Platelet Dense Granule Secretion Research Articles

Mechanism of Outside-In α IIb β 3 -Mediated Activation of Human Platelets by the Colonizing Bacterium, Streptococcus gordonii

To better understand the mechanism of platelet recruitment and activation by Streptococcus gordonii. The oral bacterium Streptococcus gordonii, is amongst the most common pathogens isolated from infective endocarditis patients, and has the property of being able to activate platelets, leading to thrombotic complications. The mechanism of platelet recruitment and activation by S. gordonii is poorly understood. Infective endocarditis is a bacterial infection of the heart valves that carries a high risk of morbidity and mortality. The oral bacterium, S gordonii, is among the most common pathogens isolated from patients with infective endocarditis and is able to activate platelets, leading to thrombotic complications. Platelets interact with S gordonii via glycoprotein Ibα- and α(IIb)β(3)-recognizing S gordonii surface proteins haemaglutitin salivary antigen (Hsa) and platelet adherence protein A, respectively. The inhibition of glycoprotein Ibα or α(IIb)β(3) using blocking antibodies or deletion of S gordonii Hsa or platelet adherence protein A significantly reduces platelet adhesion. Immunoreceptor tyrosine-based activation motif (ITAM)-containing proteins have recently played a role in transmitting activating signals into platelets. Platelet adhesion to immobilized S gordonii resulted in tyrosine phosphorylation of the ITAM-bearing receptor, FcγRIIa, and phosphorylation of downstream effectors (ie, spleen tyrosine kinase [Syk] and phospholipase C [PLC]-γ2). Tyrosine phosphorylation of FcγRIIa resulted in platelet-dense granule secretion, filopodial and lamellipodial extension, and platelet spreading. Inhibition of FcγRIIa ablated both dense granule release and platelet spreading. Streptococcus gordonii binding to the α(IIb)β(3)/FcγRIIa integrin/ITAM signaling complex results in platelet activation that likely contributes to the thrombotic complications of infective endocarditis.

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.

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.

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

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.

Platelet P2Y 12 Receptor Influences the Vessel Wall Response to Arterial Injury and Thrombosis

Platelets are believed to play an important role in atherogenesis and the vessel response to vascular injury. The P2Y(12) receptor (P2Y(12)) plays a central role in amplifying platelet aggregation, dense granule and alpha-granule secretion, P-selectin expression, microparticle formation, and procoagulant membrane changes, regardless of the activating stimulus. We hypothesized that P2Y(12) deficiency might reduce the vessel wall response to vascular injury as well as thrombosis in murine vascular injury models. P2Y(12)-deficient (-/-) mice and littermate controls (+/+) were bred on a C57 BL/6 background. In vivo murine models of arterial injury were employed alone and in combination with bone marrow transplantation to investigate the role of P2Y(12) in the vessel wall response to arterial injury and thrombosis. At 21 days after ferric chloride injury, neointima formation in P2Y(12)(-/-) arteries was significantly less than that observed in control strain arteries (P<0.025). In agreement with this, the intima-media ratio was significantly greater in femoral wire-injured arteries from P2Y(12)(+/+) compared with P2Y(12)(-/-) animals (P<0.05). Bone marrow transplantation was used to examine the importance of vessel wall P2Y(12) versus platelet P2Y(12). Analysis of arterial sections from chimeric animals at 21 days after injury revealed a smaller intima-media ratio in -/- to +/+ animals than in the positive (+/+ to +/+) control group (P<0.01). These data demonstrate a role for platelet P2Y(12) in the vessel wall response to arterial injury and thrombosis. This illustrates the manner in which platelets may contribute to atherogenesis and restenosis.

Molecular basis of platelet activation by an alphaIIbbeta3-CHAMPS peptide.

