Stimulation Of Human Platelets Research Articles

Sequential regulation of the small GTPase Rap1 in human platelets.

Rap1, a small GTPase of the Ras family, is ubiquitously expressed and particularly abundant in platelets. Previously we have shown that Rap1 is rapidly activated after stimulation of human platelets with alpha-thrombin. For this activation, a phospholipase C-mediated increase in intracellular calcium is necessary and sufficient. Here we show that thrombin induces a second phase of Rap1 activation, which is mediated by protein kinase C (PKC). Indeed, the PKC activator phorbol 12-myristate 13-acetate induced Rap1 activation, whereas the PKC-inhibitor bisindolylmaleimide inhibited the second, but not the first, phase of Rap1 activation. Activation of the integrin alpha(IIb)beta(3), a downstream target of PKC, with monoclonal antibody LIBS-6 also induced Rap1 activation. However, studies with alpha(IIb)beta(3)-deficient platelets from patients with Glanzmann's thrombasthenia type 1 show that alpha(IIb)beta(3) is not essential for Rap1 activation. Interestingly, induction of platelet aggregation by thrombin resulted in the inhibition of Rap1 activation. This downregulation correlated with the translocation of Rap1 to the Triton X-100-insoluble, cytoskeletal fraction. We conclude that in platelets, alpha-thrombin induces Rap1 activation first by a calcium-mediated pathway independently of PKC and then by a second activation phase mediated by PKC and, in part, integrin alpha(IIb)beta(3). Inactivation of Rap1 is mediated by an aggregation-dependent process that correlates with the translocation of Rap1 to the cytoskeletal fraction.

Identification of a Novel 33-kDa Ser/Thr Kinase that Phosphorylates the Cytoplasmic Tail of Protease-activated Receptor 1 (Thrombin Receptor) in Human Platelets

Stimulation of human platelets with thrombin or thrombin receptor agonist peptide (TRAP/ Ser-Phe-Leu-Leu-Arg-Asn) resulted in phosphorylation of the protease-activated receptor 1 (PAR1). However, protein kinase(s), capable of phosphorylating PAR1 upon activation of this receptor, has not been as yet identified in human platelets. The present study was undertaken to assess the presence of protein kinase(s) that may interact with PAR1 using a procedure based on the ability of protein kinase to undergo renaturation and phosphorylate a protein substrate fixed in a gel. We employed a fusion protein that was prepared using a glutathione S-transferase (GST) and the cytoplasmic tail of PARI (Pro368-Thr425)(GST-PAR1) or a reverse sequenced peptide of this domain (GST-rPAR1). The results showed that treatment of platelets with thrombin induced about 10-fold increase in the activity of the 33-kDa Ser/Thr protein kinase, which was also activated by TRAP, but not by hirudin-treated thrombin or diisopropylfluorophosphate-inactivated thrombin, suggesting that it is activated through PAR1. Furthermore, treatment of platelets with thromboxane A1 analog, STA2, led to an activation of this protein kinase and phosphorylation of PAR1. In conclusion, the present study provides evidence of homologous and heterologous activation of a novel 33-kDa Ser/Thr kinase that phosphorylates the cytoplasmic tail of PAR1.

Rap1B and Rap2B Translocation to the Cytoskeleton by von Willebrand Factor Involves FcγII Receptor-mediated Protein Tyrosine Phosphorylation

Stimulation of human platelets with von Willebrand factor (vWF) induced the translocation of the small GTPases Rap1B and Rap2B to the cytoskeleton. This effect was specifically prevented by an anti-glycoprotein Ib monoclonal antibody or by the omission of stirring, but was not affected by the peptide RGDS, which antagonizes binding of adhesive proteins to platelet integrins. Association of Rap2B with the cytoskeleton was very rapid, while translocation of Rap1B occurred in a later phase of platelet activation and was totally inhibited by cytochalasin D. vWF also induced the rapid tyrosine phosphorylation of several proteins that was prevented by the tyrosine kinases inhibitor genistein and by cAMP-increasing agents. Under these conditions, also the association of Rap1B and Rap2B with the cytoskeleton was prevented. Translocation of Rap proteins to the cytoskeleton induced by vWF, but not by thrombin, was inhibited by a monoclonal antibody against the FcgammaII receptor. The same antibody inhibited vWF-induced tyrosine phosphorylation of selected substrates with molecular masses of about 75, 95, and 150 kDa. Three of these substrates were identified as the tyrosine kinase pp72(syk), the phospholipase Cgamma2, and the inositol 5-phosphatase SHIP. Our results indicate that translocation of Rap1B and Rap2B to the cytoskeleton is regulated by tyrosine kinases and suggest a novel role for the FcgammaII receptor in the mechanism of platelet activation by vWF.

