Dense Granule Secretion Research Articles

Identification of a Novel Par1 inhibitor Using a Chemical Genetic Screen

AbstractAbstract 2018The anucleate platelet is not amenable to traditional forward genetic analysis, but is a good candidate for a chemical genetic screen. In collaboration with the Broad Institute Probe Development Center, we have performed a high throughput screen using a modified luciferin-luciferase detection system to identify inhibitors of Par1-mediated dense granule secretion. For primary screening, over 300,000 compounds were assayed in duplicate using freshly outdated platelet-rich plasma supplied by several blood banks across the United States. Computational analyses of the primary data demonstrated that approximately 0.2% of compounds showed ≥50% inhibition relative to maximally inhibitory concentrations of the known antiplatelet agent, cilostazol. Secondary screening using 8-point dose response curves and counterscreening to exclude luciferase inhibitors identified 137 small molecules that inhibited Par1-mediated ATP/ADP release without significant inhibition of luciferase. Twenty eight compounds were selected for further testing based on their IC50s in confirmatory assays (<10 μM), lack of activity in unrelated bioassays, and chemical structure. Known platelet inhibitors were excluded. Of the 28 compounds, 16 compounds potently inhibited SFLLRN-induced α-granule release from washed platelets, as monitored by P-selectin expression. IC50s for these compounds ranged from <0.3 to 1 μM. None of the compounds that failed to inhibit α-granule release demonstrated significant inhibition of SFLLRN-induced 14C-serotonin release at 10 μM. Of the 16 confirmed inhibitory compounds, 12 demonstrated >50% stimulation of cAMP levels compared to platelets exposed to PGE1 and were not further analyzed. One of the 4 remaining compounds lost activity upon resynthesis. The three compounds that demonstrated activity upon resynthesis included a diaminophenyl, an acylhydrazone, and a cyanopyridone. A series of chemical analogs for each of the three series was synthesized and/or obtained from commercial vendors. Structure activity relationships were determined by testing these compounds in the luciferin-luciferase assay. The most potent analog of each series was then further analyzed. The representative compounds (10 μM) failed to block platelet granule release induced by the Par4 activating peptide AYPGFK, PMA, calcium ionophore, or collagen, suggesting that the compounds act at or near Par1. However, only one of the three compounds, termed DAP, significantly inhibited thrombin-induced platelet activation. Determination of DAP activity in cultured cells expressing Par1 confirmed DAP-mediated inhibition of Par1 activity. Analysis of dose-response curves at multiple inhibitor concentrations were consistent with non-competitive inhibition of Par1-mediated granule secretion. In functional studies, DAP inhibited platelet aggregation and secretion, but not platelet shape change. In contrast, the orthosteric Par1 inhibitor Sch79797 blocked aggregation, secretion, and shape change. Despite its inability to inhibit activation through human Par4, DAP blocked activation through mouse Par4, demonstrating complete inhibition of AYPGFK-mediated aggregation of mouse platelets at 5 μM. Platelets obtained from mice 45 minutes after infusion of 10 mg/kg DAP failed to aggregate in response to AYPGFK. The effect of DAP on thrombus formation was evaluated using intravital microscopy. These studies showed that infusion of 10 mg/kg DAP potently inhibited platelet accumulation following laser-induced vascular injury of cremaster arterioles in mice. These studies demonstrate the feasibility of using blood-banked platelets in a large-scale chemical genetic screen. Compounds that block platelet function via elevation of cAMP are common, indicating a need to include cAMP assays early in the screening funnel. The novel antithrombotic DAP identified in this screen appears to block Par1 via an allosteric mechanism. The ability of DAP to inhibit mouse Par4, but not human Par4, indicates that DAP acts outside the ligand binding pocket of Par1. This compound will provide a useful probe to study the effect of small molecule modulation of Par1 on receptor conformation and downstream signaling. Disclosures:No relevant conflicts of interest to declare.