A novel method, known as computed helical anti-membrane protein (CHAMP), for the design of peptides that bind with high affinity and selectivity to transmembrane helices was recently described and illustrated using peptides that bind alphaIIb- and alphav-integrin subunits, which induce selective activation of integrins alphaIIbbeta3 and alphavbeta3, respectively. In the present study, we have investigated the ability of an alphaIIb-CHAMPS peptide (termed integrin-activatory-peptide or IAP) to stimulate protein tyrosine phosphorylation and aggregation in human and mouse platelets. The ability of IAP to stimulate platelet aggregation and dense granule secretion was measured in washed preparations of human and mouse platelets. Samples were taken for measurement of tyrosine phosphorylation. IAP stimulates robust tyrosine phosphorylation of the tyrosine kinase Syk and the FcR gamma-chain, but only weak phosphorylation of PLCgamma2. Aggregation to low but not high concentrations of IAP is reduced in the presence of the Src kinase inhibitor, PP1, or by inhibitors of the two feedback agonists, ADP and thromboxane A(2) (TxA(2)) suggesting that activation is reinforced by Src kinase-driven release of ADP and TxA(2). Unexpectedly, aggregation by IAP is only partially inhibited in human and mouse platelets deficient in integrin alphaIIbbeta3. Further, IAP induces partial aggregation of formaldehyde-fixed platelets. The present study demonstrates that the alphaIIb-CHAMPS peptide induces platelet activation through integrin alphaIIbbeta3-dependent and independent pathways with the former mediating tyrosine phosphorylation of FcR gamma-chain and Syk. The use of the alphaIIb-CHAMPS peptide to study integrin alphaIIbbeta3 function is compromised by non-integrin-mediated effects.

NPKC Epsilon Negatively Regulates Platelet Functional Responses

Protein Kinase C (PKC) are family of serine threonine kinases, known to regulate various platelet functional responses. Among them novel class of PKC isoforms (nPKC) including delta(δ), theta(θ), eta(η), and epsilon(ε) are expressed in platelets. Although, the role of nPKC ε and η in platelets is fairly understood, not much is known about nPKC ε and η in platelets. In this study, we investigated the role of nPKC ε in platelet functional responses using ADP-induced signaling as our stereotype. ADP causes platelet activation via Gq-coupled P2Y1 receptor and Gi-coupled P2Y12 receptor. Thus, we primarily studied the role of P2Y1 receptor in nPKC ε activation. ADP activated nPKC ε in time- and concentration- dependent manner. In the presence of P2Y1 receptor antagonist MRS-2179 and in P2Y1 knockout (KO) murine platelets ADP failed to activate nPKC ε, suggesting that ADP activates nPKC ε via P2Y1 receptor. We further investigated the functional role of nPKC ε using specific nPKC ε inhibitory RACK peptide (ε V1-2). ε V1-2 is a peptide designed to compete with native nPKC ε to bind ε-Receptors for activated C Kinase (ε-RACK) and thereby inhibits nPKC ε catalytic activity due to decreased substrate accessibility. ADP-induced thromboxane generation in human platelets pretreated with ε V1-2 peptide was more compared to the platelets pretreated with control peptide. Similarly, ADP-induced thromboxane generation in platelets derived from nPKC ε KO mouse was more compared to the wild type (WT) littermates. However, ADP- induced alpha granule secretion and aggregation in aspirin treated platelets derived from PKC ε KO mice was not significantly different from platelets derived from wild type littermates. These data suggest that nPKC e regulates an unknown pathway, which primarily regulates thromboxane generation with minimal effects on aggregation and alpha granule secretion. Furthermore, we also investigated the role of nPKC ε in PAR- and GPVI- mediated platelet aggregation and dense granule secretion. Interestingly, in both aspirin-treated and non-aspirin-treated platelets PAR- and GPVI- mediated platelet aggregation and dense granule secretion were potentiated. Consistent with ex vivo studies, FeCl3-induced arterial thrombosis was enhanced in nPKC ε KO mice compared to WT littermates.

Regulation of Platelet Dense Granule Secretion by the Ral GTPase-Exocyst Pathway

Non-hydrolyzable GTP analogues, such as guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp), induce granule secretion from permeabilized platelets in the absence of increased intracellular Ca(2+). Here, we show that the GppNHp-induced dense granule secretion from permeabilized platelets occurred concomitantly with the activation of small GTPase Ral. This secretion was inhibited by the addition of GTP-Ral-binding domain (RBD) of Sec5, which is a component of the exocyst complex known to function as a tethering factor at the plasma membrane for vesicles. We generated an antibody against Sec5-RBD, which abolished the interaction between GTP-Ral and the exocyst complex in vitro. The addition of this antibody inhibited the GppNHp-induced secretion. These data indicate that Ral mediates the GppNHp-induced dense granule secretion from permeabilized platelets through interaction with its effector, the exocyst complex. Furthermore, GppNHp enhanced the Ca(2+) sensitivity of dense granule secretion from permeabilized platelets, and this enhancement was inhibited by Sec5-RBD. In intact platelets, the association between Ral and the exocyst complex was induced by thrombin stimulation with a time course similar to that of dense granule secretion and Ral activation. Taken together, our results suggest that the Ral-exocyst pathway participates in the regulation of platelet dense granule secretion by enhancing the Ca(2+) sensitivity of the secretion.

