Diglyceride/monoglyceride lipases pathway is not essential for arachidonate release in thrombin-activated human platelets

Human platelets prelabeled with arachidonate exhibited a rapid and transient rise in arachidonoyl monoglyceride in addition to arachidonoyl diglyceride following thrombin stimulation. Substantial release of arachidonate and its metabolites also occurred at the early phase. Preincubation of labeled platelets with RHC 80267, a potent inhibitor of diglyceride lipase, prior to thrombin stimulation abolished the transient rise in monoglyceride but not the increase in diglyceride and the release of arachidonate and its metabolites. These results suggest that diglyceride does metabolize to monoglyceride and release arachidonate in intact platelets. However, the diglyceride/monoglyceride lipases pathway does not appear to be essential in releasing arachidonate during thrombin stimulation.     

Protein kinase C translocation in human blood platelets

Protein kinase C (PKC) activity and translocation in response to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA), serotonin (5-HT) and thrombin was assessed in human platelets. Stimulation with PMA and 5-HT for 10 minutes or thrombin for 1 minute elicited platelet PKC translocation from cytosol to membrane. The catecholamines, norepinephrine or epinephrine at 10 microM concentrations did not induce redistribution of platelet PKC. Serotonin (0.5-100 microM) and the specific 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (10-100 microM) but not the 5-HT1A or 5-HT1B agonists, (+/-) 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT) or 5-methoxy-3-3-(1,2,3,6-tetrahydro-4-pyridin) 1H-indole succinate (RU 24969) induced dose-dependent PKC translocations. Serotonin-evoked PKC translocation was blocked by selective 5-HT2 receptor antagonists, ketanserin and spiroperidol. These results suggest that, in human platelets, PMA, thrombin and 5-HT can elicit PKC translocation from cytosol to membrane. Serotonin-induced PKC translocation in platelets is mediated via 5-HT2 receptors.     

Properties of phosphatidylinositol kinase of human platelets

We have previously reported that phosphatidylinositol (PI) kinase of intact platelet may be activated by either elevating intracellular cAMP content or lowering cytosolic Ca2+ (Thrombos. Res. 44, 155, 1986). Further studies were conducted to elucidate properties of platelet PI-kinase, especially possible regulation by A-kinase or Ca2+. The activity of the enzyme in platelet homogenate was markedly inhibited by a very low Ca2+, while Mg2+ was absolutely required for the activity. The activity was not affected by the presence of A-kinase catalytic subunit or protein kinase inhibitor but it was inhibited by cAMP as well as other compounds containing adenosine. These results suggest that the platelet PI-kinase is not regulated directly by A-kinase but by Ca2+ and that the regulation by Ca2+ may act as a negative feedback system in activated platelets.     

Selectivity of protein kinase inhibitors in human intact platelets

The specificity of commonly used protein kinase inhibitors has been evaluated in the intact human platelet. Protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) were activated selectively by treating platelets with phorbol dibutyrate (PDBu) or prostacyclin (PGl2). PKC activity was quantitated by measuring PDBu-specific phosphorylation of a 47,000 molecular weight protein, and PKA activity monitored by measuring prostacyclin-dependent phosphorylation of a 22,000 molecular weight protein. Staurosporine and 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7) were found to be non-specific inhibitors in the intact platelet, consistent with their effects on the isolated enzymes. Tamoxifen inhibited PKC activity (IC50 = 80 microM) but increased PKA-dependent protein phosphorylation. These results support the use of human platelets for measuring the specificity of protein kinase inhibitors and indicate that tamoxifen might have value for experimental purposes as a relatively selective PKC inhibitor.     

Properties of protein kinase C subspecies in human platelets

Protein kinase C (PKC) from human platelets was resolved into two fractions by hydroxyapatite column chromatography. One of the enzymes was indistinguishable from the brain type III PKC having alpha-sequence in its kinetic and immunological properties. The other enzyme was kinetically different from any of the brain PKC subspecies so far isolated, although it resembled the brain type II PKC having beta-sequence. With H1 histone as substrate, this platelet enzyme was not very sensitive to Ca2+, and activated partly by phosphatidylserine plus diacylglycerol or by free arachidonic acid. Both platelet enzymes could phosphorylate the P47 protein in vitro, but the enzyme physiologically responsible for the P47 protein phosphorylation in the activated platelets remains to be identified.     

