Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.10.2 (focal adhesion kinase)
 44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The leflunomide metabolite analog alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)-propenamide (LFM-A13) is a rationally-designed specific inhibitor of the TEC family protein tyrosine kinase, Bruton's tyrosine kinase (BTK) which plays an important role in platelet physiology by regulating the glycoprotein GPVI-FcRgamma-coupled collagen receptor signaling pathway. At low micromolar concentrations, LFM-A13 inhibited collagen-induced ultrastructural changes indicative of activation. LFM-A13 inhibited collagen (but not thrombin, TRAP-6, or ADP)-induced platelet aggregation in a concentration-dependent fashion with an IC50 value of 2.8 microM. LFM-A13 was not toxic to mice when administered systemically at dose levels ranging from 1 to 100 mg/kg. At nontoxic dose levels, LFM-A13 prolonged the tail bleeding times of mice and improved event-free survival in two mouse models of agonist-induced invariably fatal pulmonary thromboembolism. To our knowledge, LFM-A13 is the first anti-thrombotic agent which prevents platelet aggregation by inhibiting BTK.
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PMID:The anti-leukemic Bruton's tyrosine kinase inhibitor alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl) propenamide (LFM-A13) prevents fatal thromboembolism. 1456 61

Platelet adhesion to vascular collagens is an essential step in the initiation of hemostasis and thrombosis. Several platelet receptors interact with type I and type III collagens, including GP Ia/IIa and GP VI. We recently described a new platelet receptor (TIIICBP) specific for a type III collagen-related primary binding sequence, the KOGEOGPK octapeptide. Here, we characterize platelet adhesion to the immobilized octapeptide and demonstrate that this adhesion 1) is Ca2+ and Mg2+ independent, suggesting a noninvolvement of GP Ia/IIa; 2) is not inhibited by an antibody against GP VI; and 3) triggers platelet protein tyrosine phosphorylation. Whereas TXA2 has minimal effects, released ADP via only P2Y12 potentiates platelet adhesion to the octapeptide. Octapeptide-induced platelet adhesion triggers platelet signaling through tyrosine phosphorylation of the 68 kDa subunit of TIIICBP, Syk, PLCgamma2, and FAK. Tyrosine phosphorylation of the FcR gamma-chain and LAT is also observed but to a lesser extent than with type III collagen, suggesting the requirement of GP VI for full tyrosine phosphorylation of FcR gamma-chain and LAT. The present study provides evidence for a critical role for the type III collagen-related KOGEOGPK octapeptide in mediating platelet adhesion and signaling, and consequently in platelet-collagen interactions.-
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PMID:Platelet adhesion and signaling induced by the octapeptide primary binding sequence (KOGEOGPK) from type III collagen. 1533 77

An elevated circulating level of the adipocyte-derived satiety hormone leptin is an independent risk factor for cardiovascular disease. Because thrombus formation is a major cause of acute coronary events and leptin was shown previously to facilitate ADP-induced platelet aggregation, we chose to define the signaling events involved in leptin-mediated platelet activation. Using pharmacological, biochemical, and cell biological approaches, we show that leptin-induced platelet activation required activation of a signaling cascade that included the long form of the leptin receptor, three kinases [Janus kinase 2 (JAK2), phosphatidylinositol 3-kinase (PI3K), and protein kinase B (PKB/Akt)], the insulin receptor substrate-1 (IRS-1), and the major human platelet cAMP phosphodiesterase phosphodiesterase 3A (PDE3A). Moreover, we identify a role for an intraplatelet LEPR/JAK2/IRS-1/PI3K/PKB/PDE3A molecular complex that allows for the selective leptin-mediated activation of platelets. Our data demonstrate that leptin promotes platelet activation, provides a mechanistic basis for the prothrombotic effect of this hormone, and identifies a potentially novel therapeutic avenue to limit obesity-associated cardiovascular disease.
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PMID:Leptin-mediated activation of human platelets: involvement of a leptin receptor and phosphodiesterase 3A-containing cellular signaling complex. 1588 25

