Thrombin's effects on osteoblastic cells. I. Cytosolic calcium and phosphoinositides

Thrombin, a blood coagulation factor, has been shown to be a very effective in vitro bone resorbing agent whose mechanism of action on osteoblastic cells remains to be elucidated. In the present study, the effects of highly purified human thrombin on Saos-2 and G292 cells, two human osteoblast-like osteosarcoma cell lines, were investigated. Thrombin (0.6-16 U/ml) caused a significant, dose-dependent increase in osteoblastic cell proliferation. Thrombin also elicited a dose-dependent increase in cytosolic calcium concentration in both Saos-2 and G292 cells (maximal increases were 38% and 200% over baseline, respectively). Addition of thrombin to the osteoblast-like cells resulted in significant time- and dose-dependent changes in phosphoinositide levels: the percentage of inositol monophosphate levels were decreased, whereas the percentage of inositol bisphosphate, inositol trisphosphate and inositol tetrakisphosphate levels were increased. The relative magnitude of the changes in phosphoinositide levels was similar to the changes in cytosolic calcium concentration. These results suggest that thrombin's mechanism of action on bone cells may involve increases in cytosolic calcium levels and in phosphoinositide metabolism.     

Opposing Actions of Thrombin and Protease Nexin-1 on Amyloid β-Peptide Toxicity and on Accumulation of Peroxides and Calcium in Hippocampal Neurons

Abstract: Amyloid β-peptide (Aβ) is the principal component of neuritic plaques in the brain in Alzheimer's disease (AD). Recent studies revealed that Aβ can be neurotoxic by a mechanism involving free radical production and loss of cellular ion homeostasis, thus implicating Aβ as a key factor in the pathogenesis of AD. However, other proteins are present in plaques in AD, including the protease thrombin and protease nexin-1 (PN1), a thrombin inhibitor. We therefore tested the hypothesis that thrombin and PN1 modify neuronal vulnerability to Aβ toxicity. In dissociated rat hippocampal cell cultures the toxicity of Aβ was significantly enhanced by coincubation with thrombin, whereas PN1 protected neurons against Aβ toxicity. Aβ induced an increase in levels of intracellular peroxides and calcium. Thrombin enhanced, and PN1 attenuated, the accumulation of peroxides and calcium induced by Aβ. Taken together, these data demonstrate that thrombin and PN1 have opposing effects on neuronal vulnerability to Aβ and suggest that thrombin and PN1 play roles in the pathogenesis of neuronal injury in AD.     

凝血酶诱导大鼠海马神经细胞凋亡及其与Bcl-2及Bax表达变化关系的实验研究

目的研究不同浓度凝血酶诱导海马神经元凋亡的作用及其机制.方法将原代培养新生大鼠海马神经元分为对照组,凝血酶组(1U/ml,10U/ml,20U/ml,40U/ml),凝血酶受体激活肽组.应用TUNEL及流式细胞仪检测凋亡细胞数及凋亡百分率,免疫细胞化学方法检测Bcl-2,Bax蛋白表达.结果低浓度凝血酶组(1U/ml)凋亡细胞数和凋亡率与对照组无差异,Bcl-2表达增加;随凝血酶浓度增加,TUNEL阳性细胞数及凋亡率明显增多,Bcl-2表达下调,Bax表达上调,Bcl-2/Bax比值降低.凝血酶受体激活肽的作用与大剂量凝血酶类似.结论凝血酶可能通过激活PAR-1受体诱导凋亡,凋亡呈剂量依赖性.Bcl-2的表达减少,Bax的表达增加,Bcl-2/Bax降低可能为高浓度凝血酶诱导凋亡的机制之一.     

Exposure of Astrocytes to Thrombin Reduces Levels of the Metabotropic Glutamate Receptor mGluR5

