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PubMed1997

Intimal hyperplasia following vascular injury is not inhibited by an antisense thrombin receptor oligodeoxynucleotide.

Thrombin is a multifunctional serine protease with central functions in hemostasis, but demonstration of its role in the initiation and maintenance of cell proliferation which occurs following vascular injury is still lacking. To determine the role played by thrombin and its receptor in neointimal accumulation of smooth muscle cells in a rabbit carotid artery model, we have used an 18 mer antisense phosphorothioate oligonucleotide (ODN) directed against the translation initiation region of the human thrombin receptor gene. The antisense ODN inhibited in a dose-dependent manner thrombin- or thrombin receptor activating peptide-induced human aortic smooth muscle cell proliferation. The growth-inhibitory effect of thrombin receptor antisense ODN was preventable by an excess of sense oligomer and specific for thrombin. The suppression of growth was accompanied by a marked decrease of the level of thrombin receptor expression as evidenced by [125I]-thrombin binding to smooth muscle cells. Under the same experimental conditions, the corresponding sense ODN was inactive. The effect of the antisense ODN on intimal smooth muscle hyperplasia in rabbit carotid arteries subjected to endothelial injury was then investigated. The topical application of the antisense (500 microg/artery) but not the sense ODN dissolved in F127 pluronic gel around the injured artery resulted, 2 weeks after the application, in a dramatic reduction of the expression of the thrombin receptor mRNA and protein levels as determined by in situ hybridization and immunohistochemistry. However, intimal smooth muscle cell accumulation as estimated by an intimal to medial cross-sectional area ratio was reduced only by 2.7% (vs. 10.3% for the sense ODN), whereas r-hirudin (200 microg/kg/day, s.c.), a potent direct thrombin inhibitor significantly reduced the formation of neointima in denuded carotid arteries (35.4% inhibition, P = 0.03).

Amino Acid Sequence↗
PubMed2006

The modulation of gene expression in osteoblasts by thrombin coated on biphasic calcium phosphate ceramic.

For many years, fibrin sealants were associated with bone substitutes to promote bone healing. However, the osteoblastic response to fibrin sealant components remains poorly documented. In this study, MC3T3-E1 osteoblastic cells were cultured on biphasic calcium phosphate ceramic (MBCP) coated with Tissucol components (thrombin and fibrinogen). Analysis of osteoblastic differentiation markers by RT-PCR revealed that MBCP coated with Tissucol stimulated mRNA levels for osteocalcin and alkaline phosphatase (ALP). Of all the components of Tissucol, thrombin has been reported to affect osteoblastic behavior. Our results demonstrated that low thrombin concentrations (0.5-5 U/ml) stimulated mRNA levels for ALP, whereas high thrombin concentrations (50-100 U/ml) decreased mRNA levels for ALP and PTH/PTHrP receptor and also increased mRNA level for the osteoclastogenesis inhibitor OPG. As thrombin stimulated angiogenesis, we then wondered whether thrombin could influence the expression of angiogenic factors. Low thrombin concentrations were shown to up-regulate mRNA levels for VEGF-B and VEGF-R1, suggesting an autocrine/paracrine role for VEGF-B. Higher thrombin concentrations also up-regulated mRNA for VEGF-A and neuropilin-1. In conclusion, the association of MBCP with thrombin and fibrinogen appears to be a convenient scaffold for bone cell differentiation. Thrombin could also acts at the cellular level by increasing the angiogenic potential of osteoblasts as well as their responsiveness to thrombin and VEGF.

Animals↗
PubMed2006

Arginase activity is increased by thrombin: a mechanism for endothelial dysfunction in arterial thrombosis.