Plasma leptin levels are elevated in most of obese individuals, and obesity is associated with high incidence of cardiovascular diseases. It has been reported that leptin is an independent risk factor for the coronary artery disease in obese patients and that leptin is involved in the pathogenesis of cardiovascular diseases. We previously reported that leptin promotes platelet aggregation. The present study aimed to elucidate the mechanisms underlying this effect of leptin using a megakaryoblast cell line, MEG-01 cells. Leptin receptors mRNAs expression in MEG-01 cells were analyzed by RT-PCR. Leptin-induced tyrosine-phosphorylation of proteins was analyzed by immunoblotting with an anti-phosphotyrosine antibody. ADP-induced increases in cytosolic Ca(2+) concentration ([Ca(2+)](i)) in the presence and absence of leptin were measured by dual-wavelength fura-2 microfluorometry. Both Ob-Ra and Ob-Rb, were expressed and leptin-induced tyrosine-phosphorylation of several proteins in MEG-01 cells. Leptin-potentiated increases in [Ca(2+)](i) induced by ADP. ADP at a subthreshold concentration and leptin acted synergistically in producing [Ca(2+)](i) increases. These effects of leptin on [Ca(2+)](i) were inhibited by blockers of JAK2 and tyrosine kinases. Furthermore, leptin increased the tyrosine-phosphorylation of Gq alpha-subunits. The results indicate that leptin enhances ADP-induced [Ca(2+)](i) increases via JAK2 and tyrosine kinases in a megakaryoblast cell line. This mechanism may underlie the potentiation of platelet aggregation by leptin.
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PMID:Leptin potentiates ADP-induced [Ca(2+)](i) increase via JAK2 and tyrosine kinases in a megakaryoblast cell line. 1592 98

Previous studies have implicated the immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM) superfamily member platelet endothelial cell adhesion molecule-1 (PECAM-1) in the regulation of integrin function. While PECAM-1 has been demonstrated to play a role as an inhibitory coreceptor of immunoreceptor tyrosine-based activation motif (ITAM)-associated Fcgamma receptor IIa (FcgammaRIIa) and glycoprotein VI (GPVI)/FcR gamma-chain signaling pathways in platelets, its physiologic role in integrin alpha(IIb)beta3-mediated platelet function is unclear. In this study, we investigate the functional importance of PECAM-1 in murine platelets. Using PECAM-1-deficient mice, we show that the platelets have impaired "outside-in" integrin alpha(IIb)beta3 signaling with impaired platelet spreading on fibrinogen, failure to retract fibrin clots in vitro, and reduced tyrosine phosphorylation of focal adhesion kinase p125 (125FAK) following integrin alpha(IIb)beta3-mediated platelet aggregation. This functional integrin alpha(IIb)beta3 defect could not be attributed to altered expression of integrin alpha(IIb)beta3. PECAM-1-/- platelets displayed normal platelet alpha granule secretion, normal platelet aggregation to protease-activated receptor-4 (PAR-4), adenosine diphosphate (ADP), and calcium ionophore, and static platelet adhesion. In addition, PECAM-1-/- platelets displayed normal "inside-out" integrin alpha(IIb)beta3 signaling properties as demonstrated by normal agonist-induced binding of soluble fluoroscein isothiocyanate (FITC)-fibrinogen, JON/A antibody binding, and increases in cytosolic-free calcium and inositol (1,4,5)P3 triphosphate (IP3) levels. This study provides direct evidence that PECAM-1 is essential for normal integrin alpha(IIb)beta3-mediated platelet function and that disruption of PECAM-1 induced a moderate "outsidein" integrin alpha(IIb)beta3 signaling defect.
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PMID:The Ig-ITIM superfamily member PECAM-1 regulates the "outside-in" signaling properties of integrin alpha(IIb)beta3 in platelets. 1608 92