Abstract: Thrombin is one of the first regulatory molecules present at sites of CNS trauma or injury. Exposure of neuronal and glial cells to thrombin produces potent morphological as well as cytoprotective and cytotoxic effects, but little is known about how this important modulator affects neurotransmitter signaling. In astrocyte cultures that have been morphologically differentiated by exposure to transforming growth factor-α, addition of thrombin induced a retraction of astrocytic processes and suppressed the stimulation of phosphoinositide hydrolysis by the selective metabotropic glutamate receptor (mGluR) agonist 1-aminocyclopentane-1 S ,3 R -dicarboxylic acid. In addition to the suppression of phosphoinositide hydrolysis, thrombin treatment produced a corresponding reduction in level of mGluR5 mRNA as demonstrated with ribonuclease protection assay and reduced content of mGluR5 receptor protein as seen with western blotting. In contrast, thrombin exposure up-regulated astrocyte β-actin mRNA levels. A synthetic hexapeptide with a sequence corresponding to the amino-terminus of the thrombin receptor's tethered ligand also mimicked the ability of thrombin to suppress mGluR5 levels and to increase β-actin mRNA content, suggesting that these effects of thrombin are mediated by proteolytically activated cell surface thrombin receptors. Thrombin's suppressive effect on mGluR5 was resistant to pretreatment with pertussis toxin or various protein kinase and protein phosphatase inhibitors. However, the serine/threonine protein kinase inhibitor H-7 did prevent thrombin-induced reversal of astrocyte stellation and induction of β-actin mRNA levels, indicating that these effects of thrombin involve a signaling pathway distinct from the one that mediates the suppressive effects of thrombin on mGluR5.     

Effect of thrombin on survival of hippocampal neurons

The effect of thrombin on the rat hippocampal neurons death in model of neurotoxicity induced by hemoglobin or glutamate, was studied. Thrombin (10 nM) was shown to inhibit 100-mkM glutamate--or 10-mkM hemoglobin-induced apoptosis of the rat hippocampal neurons. With the aid of PAR1 (protease-activated receptor1) agonist peptide and PAR1 antagonist, the PAR1 was found to be necessary for protective action of thrombin in hippocampal neurons in models of neurotoxicity induced by hemoglobin or glutamate. Because the prolonged elevation [Ca2+] ib neurons is a critical part of neurodestructive processes in CNS, the effect of thrombin on Ca2+-homeostatis of neurons after its injury by the inducer of neuronal apoptosis: a synthetic agonist of the NMDA receptors N-methyl-D-aspartate (NMDA), was studied. We hypothesized that thrombin via receptors PAR may prove to be neuroprotective for the hippocampus. Thrombin was shown to stimulate via PAR1 a transient increase in [Ca2+] in neurons in a concentration-dependent manner. Thrombin (1 nM) decreased the [Ca2+] signal induced by activation of the NMDA-subtype of glutamate receptors. This thrombin effect may be one of the reasons of the protective action of thrombin in hippocampal neurons.     

Reorganization of microtubule system in pulmonary endothelial cells in response to thrombin treatment

Thrombin induces rapid and reversible increase of endothelial (EC) barrier permeability associated with actin cytoskeleton remodeling and contraction. The role of microtubules (Mts) in EC barrier regulation compared with actin systems is poorly understood. In this work we studied pathways of Mt and actin regulation in response to thrombin treatment in cultured EC, and the involvement of trimeric G-proteins and in this process. Cells were treated with thrombin, and further analysed using immunofluorescent staining of actin and Mts, digital microscopy and morphometric analysis. In normal cells actin network consists of thin bundles basically located in the cell periphery, Mt density decreases from the cell center to the cell edge. Thrombin (25 nM) induced endothelial dysfunction associated with a rapid (within 5 min) decrease of peripheral Mt network and a slower actin stress fiber formation in the cytoplasm. Pretreatment with Pertussis toxin, which is Gi protein inhibitor, attenuated thrombin-induced stress fiber formation and Mt disassembly. Overexpression of activated G12, G13, Gi and Gq proteins, which are involved in thrombin receptor-mediated signaling, resulted in increasing stress fibers thickness and density and complete Mt disassembly. From the results obtained we suggest that thrombin regulates actin cytoskeleton of EC using local Mt depolymerization at the cell edge.     