A selective serotonin reuptake inhibitor, citalopram, inhibits collagen-induced platelet aggregation and activation

Clinical depression is a significant risk factor for cardiovascular diseases and confers an increased risk of mortality. Increased platelet reactivity may predispose depressed patients to cardiovascular diseases. The antidepressants selective serotonin reuptake inhibitors (SSRIs) have been found to have cardioprotective effects probably via the attenuation of platelet activation independently in addition to treatment of depression itself. However, the characters of the inhibitory effect of SSRIs on platelets remain largely unknown. Here we show that an SSRI, citalopram, specifically inhibited collagen-induced platelet aggregation. Citalopram, however, revealed only little inhibitory effect on platelet aggregation induced by thrombin, U46619, and ionomycin, and failed to inhibit reversible platelet aggregation induced by adenosine diphosphate with fibrinogen. Collagen-induced of α IIbβ 3 integrin activation in platelets under a static condition was not influenced by citalopram. Citalopram inhibited convulxin-induced platelet aggregation and α IIbβ 3 integrin activation. In the experiments with fibrinogen-induced aggregation in elastase-treated platelets, citalopram inhibited only collagen-induced α IIbβ 3 activation but not the binding activities between activated α IIbβ 3 integrin and fibrinogen. Moreover, citalopram inhibited α-granule and dense granule secretion from platelets in response to collagen, as determined by a reduced expression of P-selectin and adenosine triphosphate release, respectively. In addition, collagen-induced thromboxane A 2 release in platelets was attenuated by citalopram pretreatment. These findings might specify the mechanisms of inhibitory effects of citalopram on collagen mediated platelet activation and aggregation, and further support the cardioprotective effect of SSRIs.

Differential phosphorylation of myosin light chain (Thr)18 and (Ser)19 and functional implications in platelets.

Myosin IIA is an essential platelet contractile protein that is regulated by phosphorylation of its regulatory light chain (MLC) on residues (Thr)18 and (Ser)19 via the myosin light chain kinase (MLCK). The present study was carried out to elucidate the mechanisms regulating MLC (Ser)19 and (Thr)18 phosphorylation and the functional consequence of each phosphorylation event in platelets. Induction of 2MeSADP-induced shape change occurs within 5s along with robust phosphorylation of MLC (Ser)19 with minimal phosphorylation of MLC (Thr)18. Selective activation of G(12/13) produces both slow shape change and comparably slow MLC (Thr)18 and (Ser)19 phosphorylation. Stimulation with agonists that trigger ATP secretion caused rapid MLC (Ser)19 phosphorylation while MLC (Thr)18 phosphorylation was coincident with secretion. Platelets treated with p160(ROCK) inhibitor Y-27632 exhibited a partial inhibition in secretion and had a substantial inhibition in MLC (Thr)18 phosphorylation without effecting MLC (Ser)19 phosphorylation. These data suggest that phosphorylation of MLC (Ser)19 is downstream of Gq/Ca(2+) -dependent mechanisms and sufficient for shape change, whereas MLC (Thr)18 phosphorylation is substantially downstream of G(12/13) -regulated Rho kinase pathways and necessary, probably in concert with MLC (Ser)19 phosphorylation, for full contractile activity leading to dense granule secretion. Overall, we suggest that the amplitude of the platelet contractile response is differentially regulated by a least two different signaling pathways, which lead to different phosphorylation patterns of the myosin light chain, and this mechanism results in a graded response rather than a simple on/off switch.

Platelet function and relationship to coronary artery disease (CAD) risk in early menopausal women