Protease-activated receptors (PARs) activate Gq and G(12/13) pathways, as well as Akt (protein kinase B [PKB/Akt]) in platelets. However, the relative contribution of different G-protein pathways to Akt phosphorylation has not been elucidated. We investigated the contribution of Gq and G(12/13) to Gi/Gz-mediated Akt phosphorylation downstream of PAR activation. Selective G(12/13) activation failed to cause Akt phosphorylation in human and Galpha q-deficient mouse platelets. However, supplementing Gi/Gz signaling to G(12/13) caused significant increase in Akt phosphorylation, confirming that G(12/13) potentiates Akt phosphorylation. Inhibition of PAR-mediated Akt phosphorylation in the presence of the Gq-selective inhibitor YM-254890 was restored to the normal extent achieved by PAR agonists if supplemented with Gi signaling, indicating that Gq does not have any direct effect on Akt phosphorylation. Selective G(12/13) activation resulted in Src kinase activation, and Akt phosphorylation induced by costimulation of G(12/13) and Gi/Gz was inhibited by a Src kinase inhibitor but not by a Rho kinase inhibitor. These data demonstrate that G(12/13), but not Gq, is essential for thrombin-induced Akt phosphorylation in platelets, whereas Gq indirectly contributes to Akt phosphorylation through Gi stimulation by secreted ADP. G(12/13) activation might mediate its potentiating effect through Src activation, and Src kinases play an important role in thrombin-mediated Akt phosphorylation.
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PMID:Relative contribution of G-protein-coupled pathways to protease-activated receptor-mediated Akt phosphorylation in platelets. 1622 79

AKT/PKB is a phosphoinositide-dependent serine/threonine protein kinase that plays a critical role in the signal transduction of receptors. It also serves as an oncogene in the tumorigenesis of cancer cells when aberrantly activated by genetic lesions of the PTEN tumor suppressor, phosphatidylinositol 3-kinase, and receptor tyrosine kinase overexpression. Here we have characterized and compared kinetic mechanisms of the three AKT isoforms. Initial velocity studies revealed that all AKT isozymes follow the sequential kinetic mechanism by which an enzyme-substrate ternary complex forms before the product release. The empirically derived kinetic parameters are apparently different among the isoforms. AKT2 showed the highest Km value for ATP, and AKT3 showed the highest kcat value. The patterns of product inhibition of AKT1, AKT2, and AKT3 by ADP were all consistent with an ordered substrate addition mechanism with ATP binding to the enzymes prior to the peptide substrate. Further analysis of steady state kinetics of AKT1 in the presence of dead-end inhibitors supported the finding and suggested that the AKT family of kinases catalyzes reactions via an Ordered Bi Bi sequential mechanism with ATP binding to the enzyme prior to peptide substrate and ADP being released after the phosphopeptide product. These results suggest that ATP is an initiating factor for the catalysis of AKT enzymes and may play a role in the regulation AKT enzyme activity in cells.
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PMID:Kinetic mechanism of AKT/PKB enzyme family. 1654 Apr 65

We have used HeLa cells without mitochondrial DNA (rho0-cells) and transient rho0-phenocopies, obtained from wild-type cells by short-term treatment with ethidium bromide, to analyze how the absence of a functional mitochondrial respiratory chain slows down proliferation. We ruled out an energetic problem (ATP/ADP content) as well as defective synthesis of pyrimidine, iron-sulfur clusters or heme as important causes for the proliferative defect. Flow cytometric analysis revealed that reactive oxygen species were reduced in rho0-cells and in rho0-phenocopies, and that, quite unusually, all stages of the cell cycle were slowed down. Specific quenching of mitochondrial ROS with the ubiquinone analog MitoQ also resulted in slower growth. Some important cell-cycle regulators were reduced in rho0-cells: cyclin D3, cdk6, p18INK4C, p27KIP1, and p21CIP1/WAF1. In the rho0-phenocopies, the expression pattern did not fully duplicate the complex response observed in rho0-cells, and mainly p21CIP1/WAF1 was downregulated. Activities of the growth regulatory PKB/Akt and MAPK/ERK-signaling pathways did not correlate with proliferation rates of rho0-cells and rho0-phenocopies. Our study demonstrates that loss of a functional mitochondrial electron transport chain inhibits cell-cycle progression, and we postulate that this occurs through the decreased concentration of reactive oxygen species, leading to downregulation of p21CIP1/WAF1.
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PMID:Respiratory chain deficiency slows down cell-cycle progression via reduced ROS generation and is associated with a reduction of p21CIP1/WAF1. 1677 40

