Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The protein tyrosine phosphatase (PTPase) inhibitor pervanadate (vanadyl hydroperoxide) stimulated protein tyrosine phosphorylation 29-fold more than did thrombin in intact and saponin-permeabilized platelets. Increased tyrosine phosphorylation preceded, or was coincident with, a fall in PtdIns(4,5)P2 levels, production of PtdIns(3,4)P2 and phosphatidic acid, mobilization of intracellular Ca2+, stimulation of protein kinase C-dependent protein phosphorylation, secretion of dense and alpha-granules, increased actin polymerization, shape change and aggregation which required fibrinogen and was mediated by increased surface expression of GPIIb-IIIa. The tyrosine kinase inhibitor RG 50864 totally prevented induction of tyrosine phosphorylation by pervanadate, as well as all other responses measured; in contrast, the inactive structural analogue, tyrphostin #1, had no effect. Dense-granule secretion induced by pervanadate required protein kinase C activity; however, aggregation and alpha-granule secretion were independent of protein kinase C. In saponin-permeabilized platelets pervanadate and thrombin stimulated phospholipase C activity by GTP-independent and GTP-dependent mechanisms respectively. We conclude that PTPases are important regulators of signal transduction in platelets.
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PMID:Activation of signal transduction in platelets by the tyrosine phosphatase inhibitor pervanadate (vanadyl hydroperoxide). 153 May 76

Platelet membrane glycoprotein (GP IIb-IIIa), besides its activity as adhesive protein receptor, displays a number of properties supporting its involvement in the mechanisms of transduction of the activation signal. Recently we have observed that GP IIb-IIIa ligands, mostly fibrinogen, inhibit Ca2+ movement and cytoskeleton reorganization caused by mild platelet activation. These findings led us to investigate the effect of GP IIb-IIIa ligands on agonist-induced platelet responses, with particular attention to the two major messenger generating systems, involving the activation of phospholipase C and the inhibition of cAMP production. In this paper we demonstrate that the occupancy of the major adhesive protein receptor on the platelet surface modulates the phosphatidylinositol cycle decreasing the amount of IP3, IP2 and IP produced after mild platelet activation as well as the pattern of protein phosphorylation. The platelet cAMP content of activated platelets was also affected and kept higher when evaluated under the same experimental conditions. Our data provide evidence for a role of fibrinogen binding in regulating the degree of activation of circulating platelets.
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PMID:The occupancy of glycoprotein IIb-IIIa complex modulates thrombin activation of human platelets. 254 25

KRDS, a tetrapeptide from human lactotransferrin, inhibits thrombin-induced platelet aggregation, secretion and thromboxane (TX) synthesis without interfering with phospholipase C (PLC) beta activation, since in previous work we have shown that Ca2+ mobilization and phosphorylation of the myosin light chain kinase (20 kDa) and pleckstrin (47 kDa) were normal. However, the inhibition of arachidonic acid-induced aggregation in the presence of KRDS is accompanied by normal TX synthesis suggesting that it does not interfere with the cyclooxygenase activity. To elucidate further the mechanisms of action of this peptide we tested its effect on U46619-induced platelet activation. KRDS inhibits U46619-induced platelet aggregation time- and dose-dependently without inhibiting the phosphorylation of pleckstrin. This suggests that the PLC pathway is not affected and that the inhibitory effect of KRDS is not due to and uncoupling of TXA2 from its receptor. In addition to the PLC pathway, protein tyrosine kinases play a major role in platelet signal transduction mechanisms. At least 7 tyrosine-phosphorylated proteins are detected upon stimulation of platelets by thrombin. KRDS strongly inhibits the tyrosine-phosphorylated substrates, in particular two 100-105 kDa substrates which are related to GP IIb/IIIa activation and platelet aggregation. The absence of TX synthesis observed in the presence of KRDS could be due to the inactivation of cPLA2 since the latter needs tyrosine phosphorylation to be activated, thus explaining the inhibitory action of KRDS on platelet functions.
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PMID:KRDS, a peptide derived from human lactotransferrin, inhibits thrombin-induced thromboxane synthesis by a cyclooxygenase-independent mechanism. 748 16