Risk of CAD increases at menopause and platelets contribute to the etiology of CAD. We hypothesized that individual variation in platelet procoagulant activity at menopause correlates with the extent of CAD as reflected by coronary artery calcification (CAC). We developed several novel and global assays of platelet procoagulant activity: dense (ATP release) and alpha (P‐selectin expression) granule secretion and GPαIIbβ3 fibrinogen‐binding (PAC1 binding) to ADP stimulation, and prostaglandin E1 (PEG1) sensitivity as a surrogate for prostacyclin sensitivity. Platelets were prepared from venous blood collected from women screened for the Kronos Early Estrogen Prevention Study (n=125; mean±SD age=52.5±2.4 years) within 19.4±9.2 months of last menses. Baseline CAC score was determined. Of conventional CAD risk factors, only systolic blood pressure correlated with positive CAC score (n=23). ATP secretion tended to be less in women with positive CAC scores and was negatively correlated with blood glucose (P&lt;0.001). P‐selectin expression and PAC1 binding increased with total cholesterol (P&lt;0.05). PEG1 sensitivity increased with increasing age and waist circumference (P&lt;0.05). These results indicate that platelet functions change with age and in association with conventional risk factors for the disease in women at menopause. Supported by the Kronos Longevity Research Institute and NIH HL90639.

Phorbol ester-evoked Ca2+ signaling in human platelets is via autocrine activation of P(2X1) receptors, not a novel non-capacitative Ca2+ entry.

Platelets are reported to possess a protein kinase C (PKC)-dependent non-capacitative Ca(2+)entry (NCCE) pathway. The phorbol ester, phorbol, 12-myristate, 13-acetate (PMA) has been suggested to stimulate platelet NCCE. Recently we demonstrated important roles in store-operated Ca(2+)entry in human platelets for Na(+)/Ca(2+) exchangers (NCXs) and autocrine signaling between platelets after dense granule secretion. As PMA evokes dense granule secretion, we have investigated the role of NCXs and autocrine signaling in PMA-evoked Ca(2+)entry. To investigate the roles of NCXs and dense granule secretion in PMA-evoked Ca(2+)signaling in human platelets. Fura-2- or sodium-binding benzofuran isophthalate (SBFI)-loaded platelets were used to monitor cytosolic Ca(2+)or Na(+) concentrations. Dense granule secretion was monitored as ATP release using luciferin-luciferase. The NCX inhibitors KB-R7943 or SN-6, and removal of extracellular Na(+), significantly reduced PMA-evoked Ca(2+)entry. PMA-evoked dense granule secretion was almost abolished by pretreatment with the PKC inhibitor Ro-31-8220 and significantly slowed by KB-R7943. The P(2X1) antagonists Ro-0437626 or MRS-2159, or desensitization of P(2X1) receptors by prior treatment with alpha,beta-Methylene-ATP or omitting apyrase from the medium, reduced PMA-evoked Ca(2+)entry. Ro-0437626 or chelation of extracellular Ca(2+) slowed but did not abolish PMA-evoked ATP release, indicating that PMA-evoked dense granule secretion does not require P(2X1) receptor activation but is accelerated by P(2X1)-mediated Ca(2+)entry. The presence of NCX3 in human platelets was demonstrated by Western blotting. PMA-evoked Ca(2+)entry results from an NCX3-dependent dense granule secretion and subsequent P(2X1) receptor activation by secreted ATP, rather than activation of a novel NCCE pathway.

New P2Y 12 Inhibitors

A denosine diphosphate (ADP) plays a key role in the genesis of physiological platelet-rich hemostatic plugs and of pathological arterial thrombi.1 The transduction of the ADP signal involves its interaction with 2 platelet receptors, the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor, which belong to the family of purinergic P2 receptors. Concomitant activation of both the Gq and Gi pathways by ADP is necessary to elicit normal platelet aggregation.2 In addition to its role in ADP-induced platelet aggregation, P2Y12 (Figure 1) also mediates the potentiation of platelet secretion induced by strong agonists, which is independent of the formation of large aggregates and thromboxane A2 synthesis2; the stabilization of thrombin-induced platelet aggregates2; shear-induced platelet aggregation2; and the inhibition of the antiplatelet effects of the natural regulator of platelet function, prostacyclin.3 In contrast to P2Y1, P2Y12 has a very selective tissue distribution, making it an attractive molecular target for therapeutic intervention. Indeed, P2Y12 is the target of efficacious antithrombotic agents.1 Figure 1. Central role of P2Y12 in platelet aggregation. ADP, by interacting with P2Y12, a 7-transmembrane receptor that is coupled to the inhibitory G protein Gi, induces platelet aggregation and amplifies the aggregation response that is induced by other agonists or by ADP itself, interacting with its other platelet receptor, P2Y1. In addition, P2Y12 stabilizes the platelet aggregates and amplifies the secretion of platelet dense granules stimulated by secretion-inducing agonists (which are coupled to Gq). Although P2Y12 is coupled to inhibition of adenylyl cyclase (AC) through Gi, this function does not appear to be directly related to P2Y12-mediated platelet activation. However, it could have important implications in vivo, …