Functional Expression of the High Affinity Receptor for IgE (FcɛRI) in Human Platelets and Its' Intracellular Expression in Human Megakaryocytes

AbstractThe high affinity IgE receptor (FcɛRI) expressed on the cell surface of mast cells and basophils is the key molecule in triggering the IgE-mediated allergic reaction. Recently, it was elucidated that the FcɛRI is expressed on a variety of other cells like Langerhans cells, monocytes, and eosinophils, and the functional importance of the FcɛRI expression in Langerhans cells was also shown. Some studies suggest that human platelets may play important roles in allergic inflammation through the cell-surface expression of the FcɛRII and FcγRII. Here, we report that human platelets and megakaryocytes constitutively express the messenger RNA and protein for the FcɛRI. Although the FcɛRI is expressed on the cell surface of human platelets, it is only detected in the cytoplasm of human megakaryocytes. We also confirmed that human platelets express the genes for the α, β, and γ chains of the FcɛRI without any defined mutations. Furthermore, stimulation of human platelets via the FcɛRI induced the release of serotonin and RANTES (Regulated on Activation, Normal T Expressed, and presumably Secreted). Taken together, these results suggest a novel and important role for human platelets in perpetuating allergic inflammation through the expression of and activation via the FcɛRI.

Functional Expression of the High Affinity Receptor for IgE (FcɛRI) in Human Platelets and Its’ Intracellular Expression in Human Megakaryocytes

The high affinity IgE receptor (FcepsilonRI) expressed on the cell surface of mast cells and basophils is the key molecule in triggering the IgE-mediated allergic reaction. Recently, it was elucidated that the FcepsilonRI is expressed on a variety of other cells like Langerhans cells, monocytes, and eosinophils, and the functional importance of the FcepsilonRI expression in Langerhans cells was also shown. Some studies suggest that human platelets may play important roles in allergic inflammation through the cell-surface expression of the FcepsilonRII and FcgammaRII. Here, we report that human platelets and megakaryocytes constitutively express the messenger RNA and protein for the FcepsilonRI. Although the FcepsilonRI is expressed on the cell surface of human platelets, it is only detected in the cytoplasm of human megakaryocytes. We also confirmed that human platelets express the genes for the alpha, beta, and gamma chains of the FcepsilonRI without any defined mutations. Furthermore, stimulation of human platelets via the FcepsilonRI induced the release of serotonin and RANTES (Regulated on Activation, Normal T Expressed, and presumably Secreted). Taken together, these results suggest a novel and important role for human platelets in perpetuating allergic inflammation through the expression of and activation via the FcepsilonRI.

Secreted Dense Granule Adenine Nucleotides Promote Calcium Influx and the Maintenance of Elevated Cytosolic Calcium Levels in Stimulated Human Platelets

Evidence that secreted dense granule adenine nucleotides mediate part of the agonist-induced cytosolic calcium ([Ca2+]i) responses in human platelets was obtained from comparisons of fura-2-loaded platelets from normal subjects and from patients with a form of platelet storage pool deficiency (SPD) in which the secretory dense granules and their contents are virtually absent. SPD platelets had normal initial [Ca2+]i increases induced by thrombin and the endoperoxide analog U46619, but a significantly enhanced decay of elevated [Ca2+]i levels following the initial increases. With thrombin, this enhanced [Ca2+]i decay was associated with decreased Ca2+ influx, as measured by Mn2+ quench of fura-2 fluorescence. Addition of micromolar concentrations of ADP, alone or together with ATP, after stimulation reversed the enhanced [Ca2+]i decay and increased Mn2+ quench in SPD platelets, but had no effect on these responses in normal platelets, while addition of 100-fold higher concentrations of ATP or apyrase before stimulation increased [Ca2+]i decay and decreased Mn2+ quench in normal platelets, but had little effect in SPD platelets. ATP and alpha,beta-methylene ATP, a specific agonist for P2X1 receptors, at micromolar concentrations also increased Mn2+ quench, but to lesser extents than did ADP, in SPD platelets isolated and loaded with fura-2 in the presence of apyrase. Similar effects of ADP and excess ATP were seen in U46619-stimulated platelets, but decreased Ca2+ influx could not be measured directly in SPD platelets, presumably due to the very transient influx response seen with U46619. These results suggest that secreted dense granule ADP and ATP contribute to the maintenance of elevated [Ca2+]i levels, but not to the initial [Ca2+]i increases, in stimulated human platelets, most likely via a nucleotide-specific component of Ca2+ influx which may be mediated by interactions with both P2X1 and P2Y1 purinoceptors.

