Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the accompanying paper (Nemoto, Y., Namba, T., Teru-uchi, T., Ushikubi, F., Morii, N., and Narumiya, S. (1992) J. Biol. Chem. 267, 20916-20920), we have identified rhoA protein as the sole substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human blood platelets. Here we examined the role of rhoA protein in platelet functions. C3 exoenzyme added to washed platelets dose- and time-dependently ADP-ribosylated rhoA protein in situ in the cells. Concomitant with this modification, inhibition of thrombin-induced platelet aggregation was observed. This inhibition was not reversed by washing the treated platelets, but was not found when C3 exoenzyme was pretreated with mouse monoclonal anti-C3 exoenzyme antibody. C3 exoenzyme treatment did not affect thrombin-induced inositol 1,4,5-trisphosphate production. Secretion of preloaded [14C]serotonin was delayed by the enzyme treatment, but the extent of the secretion was not influenced. In addition, the enzyme treatment did not change the expression of the glycoprotein IIb-IIIa complex on the platelet surface. The enzyme treatment also suppressed platelet aggregation induced by phorbol myristate acetate. These results suggest that rhoA protein plays a role mainly in the aggregation process downstream from receptor-phospholipase C coupling. This, together with the previous finding that rhoA protein modulates stress fiber formation in cultured fibroblasts (Paterson, H. F., Self, A. J., Garrett, M. D., Just, I., Aktories, K., and Hall, A. (1990) J. Cell Biol. 111, 1001-1007), suggests that rhoA protein regulates the assembly of actin filaments and the avidity of the platelet integrin (glycoprotein IIb-IIIa) in the aggregation process.
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PMID:A rho gene product in human blood platelets. II. Effects of the ADP-ribosylation by botulinum C3 ADP-ribosyltransferase on platelet aggregation. 140 Apr 7

The vav proto-oncogene product, p95vav or Vav, is primarily expressed in hematopoietic cells and has been shown to be a substrate for tyrosine kinases. Although its function is unknown, Vav shares a region of homology with DBL, an exchange factor for the Rho family of GTP-binding proteins. The presence of this domain and the observation that cells transformed with Vav display prominent stress fibers and focal adhesions similar to those that are observed in RhoA transformed cells suggests that Vav may play a role in regulating the actin cytoskeleton. We have, therefore, examined Vav phosphorylation in platelets, which undergo dramatic cytoskeletal reorganization in response to agonists. Two potent platelet agonists, thrombin (via its G protein-coupled receptor) and collagen (via its interaction with the alpha2beta1 integrin), caused Vav to become phosphorylated on tyrosine. Weaker platelet agonists, including ADP, epinephrine and the thromboxane A2 analog, U46619, did not. The phosphorylation of Vav in response to thrombin was maximal within 15 s and was unaffected by aspirin, inhibitors of aggregation, or the presence of the ADP scavenger, apyrase. Vav phosphorylation was also observed when platelets became adherent to immobilized collagen (via integrin alpha2beta1), fibronectin (via integrin alpha5beta1), and fibrinogen (via integrin alphaIIbbeta3). These results show that Vav phosphorylation by tyrosine kinases 1) occurs during platelet activation by potent agonists, 2) also occurs when platelets adhere to biologically relevant matrix proteins, 3) requires neither platelet aggregation nor the release of secondary agonists such as ADP and TxA2, and 4) can be initiated by at least some members of two additional classes of receptors, G protein-coupled receptors and integrins, providing further evidence that both of these can couple to tyrosine kinases.
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PMID:Thrombin receptor activation and integrin engagement stimulate tyrosine phosphorylation of the proto-oncogene product, p95vav, in platelets. 863 86