Thrombin activates envelope glycoproteins of HIV type 1 and enhances fusion

To elucidate the roles of serine proteases, including thrombin, in HIV infection, we treated H9 cells infected with HIV-1 LAI virus (H9/IIIB) with four different proteases (thrombin, cathepsin G, trypsin and chymotrypsin) and observed their effects on functional epitopes on both gp120 and gp41 by using flow cytometry. Monoclonal antibodies (MAbs) against the V3 loop, V2 loop, CD4 binding site, coreceptor binding site and gp41 were used. It was found that trypsin decreased the binding of all MAbs except for one MAb against the V3 loop (IIIB-V3-21). Chymotrypsin and cathepsin G did not show any remarkable effect on the antigen expression. On the other hand, thrombin decreased the reactivities of two out of four anti-V3 MAbs and increased the exposure of functional gp120 epitopes including the coreceptor binding site and CD4 binding site. Thrombin also increased the expression of 2F5 antigen (a neutralizing epitope of gp41) but had no effect on other gp41 epitopes. The effect of trypsin or thrombin on HIV-induced cell fusion was examined through co-culturing H9/IIIB and MAGI cells. Trypsin slightly inhibited fusion. Fusion was significantly enhanced in a dose-dependent manner by thrombin, and a 280% increase at 5 U/ml (P < 0.001) was observed. In conclusion, thrombin, one of the major inflammatory molecules in blood, facilitates HIV-induced cell fusion, probably by activating gp120.     

Thrombin stimulates dissociation and induction of HSP27 via p38 MAPK in vascular smooth muscle cells

We investigated the effects of thrombin on the induction of heat shock proteins (HSP) 70 and 27, and the mechanism behind the induction in aortic smooth muscle A10 cells. Thrombin increased the level of HSP27 but had little effect on the level of HSP70. Thrombin stimulated the accumulation of HSP27 dose dependently between 0.01 and 1 U/ml and cycloheximide reduced the accumulation. Thrombin stimulated an increase in the level of HSP27 mRNA and actinomycin D suppressed the thrombin-increased mRNA level. Thrombin induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The HSP27 accumulation by thrombin was reduced by SB-203580 and PD-169316 but not by SB-202474. SB-203580 and PD-169316 suppressed the thrombin-induced phosphorylation of p38 MAPK. SB-203580 reduced the thrombin-increased level of HSP27 mRNA. Dissociation of the aggregated HSP27 to the dissociated HSP27 was induced by thrombin. Dissociation was inhibited by SB-203580. Thrombin induced the phosphorylation of HSP27 and the phosphorylation was suppressed by SB-203580. These results indicate that thrombin stimulates not only the dissociation of HSP27 but also the induction of HSP27 via p38 MAPK activation in aortic smooth muscle cells.     

Cyclin D1, cdk4, and Bim are involved in thrombin-induced apoptosis in cultured cortical neurons

Thrombin, a multifunctional serine protease, is neurotoxic in vitro and in vivo. Thrombin has been shown to be increased in Alzheimer's disease (AD) and other neuropathological conditions and could be a mediator of pathological neuronal cell death in the brain. The mechanisms of thrombin-induced neuronal cell death are incompletely understood. The objective of this study is to explore mechanisms that contribute to thrombin-induced neuronal apoptosis focusing on the role of cell cycle regulators and the pro-apoptotic protein Bim (Bcl-2-interacting mediator of cell death) in this process. Our data show that thrombin treatment of primary cerebral cortical cultures results in dose-dependent apoptotic cell death. Exposure of neuronal cultures to thrombin leads to induction of cell cycle proteins cyclin D1 and cyclin E, at both mRNA and protein levels. In addition, thrombin treatment causes the appearance of cyclin-dependent kinase 4 (cdk4) and expression of the pro-apoptotic protein Bim. Inhibition of cdk4 prevents both induction of Bim expression and thrombin-induced neuronal apoptosis. These data demonstrate that thrombin-induced apoptosis proceeds via cell cycle activation involving cdk4 resulting in induction of Bim. Thus, cell cycle proteins could be therapeutic targets in diseases such as AD where thrombin has been implicated.     

Induction of KDR expression in bovine arterial endothelial cells by thrombin: involvement of nitric oxide.

Thrombin, a multifunctional serine protease, is generated at the site with vascular injuries. It not only participates in the coagulation cascade, but also can induce a lot of events related to cell mitogenesis and migration. In this study, we investigated the effect of thrombin on endothelial cell proliferation induced by vascular endothelial growth factor (VEGF). Thrombin promoted proliferation of cultured bovine carotid endothelial cells in a time- and dose-dependent manner. Moreover, it drastically enhanced the cell growth stimulated by VEGF. This stimulatory effect was reduced by inhibitors of either protein kinase C (PKC) or mitogen-activated protein kinase kinase (MAPKK). Thrombin induced a significant increase in the level of mRNA of the kinase domain-containing receptor (KDR), but not tms-like tyrosine kinase (Flt-1), in a time-dependent manner, which reached the maximum after 24 h of stimulation. This increase coincides well with the KDR protein expression. The luciferase assay showed that thrombin induced an about 7.5-fold increase in the KDR promoter activity compared with the control. This enhanced KDR promoter activity was also abolished by inhibitors of either PKC or MAPKK. The deletion analyses indicated that the region between -115 and -97 (containing Sp1 binding region) within the KDR promoter gene was required for the enhanced KDR expression induced by thrombin and VEGF. Moreover, the nitric oxide synthase (NOS) inhibitor abolished both the accelerated cell proliferation and the increased KDR expression induced by thrombin and VEGF. This inhibition was abrogated by DETA NONOate, a NO donor with long half-life. These findings suggest that thrombin might potentiate the VEGF-induced angiogenic activity through increasing the level of the VEGF receptor KDR, in which production of NO is involved.     