ADP, adrenaline and serotonin stimulate inositol 1,4,5-trisphosphate production in human platelets

Although adenosine diphosphate (ADP) is a well-known stimulus of platelet aggregation, it is not the generally accepted view that ADP stimulates phosphatidylinositolbisphosphate (PtdIns(4,5)P 2) hydrolysis. Using a very sensitive competitive receptor binding assay for inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3), we have detected Ins(1,4,5)P 3 production at early (<10 s) time points after stimulation of human platelets by the weak agonists ADP, adrenaline and serotonin (5-hydroxytryptamine, 5-HT). When adrenaline or 5-HT was combined with ADP in the presence of aspirin, there was a significant potentiation of ADP-induced platelet aggregation, but there was no potentiation of Ins(1,4,5)P 3 generation. Also, the increases in intracellular calcium (Ca 2+) concentrations stimulated by ADP were not potentiated by adrenaline in the presence of aspirin. Therefore, the synergism between the purinergic and adrenergic pathways of platelet activation occurs downstream from PtdIns(4,5)P 2 hydrolysis and intracellular Ca 2+ mobilization, although prior to platelet aggregation.

Cytoskeleton-dependent inhibition of the ADP-ribosyl cyclase activity of CD38 in thrombin-stimulated platelets

Stimulation of human platelets with thrombin caused a 42% inhibition of the ADP-ribosyl cyclase activity of membrane CD38. This effect was mediated by the activation of the platelet thrombin receptor rather than by proteolysis of CD38, and was not due to a different distribution of the synthesised nucleotide or to a reduced accessibility of CD38 to the substrate. The inhibitory effect of thrombin required actin polymerisation and was not observed when interaction of CD38 with the cytoskeleton was prevented by cytochalasin D. Finally, we analysed whether cADPR could play a role as a Ca 2+-mobilising agent in human platelets. Using saponin-permeabilised cells, we found that unlike IP 3, cADPR did not induce any release of Ca 2+ from intracellular stores. These results indicate that the enzymatic activity of membrane CD38 can be modulated by platelet activation, and that the function of this glycoprotein is probably not related to Ca 2+ mobilisation.

Oxidative Stress and the Mobilisation of Arachidonic Acid in Stimulated Human Platelets: Role of Hydroxyl Radical

Platelet functions, including eicosanoid biosynthesis, can be significantly altered by exposure to reactive oxygen species. We utilised the redox properties of the phenazine derivative, pyocyanin, to generate low micromolar levels of reactive oxygen species in order to investigate the metabolism of arachidonic acid by human platelets under oxidative stress. Eicosanoid production by platelets, pre-labelled with [ 3H]arachidonic acid (AA) and stimulated with the calcium ionophore A23187, was inhibited in the presence of pyocyanin. In contrast, platelets pre-treated with pyocyanin and concurrently exposed to A23187 and AA showed no evidence of inhibition. Analysis of the free label content of labelled, pyocyanin-treated platelets after stimulation revealed diminished levels of total free label and a corresponding increase in labelled phospholipid. Prior treatment with the antioxidants, superoxide dismutase, catalase or the hydroxyl radical scavenger, mannitol, before the addition of pyocyanin afforded protection against loss of eicosanoid production and restored AA release. We conclude that hydroxyl radicals inhibit one or more steps in the cascade leading to phospholipase A 2 activation and release of arachidonic acid from platelet phospholipid stores.