BACKGROUND: Earlier observations implicate arterial thrombosis causing endothelial dysfunction by decreasing nitric oxide (NO) levels. NO levels are restored by regional L-arginine supplementation in animal models. The purpose of this study was to investigate the roles of thrombus components in NO generation. STUDY DESIGN: Human umbilical vein endothelial cells were harvested and cultured. The thrombus components thrombin, thrombin receptor agonist peptide (TRAP), and fibrin were added to a media of confluent human umbilical vein endothelial cells. Endothelial nitric oxide synthase (eNOS) activity was assayed by measuring conversion of L-arginine to L-citrulline. Endothelial NOS mRNA levels were quantitated using real-time polymerase chain reaction. Cellular membrane transport of L-arginine through the y+ channel was assayed with (14)C-labeled L-arginine. Arginase activity was determined as the conversion of (14)C L-arginine to (14)C urea and trapped as Na(2)(14)CO(3) for scintillation counting. Arginase protein amounts were assessed using Western blotting. RESULTS: Endothelial cells exposed to thrombin for 4 hours led to increased arginase activity. Thrombin (10 U/mL) caused a 1.6-fold increase compared with that in controls (320+/-29 microM urea/min versus 194+/-10 microM urea/min, p=0.03), and thrombin (30 U/mL) increased arginase activity 2.1-fold (398+/-27 microM urea/min, p < 0.001, versus controls); thrombin at 1 U/mL and fibrin had no effect. TRAP (50 microM) had an effect similar to that of thrombin 10 U/mL (316+/-21 microM urea/min, p < 0.01, versus controls). Protein amounts of arginase corresponded with activity levels. Neither eNOS nor inducible nitric oxide synthase (iNOS) activities were affected by exposure to thrombin and TRAP for 4 hours. Similarly, quantification of eNOS, iNOS, and endothelin-1 mRNA did not change, although CL-100, a known thrombin-inducible gene, was upregulated. Finally, transport of L-arginine into endothelial cells was unaffected by thrombin, TRAP, and fibrin exposure. CONCLUSIONS: Endothelial cells exposed to thrombin have increased arginase enzymatic activity, and the remainder of NO generation capability is unaffected. L-arginine supplementation or arginase blockade may counteract endothelial dysfunction in the setting of acute arterial thrombosis.

Arginase↗
PubMed1997

Nonproteolytic activation of the thrombin receptor promotes human umbilical vein endothelial cell growth but not intracellular CA2+, prostacyclin, or permeability.

Both thrombin and the synthetic tetracapeptide thrombin receptor-activating peptide (TRAP), recently described as a peptide mimicking the new amino terminus created by cleavage of the thrombin receptor, stimulated the proliferation of human umbilical vein endothelial cells (HUVEC) in culture. Although to a lesser extent, F-14, a tetradecapeptide representing the residues 365-378 of human prothrombin, also promoted HUVEC growth, thereby demonstrating that thrombin can stimulate HUVEC growth via both a proteolytic and a nonenzymatic pathway. Thrombin-TRAP, and F-14-induced HUVEC growth were inhibited by a thrombin receptor oligodeoxynucleotide antisense, showing that the growth-inducing effects of all 3 compounds were mediated through the same thrombin receptor. Thrombin and TRAP also stimulated intracellular Ca2+ increase, monolayer permeability increase, and prostacyclin release in HUVEC. None of these effects was observed with F-14 suggesting that thrombin-induced intracellular Ca2+ release, permeability increase, and prostacyclin release in HUVEC required catalytic cleavage of the receptor, whereas thrombin-induced growth might also be due to activation of the thrombin receptor through a nonproteolytic pathway.

Calcium↗
PubMed1998

Therapeutic inhibition of thrombin activities, receptors, and production.