The use of agonist monoclonal antibodies (mAbs) to probe the signalling function of platelet membrane proteins is severely limited by the dependence of the mAb effect on Fc-FcgammaRII interaction. Furthermore, in addition to its anchoring role, the FcgammaRII receptor itself generates a stimulation signal resulting in granule secretion. Platelet stimulation by the released granule contents can then further obscure the original activation signal. Here we demonstrate that these problems are largely overcome by the use of platelets which had been degranulated with thrombin prior to stimulation with mAbs. We found that, like intact cells, degranulated platelets could also be activated and induced to aggregate by mAbs against a 67 kD membrane protein (known as PTA1) and CD9, and by crosslinked CD32 (FcgammaRII). However, the signal generated by crosslinked FcgammaRII was weak compared with that induced by the other monoclonal antibodies. Thus, by diminishing the FcgammaRII signal contribution, we have succeeded for the first time to clearly dissect the target antigen signal from that generated by FcgammaRII. In addition to differences in the degree of aggregation, analysis of the signals generated by each mAb showed differences in Ca2+ fluxes and protein phosphorylation. Moreover, the signals generated by CD9 and PTA1 antigens differed significantly in their sensitivity to PKC inhibition or ADP-ribosylation of the small GTP-binding protein rhoA. Despite these differences, the signals initiated by all three antigens converged to a common signalling pathway which included activation of tyrosine kinase(s). The pattern of protein phosphorylation strongly resembled that induced by gpIIb/IIIa-mediated platelet interaction with macromolecular ligands and by mutual cell contact. The multiple intercellular links formed by mAb would have a similar effect since the Fc-receptor anchorage required for antigen stimulation is already known to be provided by adjacent cells. The present findings suggest that the function of both CD9 and PTA1 antigens is closely associated with gpIIb/IIIa activation.
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PMID:Analysis of CD9, CD32 and p67 signalling: use of degranulated platelets indicates direct involvement of CD9 and p67 in integrin activation. 902 13

The aim of our study was to evaluate the effect of ADP and the role of cytoskeleton reorganization during reversible and irreversible platelet aggregation induced by ADP and thrombin, respectively, on the heterodimeric (p85alpha-p110) phosphoinositide 3-kinase translocation to the cytoskeleton and its activation. Reversible ADP-induced aggregation was accompanied by a reversible reorganization of the cytoskeleton and an increase in levels of the regulatory subunit p85alpha in this cytoskeleton similar to the increase observed in thrombin-activated platelets. This translocation followed a course parallel to the amplitude of aggregation. No increase in levels of both phosphatidylinositol (3, 4)-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol-(3,4,5)P3 could, however, be detected even at the maximum aggregation and PI 3-kinase alpha translocation. Moreover, in contrast to the situation for thrombin stimulation, the GTP-binding protein RhoA was hardly translocated to the cytoskeleton when platelets were stimulated with ADP, whereas translocation of pp60(c-)src and focal adhesion kinase did occur. These results suggest (i) translocation of signaling enzymes does not necessarily imply their activation, (ii) the reversibility of ADP-induced platelet aggregation may be the cause or the result of a lack of PI 3-kinase activation and hence of PtdIns(3,4)P2 production, and (iii) RhoA does not seem to be involved in the ADP activation pathway of platelets. Whether PtdIns(3,4)P2 or RhoA may contribute to the stabilization of platelet aggregates remains to be established.
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PMID:Reversible translocation of phosphoinositide 3-kinase to the cytoskeleton of ADP-aggregated human platelets occurs independently of Rho A and without synthesis of phosphatidylinositol (3,4)-bisphosphate. 903 May 42