Protein Tyrosine Phosphatase SHP-1 Fails to Associate with Cytoskeleton but is Normally Phosphorylated upon Thrombin Stimulation of Thrombasthenic Platelets

SHP-1 is a cytoplasmic protein tyrosine phosphatase predominantly expressed in hematopoietic cells. Upon thrombin stimulation of human platelets, SHP-1 is rapidly phosphorylated on both serine and tyrosine residues, and becomes associated with the cytoskeleton, where it could participate in the formation of multiprotein signalling complexes. In order to discriminate between signalling events occurring downstream of G-protein-coupled thrombin receptor and those subsequent to integrin alpha IIb beta 3 engagement, SHP-1 behaviour was examined in platelets from two patients lacking integrin alpha IIb beta 3 (Glanzmann's thrombasthenia). Upon thrombin stimulation, phosphorylation of SHP-1 occurred normally in thrombasthenic platelets, whereas association with the cytoskeleton was abolished. Moreover, inhibition of normal platelet aggregation with the tetrapeptide arg-gly-asp-ser (RGDS) which impairs fibrinogen binding to integrin alpha IIb beta 3, did not alter significantly SHP-1 phosphorylation. It is concluded that SHP-1 phosphorylation is not a consequence of integrin signalling but might rather occur downstream of thrombin receptor and heterotrimeric G-proteins.

Phosphorylation of Platelet Pleckstrin Activates Inositol Polyphosphate 5-Phosphatase I

Pleckstrin is the major substrate phosphorylated on serine and threonine in response to stimulation of human platelets by thrombin (Abrams, C. S., Zhao, W., Belmonte, E., and Brass, L. F. (1995) J. Biol. Chem. 270, 23317-23321). We now show that pleckstrin in platelets is in a complex with inositol polyphosphate 5-phosphatase I (5-phosphatase I). This enzyme hydrolyzes the 5-phosphate from inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate and thus serves as a calcium signal-terminating enzyme, since the substrates but not the products mobilize intracellular calcium. Pleckstrin co-immunoprecipitates with 5-phosphatase I in homogenates of platelets. Platelet homogenates fractionated by anion exchange chromatography show co-elution of pleckstrin and 5-phosphatase I. Fractions containing phosphorylated pleckstrin have 7-fold greater 5-phosphatase activity than those containing unphosphorylated pleckstrin. Mixing experiments with recombinant 5-phosphatase I and pleckstrin in vitro show that they form a stoichiometric complex. A mutant form of pleckstrin, in which the serine and threonine residues that are phosphorylated by protein kinase C are substituted with glutamic acid (pseudophosphorylated pleckstrin), activates recombinant 5-phosphatase I 2-3-fold while native unphosphorylated pleckstrin does not stimulate the enzyme. Thus pleckstrin functions to terminate calcium signaling in platelets when it is phosphorylated by binding to and activating 5-phosphatase I.

Activation of Protein-Tyrosine Kinase Syk in Human Platelets Stimulated with Lysophosphatidic Acid or Sphingosine 1-Phosphate

It has been reported that not only lysophosphatidic acid (LPA) but also its sphingolipid counterpart, sphingosine 1-phosphate (Sph-1-P), induce platelet functional responses. We report here Syk activation in human platelets stimulated with these lysophospholipids. LPA rapidly induced platelet protein-tyrosine phosphorylation, including that of Syk, and Syk activation, assessed by immunoprecipitation kinase assay. Sph-1-P, although rather weaker, mimicked LPA in inducing these tyrosine kinase-related events. Pretreatment of platelets with staurosporine, a potent protein kinase inhibitor, diminished LPA-induced Syk phosphorylation and activation, but not intracellular Ca2+mobilization. These results demonstrate that, in platelets, the bioactive lysophospholipids induce Syk activation, which, however, may not be related to Ca2+mobilization.