Caldesmon phosphorylation in intact human platelets by cAMP-dependent protein kinase and protein kinase C

Caldesmon is a calmodulin- and actin-binding protein present in both smooth and non-muscle tissue. The present study demonstrates that platelet caldesmon is a substrate for cAMP-dependent protein kinase (protein kinase A). Purified platelet caldesmon has an apparent molecular mass of 82 kDa on sodium dodecyl sulfate-polyacrylamide gels and can be phosphorylated in vitro by the catalytic subunit of protein kinase A to a level of 2 mol of phosphate/mol of caldesmon. Phosphorylation of caldesmon by protein kinase A results in a shift in the apparent molecular mass of the protein to 86 kDa. When caldesmon was immunoprecipitated from intact platelets treated with prostacyclin (PGI2) the same shift in apparent molecular mass of caldesmon was observed. Comparison of two-dimensional tryptic phosphopeptide maps of caldesmon phosphorylated in vitro by protein kinase A with caldesmon immunoprecipitated from intact platelets verified that protein kinase A was responsible for the observed increase in caldesmon phosphorylation in PGI2-treated platelets. The present study demonstrates that although caldesmon is basally phosphorylated in the intact platelet, activation of protein kinase A by PGI2 results in the significant incorporation of phosphate into two new sites. In addition, the effects of phorbol ester, collagen, and thrombin on caldesmon phosphorylation were also examined. Although phorbol ester treatment results in a significant increase in caldesmon phosphorylation apparently by protein kinase C, treatment of intact platelets with thrombin or collagen does not result in an increase in caldesmon phosphorylation.     

Protein kinase C phosphorylation of syntaxin 4 in thrombin-activated human platelets

We postulated that the syntaxins, because of their key role in SNARE complex formation and exocytosis, could be important targets for signaling by intracellular kinases involved in secretion. We found that syntaxin 4 was phosphorylated in human platelets treated with a physiologic agent that induces secretion (thrombin) but not when they were treated with an agent that prevents secretion (prostacyclin). Syntaxin 4 phosphorylation was blocked by inhibitors of activated protein kinase C (PKC), and, in parallel assays, PKC inhibitors also blocked secretion from thrombin-activated platelets. In platelets, cellular activation by thrombin or phorbol 12-myristate 13-acetate decreased the binding of syntaxin 4 with SNAP-23, another platelet t-SNARE. Phosphatase inhibitors increased syntaxin 4 phosphorylation and further decreased syntaxin 4-SNAP-23 binding induced by cell activation. Conversely, a PKC inhibitor blocked syntaxin 4 phosphorylation and returned binding of syntaxin 4-SNAP-23 to that seen in nonstimulated platelets. In vitro, PKC directly phosphorylated platelet syntaxin 4 and recombinant syntaxin 4. PKC phosphorylation in vitro inhibited (71 +/- 8%) the binding of syntaxin 4 to SNAP-23. These results provide evidence that extracellular activation can be coupled through intracellular PKC signaling so as to modulate SNARE protein interactions involved in platelet exocytosis.     

Characterization of a novel focal adhesion kinase inhibitor in human platelets

Focal adhesion kinase (FAK) is activated in human platelets downstream of integrins, e.g. α_IIbβ₃, and other adhesion receptors e.g. GPVI. Mice in which platelets lack FAK have been shown to exhibit extended bleeding times and their platelets have been shown to display decreased spreading on fibrinogen-coated surfaces. Recently, a novel FAK inhibitor (PF-573,228) has become available, its selectivity for FAK shown in vitro and in cell lines. We determined the effect of this inhibitor on platelet function and signaling pathways. Like murine platelets lacking FAK, we found that PF-573,228 was effective at blocking human platelet spreading on fibrinogen-coated surfaces but did not affect the initial adhesion. We also found a reduced spreading on CRP-coated surfaces. Further analysis of the morphology of platelets adhered to these surfaces showed the defect in spreading occurred at the transition from filopodia to lamellipodia. Similar to that seen with murine neutrophils lacking FAK, we also observed an unexpected defect in intracellular calcium release in human platelets pre-treated with PF-573,228 which correlated with impaired dense granule secretion and aggregation. The aggregation defect could be partially rescued by addition of ADP, normally secreted from dense granules, suggesting that PF-573,228 has effects on FAK downstream of α_IIbβ₃ and elsewhere. Our data show that PF-573,228 is a useful tool for analysis of FAK function in cells and reveal that in human platelets FAK may regulate a rise in cell calcium and platelet spreading.     

