Glycoprotein VI agonists have distinct dependences on the lipid raft environment.

BACKGROUND: It has been reported that the association of glycoprotein VI (GPVI) with lipid rafts regulates GPVI signaling in platelets. OBJECTIVE: Secreted adenosine 5'-diphosphate (ADP) potentiates GPVI-induced platelet aggregation at particular agonist concentrations. We have investigated whether the decrease in GPVI signaling, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. METHODS: We disrupted platelet lipid rafts with methyl-beta-cyclodextrin and measured signaling events downstream of GPVI activation. RESULTS: Lipid raft disruption decreases aggregation induced by low concentrations of convulxin, but this decrease is almost eliminated in the presence of ADP antagonists. Signaling indicators, such as protein phosphorylation and calcium mobilization, were not affected by raft disruption in collagen or convulxin stimulated platelets. Interestingly, however, raft disruption directly reduced GPVI signaling induced by collagen-related peptide. CONCLUSIONS: Lipid rafts do not directly contribute to signaling by the physiologic agonist collagen. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates GPVI signaling.

Lipid rafts are required in Galpha(i) signaling downstream of the P2Y12 receptor during ADP-mediated platelet activation.

ADP is important in propagating hemostasis upon its secretion from activated platelets in response to other agonists. Lipid rafts are microdomains within the plasma membrane that are rich in cholesterol and sphingolipids, and have been implicated in the stimulatory mechanisms of platelet agonists. We sought to determine the importance of lipid rafts in ADP-mediated platelet activation via the G protein-coupled P2Y1 and P2Y12 receptors using lipid raft disruption by cholesterol depletion with methyl-beta-cyclodextrin. Stimulation of cholesterol-depleted platelets with ADP resulted in a reduction in the extent of aggregation but no difference in the extent of shape change or intracellular calcium release. Furthermore, repletion of cholesterol to previously depleted membranes restored ADP-mediated platelet aggregation. In addition, P2Y12-mediated inhibition of cAMP formation was significantly decreased upon cholesterol depletion from platelets. Stimulation of cholesterol-depleted platelets with agonists that depend upon Galpha(i) activation for full activation displayed significant loss of aggregation and secretion, but showed restoration when simultaneously stimulated with the Galpha(z)-coupled agonist epinephrine. Finally, Galpha(i) preferentially localizes to lipid rafts as determined by sucrose density centrifugation. We conclude that Galpha(i) signaling downstream of P2Y12 activation, but not Galpha(q) or Galpha(z) signaling downstream of P2Y1 or alpha2A activation, respectively, has a requirement for lipid rafts that is necessary for its function in ADP-mediated platelet activation.

Different G protein-coupled signaling pathways are involved in alpha granule release from human platelets.

Alpha granule release plays an important role in propagating a hemostatic response upon platelet activation. We evaluated the ability of various agonists to cause alpha granule release in platelets. Alpha granule release was measured by determining P-selectin surface expression in aspirin-treated washed platelets. ADP-induced P-selectin expression was inhibited both by MRS 2179 (a P2Y1 selective antagonist) and AR-C69931MX (a P2Y12 selective antagonist), suggesting a role for both Galpha(q) and Galpha(i) pathways in ADP-mediated alpha granule release. Consistent with these observations, the combination of serotonin (a Galpha(q) pathway stimulator) and epinephrine (a Galpha(z) pathway stimulator) also caused alpha granule release. Furthermore, U46619-induced P-selectin expression was unaffected by MRS 2179 but was dramatically inhibited by AR-C69931, indicating a dominant role for P2Y12 in U46619-mediated alpha granule release. Additionally, the Galpha(12/13)-stimulating peptide YFLLRNP potentiated alpha granule secretion in combination with either ADP or serotonin/epinephrine costimulation but was unable to induce secretion by itself. Finally, costimulation of the Galpha(i) and Galpha(12/13) pathways resulted in a significant dose-dependent increase in alpha granule release. We conclude that ADP-induced alpha granule release in aspirin-treated platelets occurs through costimulation of Galpha(q) and Galpha(i) signaling pathways. The P2Y12 receptor plays an important role in thromboxane A(2)-mediated alpha granule release, and furthermore activation of Galpha(12/13) and Galpha(q) signaling pathway can cause alpha granule release.