Signaling Responses to Alkyllysophosphatidic Acid: The Activation of Phospholipases A2and C and Protein Tyrosine Phosphorylation in Human Platelets

The profile of biochemical responses following stimulation of human platelets with 1-alkyl-2-lyso-sn-glycero-3-phosphate (ALPA), a derivative of platelet-activating factor (PAF), was investigated. In the presence of extracellular Ca2+, ALPA evoked a dose-dependent increase and sustained elevation of the intracellular free Ca2+concentration and stimulated the formation of phosphatidic acid. Platelets released free [3H]arachidonic and [3H]oleic acid at maximal rates between 5 and 15 s following ALPA stimulation. However, in platelets labeled with myo-[3H]inositol, ALPA induced [3H]phosphoinositide breakdown and formation of [3H]inositol phosphates with slower kinetics. Intracellular Ca2+mobilization and the release of free fatty acids and inositol phosphates were not inhibited by pretreatment of platelets with pertussis toxin (PTX) or the PAF receptor antagonist WEB 2086. Following platelet stimulation with ALPA, tyrosine phosphorylation of proteins with apparent molecular masses of 65–95, 110–130, and 145–170 kDa was increased in a time-dependent manner, while phosphorylation of 40- to 45-kDa proteins was decreased. One of the platelet proteins phosphorylated on tyrosine residues in response to ALPA was found to be PLC-γ1. Exogenous [3H]ALPA was metabolized primarily to [1-3H]alkyl-2,3-diacylglycerol. The metabolic conversion of [3H]ALPA involved a dephosphorylation reaction, and the formation of the dephosphorylated product, [1-3H]alkyl-monoglycerol, was detected within 5 s. These data demonstrate that an ether-linked lysophosphatidic acid can activate human platelets by a PTX-insensitive mechanism which does not involve the PAF receptor. Upon stimulation of platelets, ALPA induces the activation of phospholipases A2and C, and tyrosine phosphorylation of several cellular proteins including PLC-γ1. These signal transduction responses in platelets are accompanied by rapid metabolism of ALPA.

Differential Mobilization of Tyrosine Kinases in Human Platelets Stimulated with Thrombin or Thrombin Receptor Agonist Peptide

Both thrombin and thrombin receptor agonist peptide (TRAP) activated p72sykand p60c-srcwith similar magnitudes. Both thrombin and TRAP induced translocation of p60c-srcand p54/58lynto cytoskeleton in an aggregation-dependent manner. Thrombin also induced cytoskeletal association of p72syk, but independent of platelet aggregation. Furthermore, p72sykassociated with cytoskeleton underwent marked proteolysis, which was partially dependent upon calpain activation. In contrast, TRAP, even at concentrations as high as 100 μ M, did not induce p72syktranslocation to cytoskeleton. Our findings suggest that cytoskeletal translocation of p72sykinduced by thrombin is governed by a mechanism distinct from those of p60c-srcand p54/58lyntranslocation. It is also suggested that p72syktranslocation induced by thrombin requires additional signal(s) other than that mediated by the recently-cloned thrombin receptor that couples with GTP-binding proteins and interacts with TRAP.

Thrombin-Induced GPIb-IX Centralization on the Platelet Surface Requires Actin Assembly and Myosin II Activation

In resting platelets, the GPIb-IX complex, the receptor for the von Willebrand factor (vWF), is linked to underlying actin filaments by actin-binding protein (ABP-280). Thrombin stimulation of human platelets leads to a decrease in the surface expression of the GPIb-IX complex, which is redistributed from the platelet surface into the open canalicular system (OCS). Because the centralization of GPIb-IX is inhibited by cytochalasin, it is believed to be linked to actin cytoskeletal rearrangements that take place during platelet activation. We have further characterized the mechanism of GPIb-IX centralization in platelets in suspension. Following thrombin stimulation, GPIb-IX shifts from the membrane skeleton of the resting cell to the cytoskeleton of the activated cell in a reaction sensitive to cytochalasin B. The cytoskeletal association of GPIb-IX involves ABP-280, as it correlates with the incorporation of ABP-280 into the activated cytoskeleton and because no dissociation of the ABP-280/GPIb-IX complexes is detected after thrombin activation. However, the incorporation of GPIb-IX into the cytoskeleton is complete within 1 minute, whereas GPIb-IX centralization requires 5 to 10 minutes for completion. The movement of GPIb-IX to the cytoskeleton of activated platelets is therefore necessary, but not sufficient for GPIb-IX centralization. Blockage of cytosolic calcium increases induced by thrombin by loading with the cell permeant calcium chelator Quin-2 AM inhibited GPIb-IX centralization by 70%, but did not prevent its association with the activated cytoskeleton. Quin-2 loading did, however, decrease the incorporation of myosin II into the activated cytoskeleton. The role of myosin II was further probed using the myosin light chain kinase (MLCK) inhibitor wortmannin. Wortmannin prevents myosin II association to the activated cytoskeleton and inhibits GPIb-IX centralization by 50%, without affecting actin assembly or the association of GPIb-IX to the cytoskeleton. Only micromolar concentrations of wortmannin, high enough to inhibit MLCK, prevent GPIb-IX centralization. These results indicate that thrombin-induced GPIb-IX centralization requires a minimum of two steps, one associating GPIb-IX to the activated cytoskeleton and the second requiring myosin II activation. The involvement of myosin II implies that GPIb-IX/ABP-280 complexes, linked to actin filaments, are pulled into the cell center, and that platelets may exert contractile tension on vWF bound to its receptor.