Protein kinase C-mediated formation of phosphatidylinositol 3,4-bisphosphate in human platelets

The effect of phorbol 12,13-dibutyrate on the formation of phosphatidylinositol 3,4-bisphosphate in washed human platelets was studied. Platelets labelled with [32P]Pi were stimulated with phorbol 12,13-dibutyrate or thrombin in the presence or absence of staurosporine. Lipids were extracted, and deacylated, and the glycerophosphoinositol derivatives were analyzed by high performance liquid chromatography. Phorbol 12,13-dibutyrate increased formation of phosphatidylinositol 4-monophosphate and phosphatidylinositol 3,4-bisphosphate in a dose- and time-dependent manner. Thrombin also increased formation of phosphatidylinositol 3,4-bisphosphate. Staurosporine completely inhibited phorbol 12,13-dibutyrate or thrombin-stimulated production of phosphatidylinositol 3,4-bisphosphate. These data indicate that production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 4-monophosphate is mediated by protein kinase C. It is widely recognized that production of phosphatidylinositol 3,4-bisphosphate is caused by the tyrosine kinase-mediated activation of phosphatidylinositol 3-kinase. However, in platelets, production of phosphatidylinositol 3,4-bisphosphate might be related to stimulation of phosphatidylinositol 4-kinase, which is activated by protein kinase C.     

Diglyceride kinase from Escherichia coli: Modulation of enzyme activity by glycosphingolipids

Diglyceride kinase was purified from membranes of Escherichia coli K-12 using organic solvents. The enzyme apoprotein depended on lipids, such as cardiolipin (diphosphatidylglycerol), phosphatidylcholine or 1-monooleoylglycerol, for activity with 1,2-dipalmitoylglycerol. Mixed brain cerebrosides and gangliosides as well as defined ganglioside fractions and synthetic lactocerebroside were devoid of lipid cofactor activity. However, all these glycosphingolipids were strong inhibitors of activation by phosphatidylcholine. When cardiolipin was used as lipid activator with the detergent, Triton X-100, as solubilizing agent, the addition of mixed or purified gangliosides first (at about 0.4 mM) resulted in additional activation, but higher ganglioside concentrations were strongly inhibitory. Both effects were absolutely dependent on the presence of lipid-bound sialic acid and were not given by cerebrosides, by free sialic acid or by sialyl-lactose. The stimulating and inhibitory effects of glycosphingolipids could also be demonstrated when 1-monooleoylglycerol was used as substrate, lipid activator and solubilizing agent at the same time. The modulation of kinase activity by glycosphingolipids is discussed at the level of lipid/protein interactions.     

Diglyceride kinase from Escherichia coli. Modulation of enzyme activity by glycosphingolipids

Diglyceride kinase was purified from membranes of Escherichia coli K-12 using organic solvents. The enzyme apoprotein depended on lipids, such as cardiolipin (diphosphatidylglycerol), phosphatidylcholine or 1-monooleoylglycerol, for activity with 1,2-dipalmitoylglycerol. Mixed brain cerebrosides and gangliosides as well as defined ganglioside fractions and synthetic lactocerebroside were devoid of lipid cofactor activity. However, all these glycosphingolipids were strong inhibitors of activation by phosphatidylcholine. When cardiolipin was used as lipid activator with the detergent, Triton X-100, as solubilizing agent, the addition of mixed or purified gangliosides first (at about 0.4 mM) resulted in additional activation, but higher ganglioside concentrations were strongly inhibitory. Both effects were absolutely dependent on the presence of lipid-bound sialic acid and were not given by cerebrosides, by free sialic acid or by sialyl-lactose. The stimulating and inhibitory effects of glycosphingolipids could also be demonstrated when 1-monooleoylglycerol was used as substrate, lipid activator and solubilizing agent at the same time. The modulation of kinase activity by glycosphingolipids is discussed at the level of lipid/protein interactions.     

Physical and functional interaction between protein kinase C delta and Fyn tyrosine kinase in human platelets

An increasing number of tyrosine kinases have been shown to associate with isoforms of the protein kinase C (PKC) family. Here, we show evidence for physical and functional interaction between PKCdelta and the Src family kinase Fyn in human platelets activated by alboaggregin-A, a snake venom capable of activating both GPIb-V-IX and GPVI adhesion receptors. This interaction involves phosphorylation of PKCdelta on tyrosine and is specific in that other isoforms of PKC, PKCepsilon and lambda, which also become tyrosine-phosphorylated, do not interact with Fyn. In addition, PKCdelta does not interact with other platelet-expressed tyrosine kinases Syk, Src, or Btk. Stimulation also leads to activation of both Fyn and PKCdelta and to serine phosphorylation of Fyn within a PKC consensus sequence. Alboaggregin-A-dependent activation of Fyn is blocked by bisindolylmaleimide I, suggesting a role for PKC isoforms in regulating Fyn activity. Platelet activation with alboaggregin-A induces translocation of the two kinases from cytoplasm to the plasma membrane of platelets, as observed by confocal immunofluorescence microscopy. Translocation of Fyn and PKCdelta are blocked by PP1 and bisindolylmaleimide I, showing a dependence upon Src and PKC kinase activities. Although PKC activity is required for translocation, it is not required for association between the two kinases, because this was not blocked by bisindolylmaleimide I. Rottlerin, which inhibited PKCdelta activity, did not block translocation of either PKCdelta or Fyn but potentiated platelet aggregation, 5-hydroxytryptamine secretion, and the calcium response induced by alboaggregin-A, indicating that this kinase plays a negative role in the control of these processes.     