CeReS-18 inhibits growth and induces apoptosis in human prostatic cancer cells.

BACKGROUND: Polypeptide growth factors are positive and negative regulators of prostatic growth and function, and many positive regulators of growth in the prostate have been extensively studied. However, very few inhibitors of prostate cell proliferation have been identified. We have isolated a unique 18-kDa sialoglycopeptide (CeReS-18) which inhibits cell proliferation of three separate lines of human prostate cancer cells, as well as inducing cellular cytotoxicity via an apoptotic pathway unrelated to the Bcl-2 family of proteins. METHODS: Cell cycle inhibition was analyzed by direct cell counts with a Coulter (Miami, FL) cell counter. Apoptotic cells were analyzed by electron microscopy, annexin V-fluorescein isothiocyanate (FITC) staining, fluorescence microscopy, and propidium iodide uptake measured with a fluorescence-activated cell sorter. Expression of the proteins of the Bcl-2 family was detected by Western blot analysis. RESULTS: We found that CeReS-18 inhibits cell proliferation of androgen-responsive, LNCaP.FGC human prostate cancer cells, as well as of androgen-nonresponsive DU-145 and PC3 human prostate cancer cells. Furthermore a, fivefold increase over the inhibitory concentration of CeReS-18 elicited a cytotoxic response by all three cell lines. We thus characterized the cytotoxic mechanism as apoptotic in nature, and we measured the expression of several members of the Bcl-2 family in PC3 cells upon treatment with CeReS-18. CONCLUSIONS: The data indicate that CeReS-18 is a potent inhibitor of cellular progression through the cell cycle by both androgen-responsive and androgen-nonresponsive human prostate cancer cells. In addition, treatment of both types of cells with increased concentrations of CeReS-18 induces cellular cytotoxicity, characterized as apoptosis.

Inhibition of cyclin D-cdk activity in cell cycle arrest of Swiss 3T3 cells by CeReS-18, a novel cell regulatory sialoglycopeptide.

CeReS-18 is a unique negative regulator of cell proliferation with a wide array of target cells. To elucidate the mechanism by which CeReS-18 mediates cell growth inhibition, the possibility that CeReS-18 alters the function of G1 cyclins and their respective cyclin-dependent kinases (cdks) has been examined in mouse fibroblasts (Swiss 3T3) synchronized by CeReS-18. We show here that cyclin D-associated cdk activity is significantly inhibited in the CeReS-18-treated cells. Corresponding to the inhibited cdk function, we demonstrate a low expression of cyclin D in mid G1 determined by Western blot analysis, and cyclin D was greatly reduced in the immunocomplex recovered with antibody to cdk4 and cdk6. Previously, we have shown that the retinoblastoma susceptibility gene product (pRb), a key substrate of cyclin D-cdk complex, was maintained in the hypophosphorylated state in the CeReS-18-inhibited cells. We conclude here that cyclin D/cdk4,6/pRb is the major pathway by which CeReS-18 mediates cell cycle arrest.

Multiple inositol 1,4,5-trisphosphate receptor isoforms are present in platelets.

Platelets are activated by an increase in cytosolic Ca(2+), and a portion of this increase is derived from inositol 1,4,5-trisphosphate (InsP3)-mediated Ca(2+) release from internal stores via the InsP3 receptor. There is some uncertainty concerning the localization of the InsP3 receptor within platelets, and experiments were designed to help resolve this question. [3H]InsP3 binding to unphosphorylated and phosphorylated platelet internal membranes revealed both low and high affinity InsP3 binding sites, indicating the presence of more than one isoform of InsP3 receptor within the internal membranes. Phosphorylation did not significantly affect InsP3 binding. In contrast, a single class of high affinity sites was observed in plasma membranes indicating only one type of InsP3 receptor. Western blotting of platelet internal and plasma membranes with antibodies against the three major InsP3 receptor isoforms revealed that the internal membranes contain both type 1 and type 2 InsP3 receptors while the plasma membrane contains only InsP3 receptor type 2.