Toll-like receptor 2 stimulation of platelets is mediated by purinergic P2X1-dependent Ca2+ mobilisation, cyclooxygenase and purinergic P2Y1 and P2Y12 receptor activation

Toll-like receptor 2 (TLR2), which recognise and respond to conserved microbial pathogen-associated molecular patterns, is expressed on the platelet surface. Furthermore, it has recently been shown that the TLR2/1 agonist Pam3CSK4 stimulates platelet activation. The aim of the present study was to clarify important signalling events in Pam3CSK4-induced platelet aggregation and secretion. Platelet interaction with Pam3CSK4 and the TLR2/6 agonist MALP-2 was studied by analysing aggregation, ATP-secretion, [Ca2+]i mobilisation and thromboxane B2 (TxB2) production. The results show that Pam3CSK4 but not MALP-2 induces [Ca2+]i increase, TxB2 production, dense granule secretion and platelet aggregation. Preincubation of platelets with MALP-2 inhibited the Pam3CSK4-induced responses. The ATP-secretion and aggregation in Pam3CSK4-stimulated platelets was impeded by the purinergic P2X1 inhibitor MRS 2159, the purinergic P2Y1 and P2Y12 antagonists MRS 2179 and cangrelor, the phospholipase C inhibitor U73122, the calcium chelator BAPT-AM and aspirin. The calcium mobilisation was lowered by MRS 2159, aspirin and U73122 whereas the TxB2 production was antagonised by MRS 2159, aspirin and BAPT-AM. When investigating the involvement of the myeloid differentiation factor-88 (MyD88) -dependent pathway, we found that platelets express MyD88 and interleukin 1 receptor-associated kinase (IRAK-1), which are proteins important in TLR signalling. However, Pam3CSK4 did not stimulate a rapid (within 10 minutes) phosphorylation of IRAK-1 in platelets. In conclusion, the results show that Pam3CSK4-induced platelet aggregation and secretion depends on a P2X1-mediated Ca2+ mobilisation, production of TxA2 and ADP receptor activation. The findings in this study further support a role for platelets in sensing bacterial components.

Curcumin inhibits GPVI-mediated platelet activation by interfering with the kinase activity of Syk and the subsequent activation of PLCγ2

Turmeric (Curcuma longa), a herbal remedy and culinary spice, has been used in traditional Indian culture for millennia. An active ingredient found in turmeric is curcumin (diferuloylmethane). In the current study, we investigated the antiplatelet properties of this naturally occurring compound. Curcumin inhibited human platelet aggregation and dense granule secretion induced by GPVI agonist convulxin in a concentration-dependent manner. At 50 µM, it effectively inhibited the maximal extent of aggregation and dense granule secretion to as much as 75%. It also dramatically inhibited the activation-dependent tyrosine phosphorylation of Y753 and Y759 on PLCγ2, but did not affect the phosphorylation of Y145 residue on the cytosolic adaptor protein SLP-76. Interestingly, curcumin had no significant effect on the phosphorylation of Y525/Y526 present on the activation loop of Syk (spleen tyrosine kinase), but had a significant inhibitory effect on in vitro Syk kinase activity. Moreover, the inhibitory action of curcumin is not due to an inhibition of thromboxane generation because all our studies were performed using aspirin-treated platelets. We conclude that curcumin inhibits platelet activation induced by GPVI agonists through interfering with the kinase activity of Syk and the subsequent activation of PLCγ2.