Thrombin mediates acute vascular thrombosis following mechanical denuding injury or spontaneous rupture of atherosclerotic plaques. In the process of generating thrombin, factor VII/VIIa binds avidly with tissue factor exposed on cellular membranes, leading to sequential activation of coagulation serine proteases via macromolecular catalytic complexes on phospholipid surfaces. At sites of disrupted arteries thrombin activates platelets, blood leukocytes, endothelium, and vascular SMCs by cleaving G protein-coupled TRs, mediating SMC intimal proliferation in the formation of neointimal vascular lesions. Therapeutic strategies targeting thrombin include inactivation of bound thrombin, inhibition of TR activation by thrombin, and interruption of thrombin production. In patients having orthopedic surgery, inactivating bound thrombin with direct antithrombins markedly reduces venous thromboembolism as compared with heparin or its derivatives, without significant impairment of hemostasis. Antithrombotic effects in arterial thromboembolism, such as acute coronary syndrome, are not conclusively benefitted by systemic direct antithrombins when administered at safe levels, because interrupting TR-dependent platelet thrombosis demands systemic levels of direct antithrombins that compromise hemostatic function. Alternative safer strategies evolving from preclinical studies include (1) inhibiting thrombin activation of TRs, thereby abolishing platelet recruitment in arterial thrombogenesis, while sparing fibrin formation in hemostatic plugs; (2) enhancing the formation of endogenous activated protein C by protein C-selective thrombin mutants; and (3) preventing thrombin production by inhibiting precursor serine protease function and interrupting the formation of both acute thrombosis and vascular lesion formation. Tissue factor pathway antagonists are particularly promising because they exhibit both efficacy and safety in the prevention of thrombosis and vascular lesions.

Anticoagulants↗
PubMed1987

Binding of thrombin to thrombomodulin accelerates inhibition of the enzyme by antithrombin III. Evidence for a heparin-independent mechanism.

The endothelial cell surface provides a receptor for thrombin-designated thrombomodulin (TM) which regulates thrombin formation and the activity of the enzyme at the vessel wall surface by serving as a potent cofactor for the activation of protein C by thrombin. Heparin-like structures of the vessel wall have been proposed as another regulatory mechanism catalyzing the inhibition of thrombin by antithrombin III. In the present study, the interaction of antithrombin III with the thrombin-TM complex and its interference with heparin and polycations were investigated by using human components and TM isolated from the microvasculature of rabbit lung. Purified TM bound thrombin and acted as a cofactor for protein C activation. The addition of heparin (0.5 unit/mL) to the reaction mixture interfered neither with the binding of thrombin to TM nor with the activation of protein C. However, the polycations protamine (1 unit/mL) as well as polybrene (0.1 mg/mL) affected the thrombin-TM interaction. This was documented by an increase in the Michaelis constant from 8.3 microM for thrombin alone to 19.5 microM for thrombin-TM with the chromogenic substrate compound S-2238 in the presence of 1 unit/mL protamine. When the inhibition of thrombin by antithrombin III was determined, the second-order rate constant k2 = 8.4 X 10(3) M-1 s-1 increased about 8-fold in the presence of TM, implying an accelerative function of TM in this reaction. Although purified TM did not bind to antithrombin III-Sepharose, suggesting the absence of heparin-like structures within the receptor molecule, protamine reversed the accelerative effect of TM in the inhibition reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Role of thrombin receptor in breast cancer invasiveness.

Invasion, the ability of an epithelial cancer cell to detach from and move through a basement membrane, is a central process in tumour metastasis. Two components of invasion are proteolysis of extracellular matrix and cellular movement through it. A potential promoter of these two processes is thrombin, the serine proteinase derived from the ubiquitous plasma protein prothrombin. Thrombin promotes the invasion of MDA-MB231 breast tumour cells (a highly aggressive cell line) in an in vitro assay. Invasion by MDA-MB436 and MCF-7 cells, less aggressive cell lines, is not promoted by thrombin. Thrombin, added to the cells, is a stimulator of cellular movement; fibroblast-conditioned medium is the chemotaxin. Thrombin-promoted invasion is inhibited by hirudin. Stimulation of invasion is a receptor-mediated process that is mimicked by a thrombin receptor-activating peptide. Thrombin has no effect on chemotaxis in vitro. Thrombin receptor is detectable on the surface of MDA-MB231 cells, but not on the other two cell lines. Introduction of oestrogen receptors into MDA-MB231 cells by transfection with pHEO had no effect on thrombin receptor expression, in the presence or absence of oestradiol. This paper demonstrates that thrombin increases invasion by the aggressive breast cancer cell line MDA-MB231 by a thrombin receptor-dependent mechanism.