Preservation of platelet aggregation and dense granule secretion during extended storage of blood samples in the presence of a synthetic dual inhibitor of factor Xa and thrombin

The clinical significance of platelet function tests may be limited by the use of citrate anticoagulant. We examined the influence of BAPA, a dual inhibitor of factor Xa and thrombin, on platelet responsiveness to agonists when measured between 2 and 48 h after venipuncture. Blood samples from 24 healthy individuals were anticoagulated with citrate or BAPA. Impedance platelet aggregation (IPA) and adenosine triphosphate (ATP) release induced by ADP, collagen and thrombin-receptor activating peptide (TRAP) were determined in whole blood after a storage period between 2 and 48 h after venipuncture. Citrate resulted in significantly reduced collagen or TRAP-induced IPA and ATP release when measured 32 h and 48 h after blood collection. ADP-induced IPA and ATP release in citrated blood dropped significantly between 8 and 24 h. The length of storage of BAPA-anticoagulated blood samples over 48 h had no significant influence on platelet response to collagen and TRAP. In BAPA-anticoagulated blood, ADP-induced IPA and ATP secretion in whole blood were maintained over a storage period of 32 h. No difference in ADP-induced ATP secretion in whole blood anticoagulated with citrate and BAPA was observed, but it was largely suppressed in BAPA-anticoagulated platelet rich plasma. IPA and ATP release remain stable for at least 32 h when whole blood is anticoagulated with a dual inhibitor of factor Xa and thrombin. This would allow shipment of samples for platelet function testing on the following day. Platelet secretion studies in whole blood may include platelet activation by ADP when BAPA is used as anticoagulant.

SHIP-1 Differentially Regulates Dense Granule Secretion in Platelets through Its Association with Protein Kinase C δ.

Collagen-induced glycoprotein (GP) VI-mediated and thrombin-induced protease activated receptors (PAR)-mediated activation are important signaling pathways regulating dense granule secretion in platelets. Protein kinase C (PKC) isoforms play a crucial role in platelet secretion and we have previously shown that PKCδ plays a ying-yang role in dense granule release by different agonists (Murugappan et al, J. Biol. Chem. 2004). PKCδ isoform positively regulates PAR-mediated platelet dense granule release, whereas it negatively regulates GPVI-mediated dense granule release. In this study, we investigated the mechanism of such differential regulation by PKCδ downstream of PAR and GPVI pathways. We hypothesize that the differential association of PKCδ with phosphatases downstream of GPVI and PAR receptors differentially regulate dense granule secretion. More specifically, we explored the functional relevance of the interaction of PKCδ with Src homology 2-domain containing Inositol Phosphatases (SHIP), 5′-inositol phosphatases in platelets. In our studies, SHIP-1 was tyrosine phosphorylated by both PARs and GPVI receptors and its phosphorylation followed different activation kinetics. Whereas PAR-mediated SHIP-1 phosphorylation (Y1020) was delayed and occurred as late as 120 seconds, the GPVI-mediated SHIP-1 phosphorylation was rapid, starting as early as 15 seconds and peaked at 60 seconds. Co-immunoprecipitation experiments revealed that SHIP-1, and not SHIP-2, associated with PKCδ upon stimulation of platelets with GPVI agonist, convulxin. However, such association did not occur with the PAR agonists. GPVI-mediated SHIP-1 phosphorylation failed to occur in platelets from mice lacking Lyn kinase suggesting a role for Lyn in regulating SHIP-1 phosphorylation. In murine platelets lacking either Lyn or SHIP-1, dense granule secretion was potentiated by convulxin and not by thrombin. We attribute the phosphorylation and association of SHIP-1 with PKCδ to be critical for the regulation of agonist-induced dense granule secretion in platelets. Based on the above results, we conclude that the preferential association of SHIP-1 with PKCδ upon stimulation of GPVI receptor results in the negative regulation of collagen-induced dense granule release in platelets.

Reduced platelet serotonergic responsivity as assessed by dense granule secretion in first-episode psychosis

Objectives: To determine whether blunted serotonergic responsivity, indicated by decreased platelet dense granule secretion (DGS), occurs in neuroleptic-naïve patients with schizophrenia, as observed previously in chronic schizophrenia. Design and methods: Serotonin (5-HT)-amplified DGS was examined in 40 first-episode neuroleptic-naïve patients (24 with schizophrenia and 16 with mood disorders) and 24 healthy subjects. Results: Healthy controls showed robustly increased DGS. Schizophrenic patients showed very modest DGS increases; mood disorder patients showed intermediate response. Conclusions: Blunted DGS appears to a characteristic of schizophrenia that is observed in the treatment-naïve condition.