Signals ensuing from trimeric G-protein-coupled receptors synergize to induce platelet activation. At low doses, the thromboxane A2 analogue U46619 does not activate integrin alphaIIbbeta3 or trigger platelet aggregation, but it induces shape changes. In the present study, we addressed whether low doses of U46619 trigger tyrosine phosphorylation independently of integrin alphaIIbbeta3 activation and ADP secretion, and synergize with adrenaline (epinephrine) to induce aggregation in acetylsalicylic acid (aspirin)-treated platelets. Low doses of U46619 triggered tyrosine phosphorylation of different proteins, including FAK (focal adhesion kinase), Src and Syk, independently of signals ensuing from integrin alphaIIbbeta3 or ADP receptors engaged by secreted ADP. The G(12/13)-mediated Rho/Rho-kinase pathway was also increased by low doses of U46619; however, this pathway was not upstream of tyrosine phosphorylation, because this occurred in the presence of the Rho-kinase inhibitor Y-27632. Although low doses of U46619 or adrenaline alone were unable to trigger platelet aggregation and integrin alphaIIbbeta3 activation, the combination of the two stimuli effectively induced these responses. PP2, a tyrosine kinase inhibitor, and Y-27632 inhibited platelet activation induced by low doses of U46619 plus adrenaline and, when used in combination, totally suppressed this platelet response. In addition, the two inhibitors selectively blocked tyrosine kinases and the Rho/Rho-kinase pathway respectively. These findings suggest that both tyrosine phosphorylation and the Rho/Rho-kinase pathway are required to activate platelet aggregation via G(12/13) plus G(z) signalling.
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PMID:Rapid stimulation of tyrosine phosphorylation signals downstream of G-protein-coupled receptors for thromboxane A2 in human platelets. 1685 89

Patients with essential thrombocythemia (ET) and polycythemia vera (PV), complicated by microvascular ischemic or thrombotic events, have shortened platelet survival, increased beta-thromboglobulin, platelet factor 4, and thrombomodulin levels, and increased urinary thromboxane B2 excretion. These are all reversible by inhibition of platelet cyclooxygenase 1 with aspirin, and are therefore indicative of platelet activation and platelet-mediated thrombotic processes. The thrombotic tendency persists as long as platelet counts are above the upper limit of normal (400 x 10 (9)/L). Despite strong evidence of in vivo platelet activation, the ex vivo platelet function tests are impaired. Platelet dysfunction in ET and PV typically is characterized by a missing second-wave adrenaline aggregation, an increased adenosine diphosphate aggregation threshold, and reduced secretion products, but a normal arachidonic acid or collagen-induced aggregation. The proposed concept is that platelets in thrombocythemia (ET and PV) are hypersensitive. Due to the existing high shear stress in the microvasculature (end-arterial circulation), platelets spontaneously activate, secrete their products, form aggregates mediated by von Willebrand factor (vWF) that transiently plug the microcirculation, deaggregate, and then recirculate as exhausted defective platelets with secondary storage pool disease on ex vivo analysis. At increasing platelet counts from below to above 1000 x 10 (9)/L, the thrombotic condition changes into an overt spontaneous bleeding tendency as a result of a functional vWF deficiency that is caused by proteolysis of large vWF multimers. This is consistent with acquired type 2 von Willebrand syndrome (AvWS). AvWS is reversible by reduction of the platelet count to normal. The acquired JAK2 V617F gain of function mutation is the cause of trilinear myeloproliferative disease with the sequential occurrence of ET and PV. Heterozygous JAK2 V617F mutation with slightly increased kinase activity is enough for the induction of spontaneous megakaryopoiesis and erythropoiesis, and an increase of hypersensitive platelets is the cause of aspirin-sensitive, platelet-mediated microvascular ischemic and thrombotic complications in ET and early PV mimicking ET. Homozygous JAK2 mutation with pronounced increase of kinase activity is associated with pronounced trilinear megakaryocyte, erythroid, and granulocytic myeloproliferation, with the most frequent clinical picture of classical PV complicated by major thrombosis, in addition to the platelet-mediated microvascular thrombotic syndrome of thrombocythemia.
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PMID:The paradox of platelet activation and impaired function: platelet-von Willebrand factor interactions, and the etiology of thrombotic and hemorrhagic manifestations in essential thrombocythemia and polycythemia vera. 1697 69


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