RGD-ligand mimetic antagonists of integrin alphaIIbbeta3 paradoxically enhance GPVI-induced human platelet activation.

The integrin alpha(IIb)beta(3) is the major mediator of platelet aggregation and has, therefore, become an important target of antithrombotic therapy. Antagonists of alpha(IIb)beta(3), for example abciximab, tirofiban and eptifibatide, are used in the treatment of acute coronary syndromes. However, in addition to effective blockade of the integrin, binding of can induce conformational changes in the integrin and can also induce integrin clustering. This class effect of RGD-ligand mimetics might, therefore, underlie paradoxical platelet activation and thrombosis previously reported. To examine the components of signaling pathways and functional responses in platelets that may underlie this phenomenon of paradoxical platelet activation. We assessed the effect of lotrafiban, and other alpha(IIb)beta(3) antagonists including the clinically used drug tirofiban, on tyrosine phosphorylation of key signaling proteins in platelets by immunoblotting and also platelet functional outputs such as cytosolic calcium responses, phosphatidylserine exposure (pro-coagulant activity) and dense granule release. In all cases, no effect of alpha(IIb)beta(3) antagonists were observed on their own, but these integrin antagonists did lead to a marked potentiation of glycoprotein VI (GPVI)-associated FcR gamma-chain phosphorylation, activation of Src family kinases and Syk kinase. This correlated with increased dense granule secretion, cytosolic calcium response and exposure of phosphatidylserine on the platelet surface. P2Y(12) antagonism abolished the potentiated phosphatidylserine exposure and dense granule secretion but not the cytosolic calcium response. These data provide a mechanism for enhancement of platelet activity by alpha(IIb)beta(3) inhibitors, but also reveal a potentially important signaling pathway operating from the integrin to GPVI signaling.

Non-redundant Roles of Phosphoinositide 3-Kinase Isoforms α and β in Glycoprotein VI-induced Platelet Signaling and Thrombus Formation

Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase Cgamma2, which bears similarity to the signaling pathway evoked by T- and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3Kalpha or -beta strongly and selectively suppressed GPVI-induced Ca(2+) mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase Cgamma2 by PI3Kalpha/beta. That PI3Kalpha and -beta have a non-redundant function in GPVI-induced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca(2+) increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3Kalpha/beta was blocked or p85alpha was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3Kdelta or -gamma. Furthermore, PI3Kalpha/beta, but not PI3Kgamma, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3Kalpha and -beta isoforms are required for full GPVI-dependent platelet Ca(2+) signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3Kalpha an interesting new target for anti-platelet therapy.

Canonical Wnt signaling negatively regulates platelet function.

Wnts regulate important intracellular signaling events, and dysregulation of the Wnt pathway has been linked to human disease. Here, we uncover numerous Wnt canonical effectors in human platelets where Wnts, their receptors, and downstream signaling components have not been previously described. We demonstrate that the Wnt3a ligand inhibits platelet adhesion, activation, dense granule secretion, and aggregation. Wnt3a also altered platelet shape change and inhibited the activation of the small GTPase RhoA. In addition, we found the Wnt-beta-catenin signaling pathway to be functional in platelets. Finally, disruption of the Wnt Frizzled 6 receptor in the mouse resulted in a hyperactivatory platelet phenotype and a reduced sensitivity to Wnt3a. Taken together our studies reveal a novel functional role for Wnt signaling in regulating anucleate platelet function and may provide a tractable target for future antiplatelet therapy.

SHIP-1 Associates with Tyrosine Phosphorylated Protein Kinase C-Delta at 155 Residue.