Breast Neoplasms↗
PubMed1989

Stability of the thrombin-thrombomodulin complex on the surface of endothelial cells from human saphenous vein or from the cell line EA.hy 926.

Protein C activation by alpha-thrombin on the surface of endothelial cells depends on an essential membrane-glycoprotein cofactor, thrombomodulin. In the present study we have monitored the activity of thrombin-thrombomodulin complexes on human saphenous-vein endothelial cells (HSVEC) or on the endothelial cell line EA.hy 926. Cell monolayers were exposed for 5 min to 8.5 nM human alpha-thrombin and then washed to remove unbound thrombin. The cells were then incubated at 37 degrees C for 5-180 min. At the end of the respective incubation periods, purified human protein C (120 nM) was added in order to assay the activity of the thrombin-thrombomodulin complexes present on the cell surface. HSVEC pre-exposed to thrombin retained their full capacity to promote protein C activation up to 90 min after free thrombin was removed. This capacity then decreased slowly to reach 56% of control value after 180 min of incubation. Original activity was 3.8 +/- 0.9 pmol of activated protein C formed/min per ml per 10(6) cells (mean +/- S.E.M., n = 5). The capacity of protein C activation of EA.hy 926 cells remained constant for 120 min after free thrombin was removed, then decreased to 76% of control after 180 min. Original activity was 2.0 +/- 0.4 pmol of activated protein C formed/min per ml per 10(6) cells (mean +/- S.E.M., n = 3). Similar results were obtained with cells fixed with 3% paraformaldehyde. However, during the 5-180 min incubation period, non-fixed cells of both types were capable of significantly internalizing fluorescent acetylated low-density lipoprotein. In the experimental protocol used here, an eventual inhibition of thrombin internalization by protein C can be excluded, as protein C is only added at the end of the incubation period. We conclude that there is no evidence of rapid internalization of thrombin-thrombomodulin complexes on HSVEC or the EA.hy 926 cell line, as assessed by the ability of membrane-bound thrombin to activate protein C.

Cell Line↗
PubMed1993

Role of the glycosaminoglycan component of thrombomodulin in its acceleration of the inactivation of single-chain urokinase-type plasminogen activator by thrombin.

Thrombomodulin (TM), a membrane proteoglycan on endothelial cells, binds thrombin in a 1:1 complex, accelerates the protein C activation by thrombin, promotes the thrombin inactivation by antithrombin III and inhibits the procoagulant properties of thrombin. The inactivation of single-chain urokinase-type plasminogen activator (scu-PA) by thrombin is accelerated about 70-fold by TM [De Munk, Groeneveld and Rijken (1991) J. Clin. Invest. 88, 1680-1684]. The present study investigates the role of the O-linked glycosaminoglycan moiety of TM in the latter reaction. In the presence of an excess of a fully-glycosylated soluble recombinant human TM mutant (high-Mr rec-TM), 0.11 nM thrombin inactivated 50% of 4.4 nM scu-PA in 45 min at 37 degrees C. In the presence of a soluble recombinant TM mutant lacking the glycosaminoglycans (low-Mr rec-TM), 1.9 nM thrombin was needed to inactivate 50% scu-PA, as compared with 4.7 nM thrombin in the absence of TM. Using the scu-PA inactivation assay the dissociation constant for the thrombin-TM interaction was found to be 0.4 nM for high-Mr rec-TM and 14 nM for low-Mr rec-TM. Treatment of high-Mr rec-TM with chondroitinase ABC to digest the glycosaminoglycans decreased the accelerating effect to the level of low-Mr rec-TM. A similar decrease was observed after treatment of solubilized rabbit TM with chondroitinase ABC. As expected, chondroitinase ABC had no influence on the accelerating effect of low-Mr rec-TM. The free glycosaminoglycans obtained by alkaline treatment of TM or chondroitin sulphate A also accelerated the inactivation of scu-PA by thrombin, but about 1000-fold higher concentrations than with TM were needed to obtain the same acceleration. It is concluded that the major glycosaminoglycan of TM plays a pivotal role in the inactivation of scu-PA by the TM-thrombin complex, both in the formation and in the activity of the complex.