Thrombin induces tumor invasion through the induction and association of matrix metalloproteinase-9 and beta1-integrin on the cell surface

The procoagulatory serine protease, thrombin, is known to induce invasion and metastasis in various cancers, but the mechanisms by which it promotes tumorigenesis are poorly understood. Because the 92-kDa gelatinase (MMP-9) is a known mediator of tumor cell invasion, we sought to determine whether and how thrombin regulates MMP-9. The thrombin receptor, PAR-1, and MMP-9 are expressed in osteosarcomas, as determined by immunohistochemistry. Stimulation of U2-OS osteosarcoma cells with thrombin and a thrombin receptor-activating peptide induced pro-MMP-9 secretion as well as cell surface-associated pro-MMP-9 expression and proteolytic activity. This was paralleled by an increase in MMP-9 mRNA and MMP-9 promoter activity. Thrombin-induced invasion of U2-OS cells through Matrigel was mediated by the phosphatidylinositol 3-kinase signaling pathway and could be inhibited with an MMP-9 antibody. The stimulation of MMP-9 by thrombin was paralleled by an increase in beta1-integrin mRNA and beta1-integrin expression on the cell surface, which was also mediated by phosphatidylinositol 3-kinase and was required for invasion. Thrombin activation induced and co-localized both beta1-integrin and pro-MMP-9 on the cell membrane, as evidenced by co-immunoprecipitation, confocal microscopy, and a protein binding assay. The thrombin-mediated association of these two proteins, as well as thrombin-mediated invasion of U2-OS cells, could be blocked with a cyclic peptide and with an antibody preventing binding of the MMP-9 hemopexin domain to beta1-integrin. These results suggest that thrombin induces expression and association of beta1-integrin with MMP-9 and that the cell surface localization of the protease by the integrin promotes tumor cell invasion.     

Thrombin induces mast cell adhesion to fibronectin: evidence for involvement of protease-activated receptor-1

Thrombin activates mast cells to release inflammatory mediators through a mechanism involving protease-activated receptor-1 (PAR-1). We hypothesized that PAR-1 activation would induce mast cell adhesion to fibronectin (FN). Fluorescent adhesion assay was performed in 96-well plates coated with FN (20 microg/ml). Murine bone marrow cultured mast cells (BMCMC) were used after 3-5 wk of culture (>98% mast cells by flow cytometry for c-Kit expression). Thrombin induced beta-hexosaminidase, IL-6, and matrix metalloproteinase-9 release from BMCMC. Thrombin and the PAR-1-activating peptide AparafluoroFRCyclohexylACitY-NH(2) (cit) induced BMCMC adhesion to FN in a dose-dependent fashion, while the PAR-1-inactive peptide FSLLRY-NH(2) had no effect. Thrombin and cit induced also BMCMC adhesion to laminin. Thrombin-mediated adhesion to FN was inhibited by anti-alpha(5) integrin Ab (51.1 +/- 6.7%; n = 5). The combination of anti-alpha(5) and anti-alpha(4) Abs induced higher inhibition (65.7 +/- 7.1%; n = 5). Unlike what is known for FcepsilonRI-mediated adhesion, PAR-1-mediated adhesion to FN did not increase mediator release. We then explored the signaling pathways involved in PAR-1-mediated mast cell adhesion. Thrombin and cit induced p44/42 and p38 phosphorylation. Pertussis toxin inhibited PAR-1-mediated BMCMC adhesion by 57.3 +/- 7.3% (n = 4), indicating that G(i) proteins are involved. Wortmannin and calphostin almost completely inhibited PAR-1-mediated mast cell adhesion, indicating that PI-3 kinase and protein kinase C are involved. Adhesion was partially inhibited by the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 (24.5 +/- 3.3%; n = 3) and the p38 inhibitor SB203580 (25.1 +/- 10.4%; n = 3). The two inhibitors had additive effects. Therefore, thrombin mediates mast cell adhesion through the activation of G(i) proteins, phosphoinositol 3-kinase, protein kinase C, and mitogen-activated protein kinase pathways.     