Abstract 3004Poster Board II-981Protein Kinase C-delta (PKCδ) is a novel PKC isoform that differentially regulates platelet dense granule secretion. PKCδ positively regulates Protease activated receptor (PAR)-mediated dense granule secretion, whereas it negatively regulates glycoproteinVI (GPVI)-mediated dense granule secretion in platelets. PKCδ, a serine/threonine kinase is phosphorylated on its tyrosine residues. There are nine potential tyrosine phosphorylation sites in the regulatory domain of PKCδ. Phosphorylation at different tyrosine residues regulates its substrate specificity. We have previously shown that the association of PKCδ with Lyn and SHIP-1 negatively regulates GPVI-mediated dense granule secretion. However, the event leading to the association between PKCδ and SHIP-1 is not known. We hypothesize that the differential tyrosine phosphorylation of PKCδ downstream of PARs or GPVI receptors result in the preferential association with SHIP-1. In the current study, we show that PKCδ is phosphorylated at tyrosine residues Y332, Y523, Y525 and Y565 upon PAR or GPVI stimulation. Y311 residue is predominantly phosphorylated upon stimulation of PARs, whereas Y155 residue is preferentially phosphorylated upon GPVI stimulation. PAR-mediated Y311 phosphorylation peaks at later timepoint, whereas GPVI-mediated Y155 phosphorylation peaks at an early timepoint. correlating with dense granule secretion. Furthermore, we show that agarose-conjugated Y155 phosphorylated PKCδ peptide associates with SHIP-1 upon GPVI stimulation, and not PARs. These data suggest that the phosphorylation of PKCδ at distinct tyrosine residues differentially regulate its association with SHIP-1. Therefore, we conclude that the GPVI-mediated phosphorylation of PKCδ at 155 is required for its association with SHIP-1.This study is supported by pre-doctoral fellowship to Ramya Chari from American Heart Association, Pennsylvania-Delaware affiliate. Disclosures:No relevant conflicts of interest to declare.

A Chemical Genetic Analysis of Platelet Activation.

Abstract 4009Poster Board III-945Platelets are anucleate cells that are not amenable to traditional forward genetic analysis. In collaboration with the Broad Institute Probe Development Center, we have performed a chemical genetic analysis of platelet activation. Chemical genetics involves exposure of cells to a library of small molecules, identification of compounds that produce a phenotype of interest, and determination of the target of these small molecules. We have used an assay designed both to identify allosteric inhibitors of Protease Activated Receptor-1 (PAR1) and to find inhibitors that selectively target granule release. This assay monitors dense granule secretion mediated by SFLLRN, a PAR1-specific agonist, using a luciferase-based assay system to detect ADP/ATP release. For primary screening, over 300,000 compounds were assayed in duplicate using freshly outdated platelet-rich plasma supplied by several blood banks across the United States. Computational analyses of the primary data demonstrated that approximately 0.2% of compounds showed ≥50% inhibition relative to maximally inhibitory concentrations of the known antiplatelet agent, cilostazol. Secondary screening using 8-point dose response curves were performed on the 629 inhibitory compounds, 742 compounds with inconclusive activity (e.g., ambiguous duplicates in primary screening), and 213 structural analogs of active compounds. These assays identified 367 active compounds with IC50s ≤10 micromolar. Counter screening to exclude luciferase inhibitors demonstrated 137 small molecules that inhibited PAR1-mediated ATP/ADP release without significant inhibition of luciferase. Database mining using PubChem and CAS search engines was performed to assess the selectivity of active compounds. Twenty eight compounds were selected for further testing based on their IC50s in confirmatory assays, lack of activity in unrelated bioassays, and chemical structure. Known platelet inhibitors were excluded. Of the 28 compounds, 16 compounds potently inhibited SFLLRN-induced alpha-granule release from washed platelets, as monitored by P-selectin expression. IC50s for these compounds ranged from <0.3 to 1 micromolar. None of the selected compounds that failed to inhibit alpha-granule release demonstrated significant inhibition of SFLLRN-induced 14C-serotonin release at 10 micromolar. Ongoing studies are directed at selecting best candidates from among the 16 confirmed inhibitory compounds to develop as biological probes that target either PAR1 activation or distal steps in granule secretion. Disclosures:No relevant conflicts of interest to declare.