Chondroitin Lyases↗
PubMed1996

The serine protease granzyme A does not induce platelet aggregation but inhibits responses triggered by thrombin.

Granzyme A is a serine protease stored in cytoplasmic granules of cytotoxic and helper T lymphocytes. This protease seems to elicit thrombin receptor-mediated responses in neural cells, thereby triggering neurite retraction and reversal of astrocyte stellation. Here we report that granzyme A does not cause platelet aggregation even at concentrations that are more than two orders of magnitude higher than the EC50 for granzyme A in causing morphological changes in neural cells. However, granzyme A blocks thrombin-induced platelet aggregation in a dose-dependent manner without affecting the response to either ADP or to the peptide agonist of the thrombin receptor SFLLRN that corresponds in sequence to the tethered ligand domain. The inability of granzyme A to cause aggregation and its inhibition of thrombin-induced aggregation were seen in platelets from man, rat and mouse. Granzyme A does not affect the catalytic activity of thrombin in cleaving a chromogenic substrate or the macromolecular substrate fibrinogen. However, granzyme A does seem to cleave the thrombin receptor on platelets to produce a weak Ca2+ signal and reduce the response to subsequent challenge with thrombin, but does not induce a signal in thrombin-stimulated platelets. It is proposed that granzyme A interacts with the thrombin receptor found on platelets in a manner that is insufficient to cause aggregation, but sufficient to compete with thrombin for the receptor. These results suggest that granzyme A cleaves the thrombin receptor at a rate that is insufficient to cause platelet aggregation but is sufficient to cause morphological changes in neural cells. Furthermore, these observations demonstrate that granzyme A release occurring during immune responses within blood vessels would not directly cause platelet aggregation.

Amino Acid Sequence↗
PubMed1996

Thrombin stimulates production of interleukin-8 in human umbilical vein endothelial cells.

Interleukin-8 (IL-8) is regarded as an important mediator of inflammation because of its potent and specific chemotactic activity on neutrophils. In the present investigation, human umbilical vein endothelial cells (HUVEC) stimulated with thrombin were found to produce IL-8, in a dose- and time-dependent manner. After stimulation with 10 U/ml thrombin for 24 hr, the level of IL-8 in the conditioned medium was 14 ng/ml, or enough to elicit PMN chemotaxis in vitro. Northern blot analysis revealed that thrombin as well as IL-1 beta elevates the level of IL-8 mRNA preceding the formation of IL-8 protein. A synthetic peptide SFLLRN [human thrombin receptor-activating peptide (TRAP)] was found to mimic the action of thrombin. Preincubation with anti-thrombin compounds such as hirudin and antithrombin-III-heparin almost completely suppressed the action of thrombin without affecting the actions of other stimuli including IL-1 beta, phorbol 12-myristate 13-acetate (PMA) and TRAP. Diisopropylfluorophosphate-treated thrombin did not stimulate IL-8 production. Calphostin-C, a protein kinase C (PKC) inhibitor, attenuated the production of IL-8 by thrombin, TRAP and PMA, but left the action of IL-1 beta unchanged. These results strongly suggest that catalytic activation of thrombin receptor by thrombin results in PKC-dependent IL-8 production accompanied by an increase in IL-8 mRNA level.

Blotting, Northern↗
PubMed2000

Thrombin is a novel regulator of hexokinase activity in mesangial cells.