In this paper we demonstrate for the first time a mitogen-induced activation of a nuclear acting phosphatidylcholine-phospholipase D (PLD) which is mediated, at least in part, by the translocation of RhoA to the nucleus. Addition of alpha-thrombin to quiescent IIC9 cells results in an increase in PLD activity in IIC9 nuclei. This is indicated by an increase in the alpha-thrombin-induced production of nuclear phosphatidylethanol in quiescent cells incubated in the presence of ethanol as well as an increase in PLD activity in isolated nuclei. Consistent with our previous report (Wright, T. M., Willenberger, S., and Raben, D. M. (1992) Biochem. J. 285, 395-400), the presence of ethanol decreases the alpha-thrombin-induced production of phosphatidic acid without affecting the induced increase in nuclear diglyceride, indicating that the increase in nuclear PLD activity is responsible for the effect on phosphatidic acid, but not that on diglyceride. Our data further demonstrate that RhoA mediates the activation of nuclear PLD. RhoA translocates to the nucleus in response to alpha-thrombin. Additionally, PLD activity in nuclei isolated from alpha-thrombin-treated cells is reduced in a concentration-dependent fashion by incubation with RhoGDI and restored by the addition of prenylated RhoA in the presence of guanosine 5'-3-O-(thio)triphosphate. Western blot analysis indicates that this RhoGDI treatment results in the extraction of RhoA from the nuclear envelope. These data support a role for a RhoA-mediated activation of PLD in our recently described hypothesis, which proposes that a signal transduction cascade exists in the nuclear envelope and represents a novel signal transduction cascade that we have termed NEST (nuclear envelope signal transduction).
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PMID:Nuclear translocation of RhoA mediates the mitogen-induced activation of phospholipase D involved in nuclear envelope signal transduction. 903 May 50

The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein-coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116(Rip), that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116(Rip) stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116(Rip) fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein-coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116(Rip) inhibits RhoA-stimulated contractility and promotes neurite outgrowth.
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PMID:Identification of a novel, putative Rho-specific GDP/GTP exchange factor and a RhoA-binding protein: control of neuronal morphology. 919 74

Thrombin activity is a factor in acute CNS trauma and may contribute to such chronic neurodegenerative diseases as Alzheimer's disease. Thrombin is a multifunctional serine protease that catalyses the final steps in blood coagulation. However, increasing evidence indicates that thrombin also elicits a variety of cellular and inflammatory responses, including responses from neural cells. Most recently, high concentrations of thrombin were shown to cause cell death in both astrocyte and hippocampal neuron cultures. The purpose of this study was to determine the mechanisms underlying thrombin-induced cell death. Our data show that thrombin appears to cause apoptosis as evidenced by cleavage of DNA into oligonucleosomal-sized fragments, fragmentation of nuclei, and prevention of death by inhibition of protein synthesis. Synthetic peptides that directly activate the thrombin receptor also induced apoptosis, indicating that thrombin-induced cell death occurred via activation of the thrombin receptor. The signal transduction cascade involves tyrosine and serine/threonine kinases and an intact actin cytoskeleton. Additional study revealed the involvement of the small GTP-binding protein RhoA. Thrombin induced RhoA activity in both astrocytes and hippocampal neurons, and inhibition of RhoA activity with exoenzyme C3 attenuated cell death, indicating that thrombin activation of RhoA was necessary for thrombin-induced cell death. Tyrosine kinase inhibitors blocked thrombin induction of RhoA, indicating that tyrosine kinase activity was required upstream of RhoA. These data suggest a sequential linkage of cellular events from which we propose a model for the second messenger cascade induced by thrombin in neural cells that can lead to apoptosis.
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PMID:Thrombin induces apoptosis in cultured neurons and astrocytes via a pathway requiring tyrosine kinase and RhoA activities. 920 16