Binding sites on glycoprotein (GP) IIb/IIIa exposed by 0.5 unit/ml alpha-thrombin are insensitive to prostaglandin I2 (PGI2), in contrast with sites exposed by ADP or platelet-activating factor. Here we show that the thrombin receptor agonist peptide (TRAP) (SFLLRN; 15 microM) opens almost the same number of GPIIb/IIIa molecules as 0.5 unit/ml alpha-thrombin (64840 +/- 8920 compared with 81050 +/- 6030 molecules of fibronectin bound/platelet), but these sites rapidly close on addition of PGI2. To investigate whether alpha-thrombin and TRAP initiate different signalling pathways, we measured phospholipase C (PLC)-mediated control of GPIIb/IIIa and its sensitivity to cyclic AMP. Optimal concentrations of alpha-thrombin and TRAP activated PLC maximally, but TRAP induced only about 50% protein kinase C PKC) activation after 10 min stimulation compared with alpha-thrombin. These concentrations also suppressed PGI2-induced cyclic AMP accumulation, with alpha-thrombin inducing complete inhibition and TRAP about 10% less. Direct activation of PKC by phorbol 12-myristate 13-acetate confirmed earlier observations that PGI2-induced cyclic AMP accumulation is partly inhibited via PKC. Applying different concentration of alpha-thrombin, TRAP or a combination of alpha-thrombin and the thrombin receptor inhibitory peptide (TRIP) (Mpr-F-Cha-Cha-RKPNDK-NH2; 800 microM) (Mpr, 3-mercaptopropionic acid; Cha, cyclohexylalanine), we show that the different means of stimulating the thrombin receptor all suppressed PGI2-induced cyclic AMP accumulation via (i) activation of PKC and (ii) activation of the heterotrimeric G-protein, Gi. We conclude that complete inhibition of cyclic AMP accumulation requires activation of both PKC and Gi, as observed with 0.5 unit/ml alpha-thrombin. Although TRAP almost fully exposes GPIIb/IIIa, its activation of PKC is incomplete, enabling PGI2 to raise cyclic AMP concentration from 1.4 +/- 0.7 to 4.1 +/- 1.3 nmol/10(11) platelets (P < 0.005) which is sufficient to close exposed GPIIb/IIIa molecules.
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PMID:Regulation of platelet glycoprotein IIb/IIIa (integrin alpha IIB beta 3) function via the thrombin receptor. 754 72

Although the importance of protein kinases in platelet activation, particularly protein kinase C (PKC), is well established there remain many problems regarding the various phosphorylation cascades, the role of phosphatases and the importance of other serine/threonine and tyrosine kinases. A particular problem is the mechanism of activation of the fibrinogen receptor, GPIIb/IIIa, a critical step in aggregation. Although GPIIIa is phosphorylated (on threonine) neither the stoichiometry nor the minor changes on activation seem adequate to explain the response. Relatively unspecific inhibitors of PKC such as staurosporine prevent PO4 incorporation into most kinase substrates but only inhibit platelet aggregation partially. However, staurosporine does induce activation and then inhibits several renaturable serine/threonine kinases, probably via phosphatases. Staurosporine did not, however, inhibit the platelet Ca2+ signal in response to thrombin but rather enhanced it. 17-Hydroxywortmannin (HWT), a fungal metabolite, has been shown to inhibit respiratory burst in neutrophils and causes haemorrhages. It was recently reported to be a myosin light chain kinase (MLCK) inhibitor and to inhibit PKC only at much higher concentrations. In platelets, HWT inhibits aggregation and partially inhibits phosphorylation of myosin light chain and P47 in thrombin-activated platelets. It also allows the discrimination of an early and a late phase in the cytoplasmic Ca2+ signal since at lower concentrations it only inhibits the late phase. The late phase of ATP release was also inhibited in a dose-dependent manner. The activation of most of the renaturable serine/threonine kinases was also inhibited by HWT. These results support earlier conclusions that the early phase of the Ca2+ signal is phospholipase C dependent but indicate that other mechanisms must be responsible for the late phase. The relative specificity of HWT for MLCK might indicate that this has an unexpected major role in controlling these late phase reactions including activation of GPIIb/IIIa or its clustering. However, staurosporine completely inhibits phosphorylation of myosin light chain by its kinase (as well as other kinases) and has the opposite effect on Ca2+ signals. Clearly, the interactions and feed-back mechanisms between these kinases are very complex but the results suggest that phosphatases acting together with their complementary kinases should also be considered as important platelet activation regulators. P47, long considered a major PKC substrate, may also be phosphorylated by MLCK.
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PMID:Serine/threonine kinases in signal transduction in response to thrombin in human platelets. Use of 17-hydroxywortmannin to discriminate signals. 820 81