BACKGROUND: Hexokinase (HK) activity is fundamentally important to cellular glucose uptake and metabolism. Phorbol esters increase both HK activity and glucose utilization in cultured mesangial cells via a protein kinase C (PKC)- and extracellular signal-regulated kinases 1 and 2 (ERK1/2)-dependent mechanism. In adult kidneys, increased HK activity has been reported in both glomerular injury and in diabetes, but the mechanisms responsible for these changes are unknown. Thrombin, a known activator of both PKC and ERK1/2, is increased in the settings of renal injury and diabetes. Thus, thrombin may contribute to the observed changes in HK activity in vivo. METHODS: Thrombin and thrombin receptor agonists were tested for the ability to increase HK activity and glucose metabolism in murine mesangial (SV40 MES 13) cells. ERK1/2 activation was also evaluated in parallel. Thrombin inhibition (hirudins), PKC depletion, Ser-Thr kinase inhibition (H-7), MEK1/2 inhibition (PD98059), pertussis toxin (PTX), and general inhibitors of transcription or translation were then tested for the ability to attenuate these effects. RESULTS: Thrombin (>/=0.01 U/mL) mimicked the effect of phorbol esters, increasing HK activity> 50% within 12 to 24 hours (P < 0.05). This effect was inhibited by hirudins, mimicked by thrombin receptor agonists, and accompanied by increased Glc utilization. H-7, PD98059, and general inhibitors of transcription or translation-but not PTX-prevented thrombin-induced HK activity at 24 hours. PKC depletion and PD98059 also blocked the associated phosphorylation and activation of ERK1/2. CONCLUSIONS: Thrombin increases mesangial cell HK activity via a PTX-insensitive mechanism involving thrombin receptor activation, PKC-dependent activation of ERK1/2, and both ongoing gene transcription and de novo protein synthesis. As such, thrombin is a novel regulator of HK activity in mesangial cells and may play a role in coupling renal injury to metabolism.

Animals↗
PubMed1996

Thrombin, its receptor and protease nexin I, its potent serpin, in the nervous system.

The multifunctional serine protease, thrombin, the principal component of the blood coagulation cascade, is also active in nervous system growth and maintenance. In neural tissue culture, it prevents neurite outgrowth and modulates morphologic changes in both neurons and astrocytes. In recent studies, we found that it mediates polyneuronal synapse elimination, both in vivo and in vitro. Of relevance to neurologic disease, as well as to development, evidence also implicates thrombin in apoptosis of these cells. As with other serine proteases, thrombin is in "balance" with one or more endogenous protein inhibitors, members of the serpin superfamily of proteins. The most potent vertebrate inhibitor for thrombin is protease nexin I (PNI), which regulates thrombin's effect by forming post-translational, covalent complexes with the protease. We review some of the nervous system effects of the thrombin:PNI balance, and also present results of a recent study of this balance after peripheral nerve injury. We measured thrombin and prothrombin activity in extracts from adult mouse sciatic nerve using a specific chromogenic assay. We also performed reverse transcription polymerase chain reaction of RNA from nerve crush samples. We found a burst of activity at 3 days following injury distal to the crush site that was inhibited by thrombin specific inhibitors. It is possible that a significant fraction of the increased prothrombin in injured nerve was synthesized locally. Active PNI levels increased in these crush samples 6 to 9 days after the thrombin induction. These data suggest that nerve injury first induces the synthesis of prothrombin, which is subsequently converted to active thrombin. Nerve crush-induced thrombin is followed by the generation of functionally active PNI and may be directly responsible for its induction. These results suggest that the balance between serine proteases and their serpins is dysregulated during nerve injury and support a role for its reestablishment in nerve damage repair.

Adult↗
PubMed2000

The protease thrombin is an endogenous mediator of hippocampal neuroprotection against ischemia at low concentrations but causes degeneration at high concentrations.