An Arg-Gly-Asp sequence within thrombin promotes endothelial cell adhesion

Thrombin, in addition to its central role in hemostasis, possesses diverse cellular bioregulatory functions implicated in wound healing, inflammation, and atherosclerosis. In the present study we demonstrate that thrombin molecules modified either at the procoagulant or catalytic sites induce endothelial cell (EC) adhesion, spreading, and cytoskeletal reorganization. The most potent adhesive thrombin analogue (NO2-alpha-thrombin) was obtained by nitration of tyrosine residues. The cell adhesion promoting activity of NO2-alpha-thrombin was blocked upon the formation of thrombin-antithrombin III (ATIII) complexes and by antiprothrombin antibodies, but was unaffected by hirudin. Arg-Gly-Asp-containing peptides, fully inhibited EC adhesion to NO2-alpha-thrombin, while synthetic peptides corresponding to thrombin "Loop B" mitogenic site and the thrombin-derived chemotactic fragment "CB67-129", were uneffective. Immunofluorescence studies indicated that EC adhesion to NO2-alpha-thrombin was followed by cell spreading, actin microfilament assembly, and formation of focal contacts. By the use of specific antibodies, the vitronectin (vn) receptor (alpha v beta 3) was found to be localized in clusters upon cell adhesion to NO2-alpha-thrombin. An anti alpha v beta 3 antibody blocked EC adhesion and spreading while antifibronectin (fn) receptor (alpha 5 beta 1) antibodies were uneffective. While native thrombin exhibited a very low cell attachment activity, thrombin that was incubated at 37 degrees C before coating of plastic surfaces induced EC attachment and spreading. We propose that under certain conditions the naturally hindered RGD domain within thrombin is exposed for interaction with alpha v beta 3 on EC. This in turn promotes cell adhesion, spreading, and reorganization of cytoskeletal elements, which may altogether contribute to repair mechanisms in the disturbed vessel wall. This study defines a new biological role of thrombin and characterizes a new recognition mechanism on EC for this molecule.     

Upregulation and antiapoptotic role of endogenous Alzheimer amyloid precursor protein in dorsal root ganglion neurons

The amyloid precursor protein (APP) is a transmembrane protein whose abnormal processing is associated with the pathogenesis of Alzheimer's disease. In this study, we examined the expression and role of cell-associated APP in primary dorsal root ganglion (DRG) neurons. When dissociated DRG cells prepared from mouse embryos were treated with nerve growth factor (NGF), neuronal APP levels were transiently elevated. DRG neurons treated with an antibody against cell surface APP failed to mature and underwent apoptosis. When NGF was withdrawn from the cultures after a 36-h NGF treatment, virtually all neurons underwent apoptosis by 48 h. During the course of apoptosis, some neurons with intact morphology contained increased levels of APP immunoreactivity, whereas the APP levels were greatly reduced in apoptotic neurons. Furthermore, affected neurons contained immunoreactivities for activated caspase-3, a caspase-cleaved APP fragment (APPDeltaC31), and Abeta. Downregulation of endogenous APP expression by treatment with an APP antisense oligodeoxynucleotide significantly increased the number of apoptotic neurons in NGF-deprived DRG cultures. Furthermore, overexpression of APP by adenovirus vector-mediated gene transfer reduced the number of apoptotic neurons deprived of NGF. These results suggest that endogenous APP is upregulated to exert an antiapoptotic effect on neurotrophin-deprived DRG neurons and subsequently undergoes caspase-dependent proteolysis.     

Proteolytic processing of amyloid beta protein precursor (APP) by thrombin.