Association of phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities with the cytoskeleton in human platelets

The inositol lipid kinases were investigated in the cytoskeletons of human platelets. In the absence of added lipids the kinases were only barely detectable in the Triton-soluble fractions and undetectable in cytoskeletons of resting cells. However at least 30% of the total phosphatidylinositol kinase was present in the cytoskeleton as revealed by saturation of the enzyme. Phosphatidylinositol 4-phosphate kinase was also found in significant amounts in the cytoskeletons. On the other hand, both enzymes being only recovered in the particulate fraction of the cells, we suggest that inositol lipid kinases may be present near the anchoring points of the cytoskeletons at the membranes.     

Spacial isolation of protein kinase C activation in thrombin stimulated human platelets

Thrombin stimulation of human platelets is associated with turnover of inositol phospholipids, mobilization of intracellular Ca2+ stores, and activation of protein kinase C. However, within 5 minutes, the thrombin receptor desensitizes, but can be re-coupled to its effectors by stimulation of alpha 2-adrenergic receptors (Crouch and Lapetina, J. Biol. Chem. 263, 3363-3371, 1988). This effect of epinephrine was found to be inhibited by preincubation of platelets with phorbol ester, suggesting that protein kinase C was inhibitory. However, since thrombin also activated protein kinase C and epinephrine was active following thrombin stimulation of platelets, this implied that thrombin activation of protein kinase C may have been spacially isolated near the thrombin receptor and could not inactivate alpha 2-receptor activity. In the present paper, we have tested this possibility, and we present evidence which strongly favours the possibility that protein kinase C activation by receptors induces its local translocation to the cell membrane.     

Activators of protein kinase C decrease serotonin transport in human platelets.

Treatment of human platelets with activators of protein kinase C (PKC) for 5-20 min resulted in substantial reductions in the rate of platelet serotonin (5-HT) transport. The mean Vmax observed after 5 min treatment with 1 microM 4-beta-12-tetradecanoylphorbol 13-acetate (beta-TPA) was 66% (n = 16, P = 0.0001) of the control value. 5 min of treatment with 1 microM mezerein reduced uptake to 78% (n = 3, P = 0.01) of control. Both beta-TPA and mezerein had little effect on the Km of transport and had EC50 values of approx. 100 mM when a 20-min treatment period was used. The maximum effects of both were reached at approx. 20 min and could be blocked with staurospine. The beta-TPA effect was stereospecific, as alpha-TPA did not alter platelet 5-HT uptake. Although the PKC activators may have altered transmembrane ion-gradients for Na+ and Cl-, which are co-transported with 5-HT, minimizing ion-gradient changes had little effect on the observed reductions in transport. The PKC activators also had little or no effect on platelet 5-HT release or on the number (Bmax) of 5-HT transporters expressed at the platelet surface. The data indicate that PKC activation may down-regulate the activity of the 5-HT transporter in platelets. Apparently, most of this effect is mediated through mechanisms other than changes in ion-gradients, reductions in the number of available transporters, or increased 5-HT release. The apparent regulation of 5-HT transport by PKC may have important implications in platelet and neuronal functioning.     

Selective secretion of protein kinase C isozymes by thrombin-stimulated human platelets

The protein kinase C (PKC) was secreted from thrombin-stimulated human platelets in a time- and dose-dependent manner. The PKC specific inhibitors Ro31-8220 (0.05 microM) and GF 109203X (0.5 microM) totally inhibited the secreted kinase activity. Western blot analysis of the secretory components showed reactivity to PKCalpha, PKCbetaII, and PKCdelta antibodies, but not to PKCbetaI, and p42/44 MAPK, although they were present in lysed platelets. The fractionation of platelets secreted components showed that PKC activity increased in both soluble and microparticle fractions after thrombin treatments. This is the first report demonstrating that activated human platelets selectively secrete protein kinase C isozymes. Protein kinase C secreted by platelets in this unique manner may have an extracellular role in the plasma, and may regulate cellular functions, including remodeling of vascular endothelial cells.     

Phorbol ester induces differential membrane-association of protein kinase C subspecies in human platelets

Human platelets contained proteins which cross-reacted with antisera specific for brain protein kinase C-alpha and -beta. When platelets were incubated with 12-O-tetradecanoylphorbol-13-acetate there was a rapid accumulation of protein kinase C-alpha in the particulate fraction associated with a loss of this subspecies from the soluble fraction. No particulate accumulation or soluble loss of protein kinase C-beta could be detected when platelets were incubated with the phorbol ester.