We have considered the extracellular serine protease thrombin and its receptor as endogenous mediators of neuronal protection against brain ischemia. Exposure of gerbils to prior mild ischemic insults, here two relatively short-lasting occlusions (2 min) of both common carotid arteries applied at 1-day intervals 2 days before a severe occlusion (6 min), caused a robust ischemic tolerance of hippocampal CA1 neurons. This resistance was impaired if the specific thrombin inhibitor hirudin was injected intracerebroventricularly before each short-lasting insult. Thus, efficient native neuroprotective mechanisms exist and endogenous thrombin seems to be involved therein. In vitro experiments using organotypic slice cultures of rat hippocampus revealed that thrombin can have protective but also deleterious effects on hippocampal CA1 neurons. Low concentrations of thrombin (50 pM, 0.01 unit/ml) or of a synthetic thrombin receptor agonist (10 microM) induced significant neuroprotection against experimental ischemia. In contrast, 50 nM (10 units/ml) thrombin decreased further the reduced neuronal survival that follows the deprivation of oxygen and glucose, and 500 nM even caused neuronal cell death by itself. Degenerative thrombin actions also might be relevant in vivo, because hirudin increased the number of surviving neurons when applied before a 6-min occlusion. Among the thrombin concentrations tested, 50 pM induced intracellular Ca(2+) spikes in fura-2-loaded CA1 neurons whereas higher concentrations caused a sustained Ca(2+) elevation. Thus, distinct Ca(2+) signals may define whether or not thrombin initiates protection. Taken together, in vivo and in vitro data suggest that thrombin can determine neuronal cell death or survival after brain ischemia.

Animals↗
PubMed1995

Thrombin induces the activation of progelatinase A in vascular endothelial cells. Physiologic regulation of angiogenesis.

Angiogenesis requires degradation of vascular basement membrane prior to migration and proliferation of endothelial cells; proteinases are essential ingredients in this process. Because of thrombin's multiple effects on endothelium, we have examined its role in matrix metalloproteinase activation using human umbilical vein endothelial cells. Gelatin zymography of endothelial conditioned media revealed a prominent 72-kDa progelatinase A band. Addition of alpha-thrombin to endothelial cells resulted in the generation of 64 and 62 kDa gelatinolytic bands which is consistent with the activation of progelatinase A; thrombin had no effect in the absence of cells. This effect requires the proteolytic site of thrombin since progelatinase A activation was abolished by specific inhibitors of thrombin. Matrix metaloproteinase inhibitors diminished thrombin-induced activation of progelatinase A. Pretreatment of endothelial cells with excess tissue inhibitor of metalloproteinase-2 or a COOH-terminal fragment of progelatinase A abrogated thrombin-mediated activation of progelatinase A presumably by competing with the COOH terminus of native progelatinase A for interaction with an activator site on endothelial plasma membranes. Although membrane-type matrix metalloproteinase was demonstrated in endothelial cells by Northern and Western blotting, the receptor function of this molecule in thrombin-induced activation of progelatinase A needs to be clarified. Progelatinase A activation did not require intracellular signal transduction events mediated by the thrombin receptor. These data demonstrate that 1) endothelial cells express a novel activation mechanism for progelatinase A, 2) proteolytically active thrombin regulates this activation mechanism, and 3) activation occurs independently of the functional thrombin receptor.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Thrombin induces NO release from cultured rat microglia via protein kinase C, mitogen-activated protein kinase, and NF-kappa B.

Microglia, brain resident macrophages, become activated in brains injured due to trauma, ischemia, or neurodegenerative diseases. In this study, we found that thrombin treatment of microglia induced NO release/inducible nitric-oxide synthase expression, a prominent marker of activation. The effect of thrombin on NO release increased dose-dependently within the range of 5-20 units/ml. In immunoblot analyses, inducible nitric-oxide synthase expression was detected within 9 h after thrombin treatment. This effect of thrombin was significantly reduced by protein kinase C inhibitors, such as Go6976, bisindolylmaleimide, and Ro31-8220. Within 15 min, thrombin activated three subtypes of mitogen-activated protein kinases: extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase/stress-activated protein kinase. Inhibition of the extracellular signal-regulated kinase pathway and p38 reduced the NO release of thrombin-treated microglia. Thrombin also activated nuclear factor kappaB (NF-kappaB) within 5 min, and N-acetyl cysteine, an inhibitor of NF-kappaB, reduced NO release. However, thrombin receptor agonist peptide (an agonist of protease activated receptor-1 (PAR-1)), could not mimic the effect of thrombin, and cathepsin G, a PAR-1 inhibitor, did not reduce the effect of thrombin. These results suggest that thrombin can activate microglia via protein kinase C, mitogen-activated protein kinases, and NF-kappaB but that this occurs independently of PAR-1.