In the present study, we investigated the effect of high density lipoproteins 3 (HDL3) on Na+/H+ exchanger activity and cytosolic pH (pHi) in human platelets. HDL3 alone failed to affect pHi, but preincubation with HDL3 significantly enhanced the Na+/H+ antiport activation brought about by acidification with 100 mM sodium propionate or stimulation with 0.05 U/ml thrombin. the stimulatory effect of HDL3 was unaffected by indomethacin excluding a role for cyclooxygenase products. The HDL3 effect was not mediated by Ca2+/calmodulin-dependent protein kinase as HDL3 failed to increase cytosolic free calcium concentration. However, the potentiating effect of HDL3 was completely blocked in the presence of the protein kinase C inhibitor, bisindoylmaleimide and the phosphatidylcholine-specific phospholipase C inhibitor, D609. Furthermore, the effect of HDL3 was abolished after covalent modification of HDL3 with dimethylsuberimidate and was not observed in platelets from Glanzmann thrombasthenia type 1 which do not express GP IIb/IIIa, as well as in platelets preincubated with anti-GP IIb/IIIa polyclonal antibodies. We conclude that HDL3 enhances the sodium propionate- and thrombin-induced Na+/H+ antiport activity in human platelets via binding to GP IIb/IIIa and activation of protein kinase C and phosphatidylcholine-specific phospholipase C.
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PMID:High density lipoproteins enhance the Na+/H+ antiport in human platelets. 874 92

Congenital defects in platelet function are associated with bleeding manifestations of variable intensity and arise by diverse mechanisms. Defects in platelet-vessel wall interaction (disorders of adhesion) may arise because of qualitative or quantitative abnormalities in plasma von Willebrand factor (von Willebrand disease) or in platelet glycoprotein Ib, the binding site on platelets for von Willebrand factor (Bernard-Soulier syndrome). Disorders characterized by abnormal platelet-platelet interaction (disorders of aggregation) arise because of absence of plasma fibrinogen (congenital afibrinogenemia) or because of qualitative or quantitative abnormalities in platelet glycoprotein IIb-IIIa complex (Glanzmann's thrombasthenia). Patients with abnormalities in platelet secretion and signal transduction are a heterogeneous group characterized by impaired aggregation responses and secretion of granule contents. A small proportion of these patients have deficiency of granule stores (storage pool deficiency [SPD]) or impaired production of thromboxane A2; in most, the mechanisms underlying the platelet dysfunction are unknown. Evidence is accumulating that at least some patients have abnormalities in early signal transduction events. Abnormalities in phospholipase C activation, G-protein activation, and other events (eg, protein phosphorylation) have been documented. Platelets play a major role in blood coagulation, and in Scott syndrome, there is an abnormality in platelet contribution to the mechanisms leading to thrombin generation. In most patients with inherited platelet dysfunction, the underlying mechanisms remain to be delineated. Future studies of these patients should yield valuable new information on normal platelet mechanisms.
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PMID:Congenital disorders of platelet function: disorders of signal transduction and secretion. 970 60

In this study, the mechanism involved in the antiplatelet activity of rutaecarpine in human platelet suspensions was investigated. In platelet suspensions (4.5 x 10(8)/ml), rutaecarpine (100 and 200 microM) did not influence the binding of FITC-triflavin to platelet glycoprotein IIb/IIIa complex. Additionally, rutaecarpine (200 microM) did not significantly change the fluorescence of platelet membrane labeled with diphenylhexatriene (DPH). On the other hand, rutaecarpine (50 and 100 microM) dose-dependently inhibited the increase in intracellular free Ca2+ of Fura 2-AM loaded platelets stimulated by collagen. Moreover, rutaecarpine (100 and 200 microM) did not significantly affect the thromboxane synthetase activity of aspirin-treated platelet microsomes. Furthermore, retaecarpine (100 and 200 microM) significantly inhibited [3H]arachidonic acid released in collagen-activated platelets but not in unactivated-platelets. Nitric oxide (NO) production in human platelets was measured by a chemiluminesence detection method in this study. Rutaecarpine (100 and 200 microM) did not significantly affect nitrate production in collagen (10 microg/ml)-induced human platelet aggregation. On the other hand, various concentrations of rutaecarpine (50, 100, and 200 microM) dose-dependently inhibited [3H]inositol monophosphate formation stimulated by collagen (10 microg/ml) in [3H]myoinositol-loaded platelets at different incubation times (1, 2, 3, and 5 minutes). It is concluded that the antiplatelet activity of rutaecarpine may possibly be due to the inhibition of phospholipase C activity, leading to reduce phosphoinositide breakdown, followed by the inhibition of thromboxane A2 formation, and then inhibition of [Ca2+]i mobilization of platelet aggregation stimulated by agonists.
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PMID:The antiplatelet activity of rutaecarpine, an alkaloid isolated from Evodia rutaecarpa, is mediated through inhibition of phospholipase C. 979 12