Search for proteases responsible for an altered processing of APP which generates intermediates containing beta/A4 peptide is preceding to understand the formation of beta amyloid deposits characteristic of Alzheimer's disease, since many studies reveal that APP is ordinarily processed so as not to generate beta amyloid. Here, we have examined the action of thrombin, a serine protease in the blood clotting, in APP processing. Thrombin cleaved the mouse recombinant APP695 in vitro, resulting in the accumulation of 28 kDa fragment. The immunoblot analysis showed that the fragment is derived from the carboxy-terminal side of the recombinant APP695. Further, amino acid sequencing exhibited that the fragment is generated by the cleavage at Arg 510-Ile 511 and therefore includes entire beta/A4 peptide. We consider that the 28 kDa fragment is a possible intermediate for beta/A4 peptide. Thus thrombin may be involved in the altered processing of APP.     

Amyloid Precursor Protein Metabolism in Primary Cell Cultures of Neurons, Astrocytes, and Microglia

Abstract: Amyloid precursor protein (APP) gives rise by proteolytic processing to the amyloid β peptide (Aβ) found abundantly in cerebral senile plaques of individuals with Alzheimer's disease. APP is highly expressed in the brain. To assess the source of cerebral Aβ, the metabolism of APP was investigated in the major cell types of the newborn rat cerebral cortex by pulse/chase labeling and immunoprecipitation of the APP and APP metabolic fragments. We describe a novel C-terminally truncated APP isoform that appears to be made only in neurons. The synthesis, degradation, and metabolism of APP were quantified by phosphorimaging in neurons, astrocytes, and microglia. The results show that although little APP is metabolized through the amyloidogenic pathways in each of the three cultures, neurons appear to generate more Aβ than astrocytes or microglia.     

Insertion of Lysosomal Targeting Sequences to the Amyloid Precursor Protein Reduces Secretion of βA4

Abstract: The processing of the amyloid precursor protein (APP) was investigated in cells stably expressing different APP hybrid proteins. The cytoplasmic domain of APP was either deleted or replaced by the corresponding domain of the membrane protein TGN38, lamp-1, or LIMPII. The cytosolic domain of TGN38 in the APP molecule did not alter the secretion of βA4 when compared with the wild-type APP; however, APP associated with the cell surface and the nonamyloidogenic processing of APP were reduced. With the APP molecules carrying the lysosomal targeting signals of lamp-1 or LIMPII, a decrease in the secretion of βA4 was observed. Cell surface association and nonamyloidogenic processing were also impaired. This suggests increased degradation of APP and thus efficient targeting to the lysosomal system. Cells expressing the Swedish APP variant generated intracellular βA4 that accumulated after treatment with chloroquine. This effect was more dramatic with APP mutants carrying lysosomal targeting signals than with full-length APP. Our data suggest the existence of an intracellular site of βA4 generation from where βA4 is degraded rather than secreted.     

Contribution of protease-activated receptors 1 and 4 and glycoprotein Ib-IX-V in the G(i)-independent activation of platelet Rap1B by thrombin

Thrombin activates human platelets through three different membrane receptors, the protease-activated receptors PAR-1 and PAR-4 and the glycoprotein Ib (GPIb)-IX-V complex. We investigated the contribution of these three receptors to thrombin-induced activation of the small GTPase Rap1B. We found that, similarly to thrombin, selective stimulation of either PAR-1 or PAR-4 by specific activating peptides caused accumulation of GTP-bound Rap1B in a dose-dependent manner. By contrast, in PAR-1- and PAR-4-desensitized platelets, thrombin failed to activate Rap1B. Thrombin, PAR-1-, or PAR-4-activating peptides also induced the increase of intracellular Ca(2+) concentration and the release of serotonin in a dose-dependent manner. We found that activation of Rap1B by selected doses of agonists able to elicit comparable intracellular Ca(2+) increase and serotonin release was differently dependent on secreted ADP. In the presence of the ADP scavengers apyrase or phosphocreatine-phosphocreatine kinase, activation of Rap1B induced by stimulation of either PAR-1 or PAR-4 was totally inhibited. By contrast, thrombin-induced activation of Rap1B was only minimally affected by neutralization of secreted ADP. Concomitant stimulation of both PAR-1 and PAR-4 in the presence of ADP scavengers still resulted in a strongly reduced activation of Rap1B. A similar effect was also observed upon blockade of the P2Y12 receptor for ADP, as well as in P2Y12 receptor-deficient human platelets, but not after blockade of the P2Y1 receptor. Activation of Rap1B induced by thrombin was not affected by preincubation of platelets with the anti-GPIbalpha monoclonal antibody AK2 in the absence of ADP scavengers or a P2Y12 antagonist but was totally abolished when secreted ADP was neutralized or after blockade of the P2Y12 receptor. Similarly, cleavage of the extracellular portion of GPIbalpha by the cobra venom mocarhagin totally prevented Rap1B activation induced by thrombin in the presence of apyrase and in P2Y12 receptor-deficient platelets. By contrast, inhibition of MAP kinases or p160ROCK, which have been shown to be activated upon thrombin binding to GPIb-IX-V, did not affect agonist-induced activation of Rap1B in the presence of ADP scavengers. These results indicate that although both PAR-1 and PAR-4 signal Rap1B activation, the ability of thrombin to activate this GTPase independently of secreted ADP involves costimulation of both receptors as well as binding to GPIb-IX-V.     