Acetylcysteine↗
PubMed2003

Thrombin up-regulates tissue factor pathway inhibitor-2 synthesis through a cyclooxygenase-2-dependent, epidermal growth factor receptor-independent mechanism.

The serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2) inhibits the tissue factor-factor VIIa complex and thereby impairs factor Xa and subsequently thrombin generation. Here we show that thrombin itself up-regulates TFPI-2 mRNA and protein expression in human liver myofibroblasts, a cell type shown to express high levels of TFPI-2 (Neaud, V., Hisaka, T., Monvoisin, A., Bedin, C., Balabaud, C., Foster, D. C., Desmoulière, A., Kisiel, W., and Rosenbaum, J. (2000) J. Biol. Chem. 275, 35565-35569). This effect required thrombin catalytic activity, as shown by its abolition with hirudin. Although the thrombin effect could be mimicked by agonists of both protease-activated receptor (PAR)-1 and PAR-4, it was largely blocked by a PAR-1 blocking antibody. Transactivation of the epidermal growth factor (EGF) receptor has been reported as a common event in thrombin signaling. However, thrombin did not detectably transactivate the EGF receptor in liver myofibroblasts, and blocking the EGF receptor did not affect TFPI-2 induction. On the other hand, thrombin increased the expression of cyclooxygenase-2 (COX-2) mRNA via a MAPK-dependent pathway, and a specific COX-2 inhibitor abolished the effect of thrombin on TFPI-2 expression. Thus, thrombin, through PAR-1 signaling, up-regulates the synthesis of TFPI-2 via a MAPK/COX-2-dependent pathway. The up-regulation of TFPI-2 expression by thrombin could in turn down-regulate thrombin generation and contribute to limit blood coagulation.

Blotting, Northern↗
PubMed1993

Late-fibrin(ogen) fragment E modulates human alpha-thrombin specificity.

Fibrinogen contains at least two independent sites having demonstrable affinity for alpha-thrombin. One of these two sites, located in the fibrin E domain, binds to structures within the anion-binding exosite of alpha-thrombin. Taking advantage of its solubility, we have used late-fibrin(ogen) fragment E in competition experiments to examine its effect on alpha-thrombin specificity. We show that fragment E modulates alpha-thrombin enzymic activity towards small synthetic substrates, suggesting that fibrin-thrombin interaction might induce subtle changes in the conformation near the catalytic center of the enzyme. In addition, fragment E behaved as a competitive inhibitor of alpha-thrombin-catalyzed fibrinopeptide-A cleavage (Ki = 5.2 +/- 1.3 microM), indicating that alpha-thrombin interaction with the fibrin moiety of fibrinogen makes a major contribution to the efficacy of fibrinogen hydrolysis. Fragment E inhibited alpha-thrombin-induced serotonin release by platelets (concentration required to obtain 50% inhibition, IC50 = 10 microM) and alpha-thrombin binding to GPIb. Fragment E competitively inhibited alpha-thrombin binding to thrombomodulin (Ki = 18.3 +/- 0.8 microM) but did not inhibit protein-C activation in the absence of thrombomodulin. The data are consistent with the proposal that fibrin, platelet GPIb and thrombomodulin bind to overlapping, but probably non-identical sites, while protein C binds to an independent site on alpha-thrombin.

Amino Acid Sequence↗