Protein-Tyrosine Phosphorylation and p72 syk Activation in Human Platelets Stimulated with Collagen Is Dependent upon Glycoprotein Ia/IIa and Actin Polymerization

In human platelets treated with acetylsalicylic acid, collagen induced protein-tyrosine-phosphorylation of several proteins. The major 75 kDa band included cortactin and auto-phosphorylated p72syk. p72syk activity rapidly increased upon collagen stimulation, whereas p60c-src activation was below detectable levels. A combination of inhibitors to remove the effects of extracellular and intracellular Ca2+, released ADP, and fibrinogen binding to GPIIb/IIIa delayed and attenuated the major 75 kDa band. By contrast, p72syk activation was not inhibited by these treatments. Cytochalasin D completely inhibited protein tyrosine phosphorylation and p72syk activation. It also potently inhibited aggregation and [Ca2+]i elevation. Anti-GPIa/IIa MoAb in a concentration-dependent manner partially attenuated protein tyrosine phosphorylation and p72syk activation. Its inhibitory effects on intracellular Ca2+ mobilization, release of intracellular granule contents, and aggregation also were partial. No tyrosine kinase activity was coprecipitated with GPIa/IIa. These results suggest that p72syk activation lies upstream of protein tyrosine phosphorylation, Ca2+ mobilization, ADP release, thromboxane A2 production and aggregation. GPIa/IIa plays a key role in p72syk activation induced by collagen, but other collagen receptors may work in synergy to fully activate p72syk. Actin polymerization is a prerequisite for both p72syk activation and other intracellular signal transduction pathways.

Alterations in individual molecular species of human platelet phospholipids during thrombin stimulation: electrospray ionization mass spectrometry-facilitated identification of the boundary conditions for the magnitude and selectivity of thrombin-induced platelet phospholipid hydrolysis.

Although the rapid thrombin-induced release of arachidonic acid in human platelets has been known for over 20 years, the amount of arachidonic acid mass mobilized and the source of the released arachidonic acid has remained a subject of intense controversy. Herein, we exploit the analytic power and sensitivity of electrospray ionization mass spectrometry to identify plasmenylethanolamines as the largest source of arachidonic acid mass released during thrombin stimulation and to demonstrate the presence of multiple novel molecular species of plasmenylethanolamines in human platelets. Specifically, 90 s after thrombin stimulation a total of 60.1 nmol of arachidonic acid-containing phospholipids/10(9) platelets was hydrolyzed which included the loss of 31.8 nmol/10(9) platelets from ethanolamine glycerophospholipids (hydrolysis of plasmenylethanolamines represented 63% of the mass lost from the ethanolamine glycerophospholipid pool) but only 10.9 nmol/10(9) platelets from choline glycerophospholipids. Human platelet phosphatidylserine and phosphatidylinositol pools contained similar amounts of arachidonic acid mass in resting platelets (approximately equal to 20 nmol/10(9) platelets), and each pool contributed 8.7 nmol/10(9) platelets after thrombin stimulation. From these results, a lower boundary for the rate of thrombin-induced arachidonic acid mobilization in human platelets can be set at > 60 nmol/10(9) platelets, thereby identifying specific kinetic characteristics and substrate selectivities of the phospholipase(s) activated during platelet stimulation. Collectively, these results underscore the importance of plasmenylethanolamines as the major storage depot of arachidonic acid in resting platelets and as the major source of arachidonic acid mobilized after thrombin stimulation of human platelets.