Lyn, PKC-δ, SHIP-1 interactions regulate GPVI-mediated platelet-dense granule secretion

Protein kinase C-delta (PKC-delta) is expressed in platelets and activated downstream of protease-activated receptors (PARs) and glycoprotein VI (GPVI) receptors. We have previously shown that PKC-delta positively regulates PAR-mediated dense granule secretion, whereas it negatively regulates GPVI-mediated dense granule secretion. We further investigated the mechanism of such differential regulation of dense granule release by PKC-delta in platelets. SH2 domain-containing inositol phosphatase-1 (SHIP-1) is phosphorylated on Y1020, a marker for its activation, upon stimulation of human platelets with PAR agonists SFLLRN and AYPGKF or GPVI agonist convulxin. GPVI-mediated SHIP-1 phosphorylation occurred rapidly at 15 seconds, whereas PAR-mediated phosphorylation was delayed, occurring at 1 minute. Lyn and SHIP-1, but not SHIP-2 or Shc, preferentially associated with PKC-delta on stimulation of platelets with a GPVI agonist, but not with a PAR agonist. In PKC-delta-null murine platelets, convulxin-induced SHIP-1 phosphorylation was inhibited. Furthermore, in Lyn null murine platelets, GPVI-mediated phosphorylations on Y-1020 of SHIP-1 and Y311 of PKC-delta were inhibited. In murine platelets lacking Lyn or SHIP-1, GPVI-mediated dense granule secretions are potentiated, whereas PAR-mediated dense granule secretions are inhibited. Therefore, we conclude that Lyn-mediated phosphorylations of PKC-delta and SHIP-1 and their associations negatively regulate GPVI-mediated dense granule secretion in platelets.

Clinical Summaries

Clinical Summaries

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. αIIbβ3, 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 αIIbβ3 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.

Platelet Dense-Granule Secretion Plays a Critical Role in Thrombosis and Subsequent Vascular Remodeling in Atherosclerotic Mice

Platelet aggregation plays a critical role in myocardial infarction and stroke; however, the role of platelet secretion in atherosclerotic vascular disease is poorly understood. Therefore, we examined the hypothesis that platelet dense-granule secretion modulates thrombosis, inflammation, and atherosclerotic vascular remodeling after injury. Functional deletion of the Hermansky-Pudlak syndrome 3 gene (HPS3(-/-)) markedly reduces platelet dense-granule secretion. HPS3(-/-) mice have normal platelet counts, platelet morphology, and alpha-granule number, as well as maximal secretion of the alpha-granule marker P-selectin; however, their capacity to form platelet-leukocyte aggregates is significantly reduced (P<0.05). To examine the role of platelet dense-granule secretion in these processes, atherosclerosis-prone mice with combined genetic deficiency of apolipoprotein E and HPS3 (ApoE(-/-), HPS3(-/-)) were compared with congenic, atherosclerosis-prone mice with normal platelet secretion (ApoE(-/-), HPS3(+/+)). After 16 to 18 weeks on a high-fat diet, both groups of mice had similar fasting cholesterol levels and body weight. Carotid arteries of ApoE(-/-), HPS3(+/+) mice thrombosed rapidly after FeCl(3) injury, but ApoE(-/-), HPS3(-/-) mice were completely resistant to thrombotic arterial occlusion (P<0.01). Three weeks after injury, neointimal hyperplasia (from alpha-smooth muscle actin-positive cells) was significantly less (P<0.001) in arteries from ApoE(-/-), HPS3(-/-) mice. In ApoE(-/-), HPS3(-/-) mice, there were also pronounced reductions in arterial inflammation, as indicated by a 74% decrease in CD45-positive leukocytes (P<0.01) and a 73% decrease in Mac-3-positive macrophages (P<0.05). In atherosclerotic mice, reduced platelet dense-granule secretion is associated with marked protection against the development of arterial thrombosis, inflammation, and neointimal hyperplasia after vascular injury.