Thrombin effects on osteoblastic cells. II. Structure-function relationships.

Thrombin has been shown to cause in vitro bone resorption and to stimulate osteoblastic cell proliferation, phosphoinositide turnover and cytosolic calcium levels. In the present study, the role of the active site of thrombin in its action on osteoblastic cells was investigated. Either hirudin or (4-amidinophenyl)methanesulfonyl fluoride inhibited, in a dose-dependent manner, the effects of thrombin on human osteoblast-like osteosarcoma cells (G292 and Saos-2 cell lines) and on normal rat calvarial osteoblastic cells. Thrombin-induced stimulation of cell proliferation, cytosolic calcium increases, and stimulation of phosphoinositide metabolism were concomitantly, and to a proportionally similar extent, inhibited. The inhibitors, when present in the absence of thrombin, did not affect the basal levels of cell functions. Both zeta-thrombin and gamma-thrombin, forms resulting from proteolytic cleavage of alpha-thrombin, were capable of stimulating the osteoblastic cells. These data indicate that thrombin's actions on osteoblast-like cells are dependent on the availability of its catalytic site.     

Influence of thrombin on proliferation and prostaglandin production in cultured bovine endometrial cells

The control of cell proliferation by thrombin was studied in vitro in cultured epithelial and stromal cells of the endometrium. The effect of thrombin was studied after chronic treatment (72 hr) in medium containing 10% fetal bovine serum (FBS) combined or not with sex steroids. Thrombin inhibited slightly the proliferation (based on DNA measurements) only in epithelial cells (P < 0.05). 17β-estradiol (E) and progesterone (P4) had no mitogenic effects. The presence of functional thrombin receptors was estimated by stimulation of second messenger generation in response to increasing doses of thrombin (0-1,500 ng/ml). In confluent cultures of epithelial cells, the addition of thrombin for 10 min stimulated cAMP production by 50% with a maximal response at 500 ng/ml (P < 0.05). Similarly, in stromal cells, thrombin stimulated cAMP production in a dose-dependent manner (P < 0.01). Generation of inositol-phosphates was also stimulated by 50% in epithelial cells (P < 0.03), with a maximal response at 500 ng/ml, and by 45% in stromal cells (P < 0.01), with a maximal response at 50 ng/ml. The effect of thrombin on cell proliferation was investigated by ³H-thymidine incorporation in serum-free medium for 24 hr. Thrombin inhibited incorporation in epithelial cells (P < 0.0001) in a dose-dependent manner. Conversely, thrombin stimulated significantly incorporation of stromal cells (P < 0.05) at 50 ng/ml. The effect of sex steroids was also evaluated and it was found that E had no effect on cell proliferation, while P4 inhibited the incorporation in both epithelial (P < 0.001) and stromal cells (P < 0.001). The effect of a combined treatment with thrombin and E inhibited both epithelial (P < 0.001) and stromal cell (P < 0.001) growth, but a combination of thrombin and P4 had no additional effect on growth compared to P4 alone. Further investigation of the role of thrombin has been carried out by measuring prostaglandin (PG) responses. Addition of thrombin for 24 hr inhibited PGF_2α production by epithelial cells (P < 0.0001) but had no effect on PGE₂ production by stromal cells. Therefore, functional receptors for thrombin appear to be present in epithelial and stromal cells of the bovine endometrium. The minimal effect of thrombin alone or in combination with sex steroids on endometrial cell proliferation in vitro combined with the evidence of functional thrombin receptor in these cells, suggest that: (1) the effect of sex steroids in cultured endometrial cells is not modulated by the presence of thrombin, and (2) other factors are necessary for the full expression of mitogenic responses to sex steroids in vitro. © 1996 Wiley-Liss, Inc.