The biochemical basis for the functional heterogeneity of human blood platelets was investigated in terms of protein phosphorylation, cytoplasmic calcium ([Ca2+]i), the ratio of 46 and 50 kDa vasodilator-stimulated protein (VASP), and GTP-binding proteins (G-proteins). Platelets were fractionated by density. Comparing resting low-density platelets (LDP) to high-density platelets (HDP) revealed higher phosphorylation of proteins in the 47, 31, and 24 kDa ranges. A higher phosphorylation of the 20 kDa protein in LDP compared to HDP was related to an enhanced [Ca2+]i, an increased ADP-ribosylation of the inhibitory G-protein (G(i alpha1-3)) and rhoA, and a decreased ADP-ribosylation of the stimulatory G-protein (G(s alpha)). The differences in the ribosylation patterns of the subpopulations were not influenced by thrombin stimulation or exposure to prostaglandin E1 (PGE1). An 18 kDa phosphoprotein was more highly phosphorylated in resting HDP than in LDP. Thrombin exposure caused dephosphorylation of the 18 kDa phosphoprotein in the HDP, but generally increased phosphorylation of both HDP and LDP in the 47, 31, 24, and 20 kDa bands. Preincubation with prostaglandin E1 or sodium nitroprusside diminished the subsequent thrombin-induced increase in phosphorylation, particularly in HDP. In unstimulated HDP, the 50 kDa VASP phospho form was enhanced, whereas in unstimulated LDP the 46 kDa VASP dephospho form was increased. Our findings suggest that the functional heterogeneity of platelets is partly derived from differences in signal transduction mechanisms reflected in varying phosphoprotein patterns and G-protein properties of platelet stimulatory and inhibitory pathways.
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PMID:Analysis of GTP-binding proteins, phosphoproteins, and cytosolic calcium in functional heterogeneous human blood platelet subpopulations. 937 24

This study examined the signal transduction pathways involved in thrombin-induced neuroprotection and compares these results with those of a similar study of thrombin-induced neuronal death. In thrombin-induced protection of astrocytes from hypoglycemia, pretreatment of astrocytes with tyrosine or serine/threonine kinase inhibitors, cytochalasin D, or exoenzyme C3, a potent inhibitor of the small GTPase RhoA, attenuated thrombin-induced protection. These same inhibitors were previously shown to block thrombin-induced cell death, implying a similarity in the cell death and cell-protective pathways. Biochemical assays determined that thrombin increased available RhoA activity, although more slowly and to a lesser extent than occurs in thrombin-induced cell death. A clear difference in these pathways was revealed when a time course study of thrombin-induced cell death indicated that unlike thrombin-induced protection, cells must be exposed to thrombin for >16 h to irreversibly enter the cell death pathway. Addition of lower doses of thrombin every 24 h also induced cell death. These studies indicate that exposure of cells to micromolar concentrations of thrombin alone does not induce cell death, but the continued exposure to thrombin is required. Thus the cell death and protective pathways may share initial signaling proteins, but differences in the amplitude as well as the duration of the signal may result in different final pathways.
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PMID:Signaling pathways involved in thrombin-induced cell protection. 958 99

Dramatic transient changes resulting in a stellate morphology are induced in many cell types on treatment with agents that enhance intracellular cAMP levels. Thrombin fully protects cells from this inductive effect of cAMP through the thrombin receptor. The protective effect of thrombin was shown to be Rho-dependent. Clostridium botulinum C3 exoenzyme, which inactivates RhoA functions, abolished the ability of thrombin to protect cells from responding to increased cAMP levels. A constitutively activated RhoAV14 mutant protein also prevented cells from responding to cAMP. RhoA can be specifically phosphorylated at Ser-188 by the cAMP-activated protein kinase A (PKA). We demonstrate that RhoAV14A188, which cannot be phosphorylated by PKA in vitro, is more effective than RhoAV14 in preventing cells from responding to cAMP and in inducing actin stress fiber formation. This suggests that PKA phosphorylation of RhoA impairs its biological activity in vivo. ROKalpha, a RhoA-associated serine/threonine kinase can also prevent cells from responding to cAMP with shape changes. Phosphorylation of RhoA by PKA in vitro decreases the binding of RhoA to ROKalpha. These results indicate that RhoA and cAMP have antagonistic roles in regulating cellular morphology and suggest that cAMP-mediated down-regulation of RhoA binding to its effector ROKalpha may be involved in this antagonism.
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PMID:cAMP-induced morphological changes are counteracted by the activated RhoA small GTPase and the Rho kinase ROKalpha. 971 82


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