Rab27b regulates number and secretion of platelet dense granules

The Rab27 GTPase subfamily consists of two closely related homologs, Rab27a and Rab27b. Rab27a has been shown previously to regulate organelle movement and regulated exocytosis in a wide variety of secretory cells. However, the role of the more restrictedly expressed Rab27b remains unclear. Here we describe the creation of Rab27b knockout (KO) strain that was subsequently crossed with the naturally occurring Rab27a KO line, ashen, to produce double KO (Rab27a(ash/ash) Rab27b(-/-)) mice. Rab27b KO (and double KO) exhibit significant hemorrhagic disease in contrast to ashen mice. In vitro assays demonstrated impaired aggregation with collagen and U46619 and reduced secretion of dense granules in both Rab27b and double KO strains. Additionally, we detected a 50% reduction in the number of dense granules per platelet and diminished platelet serotonin content, possibly due to a dense granule packaging defect into proplatelets during megakaryocyte maturation. The presence of Rab27a partially compensated for the secretory defect but not the reduced granule number. The morphology and function of platelet alpha-granules were unaffected. Our data suggest that Rab27b is a key regulator of dense granule secretion in platelets and thus a candidate gene for delta-storage pool deficiency in humans.

PAR1-Induced Human Platelet Shape Change, Aggregation and Secretion Requires Gα13 Switch Region I Signaling.

While it is known that platelets possess multiple G protein signaling pathways that contribute to the different platelet functional responses, the relative participation of these individual pathways in platelet shape change, aggregation and secretion is not well characterized. To a large extent this is due to the lack of suitable reagents which selectively interfere with specific G protein signaling events, and which can be applied to the study of intact human platelets. With the exception of pepducins, which modulate receptor-G protein coupling (Kuliopulos, A. and Covic, L. Life Sciences 74, 255–262, 2003), the field has for the most part been limited to agents which interfere with different downstream kinases or other downstream effectors. However, the G protein pathways share many of these downstream targets, and consequently, it has been difficult to assign a specific platelet function to a certain G protein. In order to address this issue, it was reasoned that more direct information about specific G protein involvement in human platelet activation might be obtained by interfering with the initial G protein signal transduction events, rather than by interfering with the secondary downstream consequences of this transduction process. Based on this consideration, the present study used a specific Gα13 switch region I (SRI) peptide to investigate the involvement of Gα13 signaling in protease-activated receptor 1 (PAR1)-mediated human platelet function. Specifically, a myristoylated peptide representing the Gα13 SRI (Myr-G13SRIpep) was synthesized and evaluated for its effects on PAR1 activation. Initial studies using dot blot and mass spectrum analysis demonstrated that Myr-G13SRIpep, and its random sequence control (Myr-G13SRIRandom-pep), were equally taken up by intact human platelets. Radioligand binding experiments revealed that Myr-G13SRIpep did not interfere with PAR1-ligand interaction. Subsequent experiments demonstrated that G13SRIpep specifically bound to platelet p115Rho guanine nucleotide exchange factor (p115RhoGEF) and blocked PAR1-mediated RhoA activation. These results suggest a direct interaction of Gα13 SRI with p115RhoGEF, and indicate a possible mechanism for Myr-G13SRIpep inhibition of RhoA activation. Platelet function studies revealed that Myr-G13SRIpep inhibited PAR1-stimulated platelet shape change, aggregation and dense granule secretion in a dose-dependent manner. On the other hand, Myr-G13SRIpep did not inhibit platelet activation induced by ADP, A23187 or PAR4 activating peptide (AYPGKF). Taken together, these findings demonstrate that the inhibitory effects of Myr-G13SRIpep are limited to PAR1 signaling mechanisms and are not due to nonspecific effects on platelet function. These results also suggest a significant role for Gα13 SRI signaling in the process of PAR1-mediated human platelet activation. In additional studies it was found that Myr-G13SRIpep also inhibited low-dose thrombin-induced aggregation and PAR1-induced intraplatelet calcium mobilization. Collectively, these results provide evidence that: 1. interaction of Gα13 SRI with p115RhoGEF is required for G13-mediated RhoA activation in platelets; 2. signaling through the G13 pathway is critical for PAR1-mediated human platelet functional changes; 3. Gα13 SRI signaling is involved in low-dose thrombin-induced platelet aggregation as well as PAR1-mediated calcium mobilization; and 4. permeable peptides representing SRI of Gα-subunits should be a useful approach for studying individual G protein signaling pathways in intact cells.

