factor Xa generation, factor V(a) and factor VIII(a) formation/inactivation, and protein C activation. Thrombin generation initiated by low concentrations of factor VIIa.TF (1.25 pM) occurs in an explosive fashion during a propagation phase which occurs after an initiation phase of approximately 1 min in which only traces of thrombin are formed. In the absence of other inhibitors, protein C (65 nM) in combination with high concentrations of soluble thrombomodulin (10 nM) resulted in a reduced rate of thrombin generation during the propagation phase without affecting the initiation phase; the activated protein C generated failed to neutralize prothrombinase activity and did not prevent prothrombin consumption. In the presence of plasma levels of the tissue factor pathway inhibitor (2. 5 nM recombinant TFPI), the protein C pathway reduced the rate of thrombin generation, initiated by 1.25 pM factor VIIa.TF, and completely eliminated prothrombinase activity at soluble thrombomodulin concentrations of >/=1 nM. The neutralization of prothrombinase activity coincided with cleavages at Arg-506 and subsequent cleavage at Arg-306 of the factor Va heavy chain by activated protein C. Thus, the protein C pathway combined with TFPI creates a minimal inhibitory potential required to shut down TF-initiated thrombin generation. The protein C pathway constituents did not influence factor Xa generation or factor VIIIa degradation over the interval in which prothrombinase activity was neutralized. Our data thus suggest that the protein C pathway regulates thrombin generation solely by the inactivation of factor Va. At low initiating factor VIIa.TF (1.25 pM) and high thrombomodulin concentrations (10 nM), the factor Va heavy chain is cleaved before significant amounts of light chain are generated. The ability of the protein C pathway to inhibit thrombin generation was greatly reduced when the reaction was initiated in the presence of factor Va, supporting the hypothesis that effective down-regulation of thrombin generation by the protein C pathway, in reactions initiated with the procofactor, occurs by prevention of the coexistence of the factor Va heavy and light chains.

Drug Synergism↗
PubMed1999

Role of regulatory exosite I in binding of thrombin to human factor V, factor Va, factor Va subunits, and activation fragments.

The blood coagulation proteinase, thrombin, converts factor V into factor Va through a multistep activation pathway that is regulated by interactions with thrombin exosites. Thrombin exosite interactions with human factor V and its activation products were quantitatively characterized in equilibrium binding studies based on fluorescence changes of thrombin covalently labeled with 2-anilinonaphthalene-6-sulfonic acid (ANS) linked to the catalytic site histidine residue by Nalpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl ([ANS]FPR-thrombin). Exosite I was shown to play a predominant role in the binding of factor V and factor Va from the effect of the exosite I-specific ligand, hirudin54-65, on the interactions. Factor V and factor Va bound to exosite I of [ANS]FPR-thrombin with similar dissociation constants of 3.4 +/- 1.3 and 1.1 +/- 0.4 microM and fluorescence enhancements of 182 +/- 41 and 127 +/- 17%, respectively. Native thrombin and labeled thrombin bound with similar affinity to factor Va. Among factor V activation products, the factor Va heavy chain was shown to contain the site of exosite I binding, whereas exosite I-independent, lower affinity interactions were observed for activation fragments E and C1, and no detectable binding was observed for the factor Va light chain. The results support the conclusion that the factor V activation pathway is initiated by exosite I-mediated binding of thrombin to a site in the heavy chain region of factor V that facilitates the initial cleavage at Arg709 to generate the heavy chain of factor Va. The results further suggest that binding of thrombin through exosite I to factor V activation intermediates may regulate their conversion to factor Va and that similar binding of thrombin to the factor Va produced may reflect a mode of interaction involved in the regulation of prothrombin activation.

Anilino Naphthalenesulfonates↗
PubMed2001

Molecular basis for the susceptibility of fibrin-bound thrombin to inactivation by heparin cofactor ii in the presence of dermatan sulfate but not heparin.

Although fibrin-bound thrombin is resistant to inactivation by heparin.antithrombin and heparin.heparin cofactor II complexes, indirect studies in plasma systems suggest that the dermatan sulfate.heparin cofactor II complex can inhibit fibrin-bound thrombin. Herein we demonstrate that fibrin monomer produces a 240-fold decrease in the heparin-catalyzed rate of thrombin inhibition by heparin cofactor II but reduces the dermatan sulfate-catalyzed rate only 3-fold. The protection of fibrin-bound thrombin from inhibition by heparin.heparin cofactor II reflects heparin-mediated bridging of thrombin to fibrin that results in the formation of a ternary heparin.thrombin.fibrin complex. This complex, formed as a result of three binary interactions (thrombin.fibrin, thrombin.heparin, and heparin.fibrin), limits accessibility of heparin-catalyzed inhibitors to thrombin and induces conformational changes at the active site of the enzyme. In contrast, dermatan sulfate binds to thrombin but does not bind to fibrin. Although a ternary dermatan sulfate. thrombin.fibrin complex forms, without dermatan sulfate-mediated bridging of thrombin to fibrin, only two binary interactions exist (thrombin.fibrin and thrombin. dermatan sulfate). Consequently, thrombin remains susceptible to inactivation by heparin cofactor II. This study explains why fibrin-bound thrombin is susceptible to inactivation by heparin cofactor II in the presence of dermatan sulfate but not heparin.

Binding Sites↗