Synaptotagmin-like protein 1 interacts with the GTPase-activating protein Rap1GAP2 and regulates dense granule secretion in platelets

AbstractThe small guanine-nucleotide–binding protein Rap1 plays a key role in platelet aggregation and hemostasis, and we recently identified Rap1GAP2 as the only GTPase-activating protein of Rap1 in platelets. In search of Rap1GAP2-associated proteins, we performed yeast-2-hybrid screening and found synaptotagmin-like protein 1 (Slp1) as a new binding partner. We confirmed the interaction of Rap1GAP2 and Slp1 in transfected COS-1 and HeLa cells and at endogenous level in human platelets. Mapping studies showed that Rap1GAP2 binds through amino acids T524-K525-X-T527 within its C-terminus to the C2A domain of Slp1. Slp1 contains a Rab27-binding domain, and we demonstrate that Rap1GAP2, Slp1, and Rab27 form a trimeric complex in transfected cells and in platelets. Purified Slp1 dose-dependently decreased dense granule secretion in streptolysin-O–permeabilized platelets stimulated with calcium or guanosine 5′-O-[gamma-thio] triphosphate. The isolated C2A domain of Slp1 had a stimulatory effect on granule secretion and reversed the inhibitory effect of full-length Slp1. Purified Rap1GAP2 augmented dense granule secretion of permeabilized platelets, whereas deletion of the Slp1-binding TKXT motif abolished the effect of Rap1GAP2. We conclude that Slp1 inhibits dense granule secretion in platelets and that Rap1GAP2 modulates secretion by binding to Slp1.

Inhibition of Platelet-Rich Arterial Thrombus In Vivo

To test the hypothesis that statins will acutely inhibit platelet thrombus formation, intravenous lovastatin was assessed in our well-characterized porcine carotid injury model. The first carotid artery was crush-injured and harvested after 30 minutes. Pigs then received intravenous lovastatin (100 microg/kg bolus+100 microg/kg/h infusion, n=6) or saline (n=11) before injury of the second carotid artery. Thrombus size was quantified by scintillation detection of autologous (111)In-platelets. Sequential carotid injury produced a thrombus more than 50% greater in volume in the second (3149+/-2053 x 10(6)/cm(2)) relative to the first injured artery (2081+/-1552 x 10(6)/cm(2); P=0.04) in control pigs. This augmentation was inhibited by intravenous lovastatin which acutely reduced platelet deposition (944+/-246 x 10(6)/cm(2)) relative to saline control (P=0.02). Flow chamber closure times increased on average by 2.45-fold in response to whole blood lovastatin incubation. Lovastatin (P<0.05) and simvastatin (P<0.05) reduced platelet dense granule secretion in vitro. Sequential arterial injury augments the thrombotic response suggesting that the propensity for arterial thrombosis is at least partially acquired. This thrombotic augmentation can be acutely attenuated by intravenous lovastatin which may result from a pleiotropic impact on platelet function. These results appear to be a class effect of 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors.

Mechanism of Activation and Functional Role of Protein Kinase Cη in Human Platelets

The novel class of protein kinase C (nPKC) isoform eta is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCeta using pharmacological and gene knock-out approaches. nPKCeta was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1 receptor antagonist, or YM-254890, a G(q) blocker, abolished 2MeSADP-induced phosphorylation of nPKCeta. Similarly, ADP failed to activate nPKCeta in platelets isolated from P2Y1 and G(q) knock-out mice. However, pretreatment of platelets with P2Y12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCeta phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCeta was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin alpha(IIb)beta3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a alpha(IIb)beta3 receptor antagonist, nPKCeta dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cgamma, a catalytic subunit of serine/threonine phosphatase, alpha(IIb)beta3 failed to dephosphorylate nPKCeta. Thus, we conclude that ADP activates nPKCeta via P2Y1 receptor and is subsequently dephosphorylated by PP1gamma phosphatase activated by alpha(IIb)beta3 integrin. In addition, pretreatment of platelets with eta-RACK antagonistic peptides, a specific inhibitor of nPKCeta, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCeta positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.