Histidine phosphorylation of P-selectin upon stimulation of human platelets: A novel pathway for activation-dependent signal transduction

Transient phosphorylation of histidine characterizes the two-component systems in prokaryotes that control important physiological functions, but analogous events have not been implicated in signal transduction in mammalian cells. To explore histidine phosphorylation during activation of human cells, stimulated platelets were analyzed for the formation of protein phosphohistidine in a model system employing P-selectin. P-selectin, a leukocyte adhesion molecule, undergoes rapid phosphorylation and selective dephosphorylation of tyrosine, serine, and threonine. We now establish that phosphorylation following platelet activation with thrombin or collagen generates phosphohistidine at histidines on the cytoplasmic tail of P-selectin. With thrombin stimulation, the kinetics of phosphohistidine appearance and disappearance on P-selectin are very rapid. Platelets exhibit a novel ligand-induced signaling pathway to generate phosphohistidine. These results provide direct biochemical evidence for the induction of rapid and reversible histidine phosphorylation in mammalian cells upon cell activation and represent a novel paradigm for mammalian cell signaling.

Integrin αIIbβ3-mediated Translocation of CDC42Hs to the Cytoskeleton in Stimulated Human Platelets

To investigate the function of the human Ras-related CDC42 GTP-binding protein (CDC42Hs) we studied its subcellular redistribution in platelets stimulated by thrombin-receptor activating peptide (TRAP) or ADP. In resting platelets CDC42Hs was detected exclusively in the membrane skeleton (9.6 +/- 1.5% of total) and the detergent soluble fraction (90 +/- 4%). When platelets were aggregated with TRAP or ADP, CDC42Hs (10% of total) appeared in the cytoskeleton and decreased in the membrane skeleton, whereas RhoGDI (guanine-nucleotide dissociation inhibitor) and CDC42HsGAP (GTPase-activating protein) remained exclusively in the detergent-soluble fraction. Upon prolonged platelet stimulation CDC42Hs disappeared from the cytoskeleton and reappeared in the membrane skeleton. Rac translocated to the cytoskeleton with a similar time course as CDC42Hs. When platelets were stimulated under conditions that precluded the activation of the alpha IIb beta 3 integrin and platelet aggregation, cytoskeletal association of CDC42Hs was abolished. Translocation of CDC42Hs to the cytoskeleton but not aggregation was also prevented by cytochalasins B or D or the protein tyrosine kinase inhibitor genistein. Platelet secretion and thromboxane formation were not required but facilitated the cytoskeletal association of CDC42Hs. The results indicate that in platelets stimulated by TRAP or ADP, a fraction of CDC42Hs translocates from the membrane skeleton to the cytoskeleton. This process is reversible and is mediated by activation of the alpha IIb beta 3 integrin and subsequent actin polymerization and protein-tyrosine kinase stimulation. CDC42Hs might be a new component of a signaling complex containing specific cytoskeletal proteins and protein-tyrosine kinases that forms after activation of the alpha IIb beta 3 integrin in platelets.

Endothelin influences pH i of human platelets through protein kinase C mediated pathways

Stimulation of human platelets with endothelin raises cytosolic pH (pH i). In order to determine whether this effect is mediated via protein kinase C and Na +-H + linked pathways, the effects of staurosporine and calphostin C (protein kinase C inhibitors) and 5-(N,N-hexamethylene) amiloride (Na +-H + exchange blocker) on endothelin-induced pH i responses in human platelets were assessed. In addition, platelet endothelin receptor subtypes were determined pharmacologically using the selective ET A receptor antagonist BQ-123 and the ET B receptor agonist sarafotoxin S6c. pH i was measured spectrofluorometrically using the fluorescent probe BCECF-AM in platelets obtained from 15 healthy subjects. Endothelin-1 at a fixed concentration of 10 −9 M significantly increased pH i from 7.11 ± 0.01 ([H +] i = 77 ± 0.9 nM) to 7.19 ± 0.04 ([H +] i = 64 ± 0.9 nM) (p<0.01). The pH i-stimulating effect of endothelin-1 was inhibited by 10 −7 M staurosporine, calphostin C and 5-(N,N-hexamethylene) amiloride. BQ-123 (10 −7 M) abolished the pH i responses to endothelin-1, whereas sarafotoxin S6c had no effect on platelet pH i. These data suggest that in vitro effects of endothelin-1 on platelet pH i are receptor-mediated via a pathway involving protein kinase C. Platelet endothelin receptors appear to be of the ET A subtype.