Peroxynitrite can affect platelet responses by inhibiting energy production.

Peroxynitrite (ONOO-) strongly inhibits agonist-induced platelet responses. However, the mechanisms involved are not completely defined. Using porcine platelets, we tested the hypothesis that ONOO- reduces platelet aggregation and dense granule secretion by inhibiting energy production. It was found that ONOO- (25-300 microM) inhibited collagen-induced dense granule secretion (IC50 = 55 +/- 7 microM) more strongly than aggregation (IC(50) = 124 +/- 16 microM). The antiaggregatory and antisecretory effects of ONOO- were only slightly (5-10%) reduced by 1H-[1,2,4]-oxadiazolo-[4,3-alpha]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase. In resting platelets ONOO- (50-300 microM) enhanced glycolysis rate and reduced oxygen consumption, in a dose dependent manner. The ONOO- effects on glycolysis rate and oxygen consumption were not abolished by ODQ. The extent of glycolysis stimulation exerted by ONOO- was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or an uncoupler (2,4-dinitrophenol). Stimulation of platelets by collagen was associated with a rise in mitochondrial oxygen consumption, accelerated lactate production, and unchanged intracellular ATP content. In contrast to resting cells, in collagen-stimulated platelets, ONOO- (200 microM) distinctly decreased the cellular ATP content. The glycolytic activity and oxygen consumption of resting platelets were not affected by 8-bromoguanosine 3',5'-cyclic monophosphate. Blocking of the mitochondrial ATP production by antimycin A slightly reduced collagen-induced aggregation and strongly inhibited dense granule secretion. Treatment of platelets with ONOO- (50-300 microM) resulted in decreased activities of NADH : ubiquinone oxidoreductase, succinate dehydrogenase and cytochrome oxidase. It is concluded that the inhibitory effect of ONOO- on platelet secretion and to a lesser extent on aggregation may be mediated, at least in part, by the reduction of mitochondrial energy production.

Constitutive GDP/GTP Exchange and Secretion-dependent GTP Hydrolysis Activity for Rab27 in Platelets

We have previously demonstrated that Rab27 regulates dense granule secretion in platelets. Here, we analyzed the activation status of Rab27 using the thin layer chromatography method analyzing nucleotides bound to immunoprecipitated Rab27 and the pull-down method quantifying Rab27 bound to the GTP-Rab27-binding domain (synaptotagmin-like protein (Slp)-homology domain) of its specific effector, Slac2-b. We found that Rab27 was predominantly present in the GTP-bound form in unstimulated platelets due to constitutive GDP/GTP exchange activity. The GTP-bound Rab27 level drastically decreased due to enhanced GTP hydrolysis activity upon granule secretion. In permeabilized platelets, increase of Ca(2+) concentration induced dense granule secretion with concomitant decrease of GTP-Rab27, whereas in non-hydrolyzable GTP analogue GppNHp (beta-gamma-imidoguanosine 5'-triphosphate)-loaded permeabilized platelets, the GTP (GppNHp)-Rab27 level did not decrease upon the Ca(2+)-induced secretion. These data suggested that GTP hydrolysis of Rab27 was not necessary for inducing the secretion. Taken together, Rab27 is maintained in the active status in unstimulated platelets, which could function to keep dense granules in a preparative status for secretion.

Biology and Pharmacology of the Platelet P2Y12 Receptor

Platelets possess two receptors for ADP, P2Y(1) and P2Y(12). ADP is released from platelet dense granules upon platelet activation by numerous agonists and thereby amplifies platelet responses regardless of the initial stimulus. The P2Y(1) receptor is one of many platelet receptors coupled to Gq and initiates ADP-induced activation. The P2Y(12) receptor on the other hand is linked to Gi and plays a special role in the amplification of platelet activation initiated by numerous other pathways. Platelet activation leads to a range of responses that play a critical role in arterial thrombosis and the inflammatory responses associated with this, including platelet aggregation, dense and alpha granule secretion and procoagulant activity. P2Y(12) receptor activation yields powerful amplification of these processes such that P2Y(12) receptor antagonists may have dramatic inhibitory effects on platelet function regardless of the activating stimuli. This phenomenon, coupled with the restricted distribution of the P2Y(12) receptor in humans, makes the receptor an ideal target for pharmaceutical therapy. This has already been established by the therapeutic success of clopidogrel, which acts, via an active metabolite, on this receptor. However, current therapeutic regimens of clopidogrel yield variable and incomplete P2Y(12) receptor blockade and more effective strategies to block P2Y(12) receptor activation offer the potential of greater clinical efficacy.