Inositol 1,4,5-trisphosphate-mediated Ca2+ release from platelet internal membranes is regulated by differential phosphorylation.

Platelets are activated by an increase in cytosolic Ca2+, and a portion of this increase is derived from inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release from internal stores via the InsP3 receptor. Cytosolic cAMP inhibits platelet activation, and experiments were designed to determine if cAMP-dependent phosphorylation affects the rate of InsP3-mediated Ca2+ release. Western blotting of platelet internal membranes with anti-InsP3 receptor and anti-actin binding protein antibodies revealed that the platelet contains type 1 InsP3 receptor and that it is distinct from actin binding protein. The platelet InsP3 receptor was shown to be phosphorylated by endogenous, membrane-bound kinases as well as by exogenous protein kinase A. Prior phosphorylation of the insP3 receptor by endogenous kinases inhibited additional protein kinase A-dependent phosphorylation by 60%. Furthermore, endogenous phosphorylation resulted in a 2-fold increase in the InsP3-mediated Ca2+ release rate relative to dephosphorylated controls. Following endogenous phosphorylation, additional phosphorylation by protein kinase A returned the Ca2+ release rate to control values, while protein kinase A-dependent phosphorylation of dephosphorylated membranes did not affect the release rate. These results suggest that the InsP3 receptor within intact platelets is phosphorylated by endogenous kinases which results in a high InsP3-mediated Ca2+ release rate, and that increases in cAMP result in additional phosphorylation that inhibits Ca2+ release, thus contributing to inhibition of platelet activation.

Distribution of plasma membrane Ca(2+)-ATPase and inositol 1,4,5-trisphosphate receptor in human platelet membranes.

Human platelet plasma membranes were prepared by the glycerol lysis method of Harmon et al. [Harmon JT. Greco NJ. Jamieson GA. (1992) Isolation of human platelet plasma membranes by glycerol lysis. Meth. Enzymol., 215, 32-36]. The membranes were observed to contain a Ca(2+)-ATPase with different properties than those of internal membranes. The specific activity of Ca(2+)-ATPase was lower in plasma membranes (10-40 nmol ATP hydrolyzed/min/mg), but the ATPase was less sensitive to thapsigargin (41% inhibition at 500 nM) and more sensitive to vanadate (50% inhibition at 4 microM) than the Ca(2+)-ATPase in internal platelet membranes. The plasma membranes contained a Ca(2+)-ATPase detectable by monoclonal and polyclonal antibodies against erythrocyte Ca(2+)-ATPase that had a molecular mass of 144 kD. However, an anti-peptide antibody against an N-terminal sequence of the inositol 1,4,5-trisphosphate receptor recognized this protein in internal membranes, but not plasma membranes.

Cyclic AMP-dependent phosphorylation of the inositol-1,4,5-trisphosphate receptor inhibits Ca2+ release from platelet membranes.

Purified internal platelet membranes were treated with the catalytic subunit of protein kinase A to determine its effect on inositol-1,4,5-trisphosphate (IP3)-mediated Ca2+ release. Release kinetics were monitored using rhod-2, a Ca(2+)-specific fluorophore. Protein kinase A maximally inhibited the rate of IP3-mediated Ca2+ release by approximately 30% at saturating IP3 (10 microM). This inhibition was eliminated by pretreatment with a specific kinase inhibitor peptide. Partial purification of the platelet IP3 receptor showed that both endogenous kinases and added A kinase directly phosphorylate the receptor. Since the IP3 receptor is phosphorylated in the absence of added kinase, the observed inhibition (30%) by protein kinase A does not represent the maximal effect of phosphorylation.