Phospholipase C (PLC)-beta2 plays a major role in platelet activation. Previous studies have described a unique patient with impaired receptor-mediated platelet aggregation, secretion, calcium mobilization, and phospholipase C (PLC) activation associated with a selective decrease in platelet PLC-beta2 isozyme. To identify the mechanisms leading to the defect, platelet RNA from the patient and healthy subjects was subjected to reverse transcription-polymerase chain reaction (RT-PCR) and the products sequenced. The PLC-beta2 cDNA sequence in the patient showed no abnormalities. Platelet PLC-beta2 and beta-actin (internal control) mRNA levels were assessed by RT-PCR; the ratio of PLC-beta2 to beta-actin mRNA levels was 0.80 to 0.95 in 4 healthy subjects and 0.28 in the patient. PLC-beta2 mRNA levels were similarly reduced compared with GPIIb and Galphaq mRNA levels. PLC-gamma2 and platelet factor 4 mRNA levels were normal. Calcium mobilization was studied in neutrophils upon activation with formyl-Met-Leu-Phe (fMLP), adenosine diphosphate (ADP), platelet-activating factor (PAF), interleukin-8 (IL-8), C5a, and leukotriene B(4) (LTB(4)), and it was normal. Neutrophil elastase secretion upon activation with fMLP, ADP, PAF, IL-8, C5a, and LTB(4) was normal, as were neutrophil PLC-beta2 mRNA and PLC-beta2 on immunoblotting. Thus, responses to activation, PLC-beta2 protein, and PLC-beta2 mRNA are decreased in patient platelets but not in neutrophils, providing evidence for a hitherto undescribed lineage (platelet)-specific defect in PLC-beta2 gene expression. These studies provide a physiologically relevant model to delineate regulation of PLC-beta2 gene and its tissue-specific expression. (Blood. 2002;99:905-911)
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PMID:Lineage-specific defect in gene expression in human platelet phospholipase C-beta2 deficiency. 1180 93

1 The lysophospholipids, lysophosphatidic acid and sphingosine 1-phosphate, have been reported to activate platelets. Here we examined effects of the naturally occurring related sphingosylphosphorylcholine (SPC) on human platelet function. 2 Platelet activation was determined as aggregation, elevation of intracellular Ca(2+) concentrations, surface expression of P-selectin, GP 53, and GP IIb/IIIa neoepitope PAC-1, and of fibrinogen binding to the platelet surface. 3 Platelets were activated by ADP (5 and 20 micro M), the thrombin receptor-activating peptide TRAP-6 (5 and 20 micro M), the thromboxane A(2) mimetic U-46619 (1 micro M) and collagen (20 and 50 micro g ml(-1)) but not by SPC (up to 20 micro M). 4 SPC concentration-dependently (IC(50) approximately 1-10 micro M) inhibited activation of washed human platelets in response to all of the above agonists, with almost complete inhibition occurring at 20 micro M SPC. 5 The SPC stereoisomers, D-erythro SPC and L-threo SPC, exhibited similar concentration-response curves in inhibiting 20 micro M ADP-induced platelet aggregation, suggesting that SPC did not act via specific lysophospholipid receptors. 6 Although SPC slightly activated platelet protein kinase A (as assessed by VASP phosphorylation), this effect could not explain the marked platelet inhibition. Possible protein kinase C inhibition also did not explain the inhibition of platelet activation by SPC. On the other hand, SPC suppressed agonist-induced Ca(2+) mobilization and phospholipase C stimulation. 7 These results indicate that the lysophospholipid SPC is an effective inhibitor of human platelet activation, apparently primarily by uncoupling agonist-activated receptors from their effectors.
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PMID:Sphingosylphosphorylcholine, a naturally occurring lipid mediator, inhibits human platelet function. 1256 68


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