HUMAN PLATELET ACTIVATION BY BACTERIAL PHOSPHOLIPASE C: MECHANISM OF INHIBITION BY FLURAZEPAM

1987 ◽  
Author(s):  
Huzoor Akbar ◽  
David Wallace ◽  
Khursheed Anwer
Keyword(s):  
Platelet Aggregation ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Calcium Ions ◽  
Human Platelet ◽  
Calcium Ionophore ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Free System ◽  
Serotonin Secretion

We have shown earlier that flurazepam inhibits human platelet aggregation and serotonin secretion induced by bacterial phospholipase C (BPLC, Thromb. Res. 38, 361-374, 1985). This study was conducted to examine the mechanism(s) of inhibitory action of flurazepam. Only 15 uM and 11 uM flurazepam were required to inhibit platelet aggregation and serotonin secretion by 50%. In a platelet free system, BPLC hydrolyzed 14C-phosphatidylcholine (14C-PC> in a time- and concentration-dependent manner in the presence of calcium ions. Flurazepam had no effect on BPLC-induced hydrolysis of 14C-PC. Incubation of 14C-arachidonic acid labelled platelets with BPLC produced diacylglycerol(DAG) in a time- and concentration-dependent manner. Flurazepam did not inhibit DAG production by BPLC. However, prostaglandin E1 and paranitrophenolphosphorylcholine inhibited DAG production by 20% and 75% respectively. Platelet cytosolic fraction,containing phosphatidylinositol-specific PLC (PI-PLC), hydrolyzed 3H -phosphatidylinositol (3H-PI) in a concentration-dependent manner. Flurazepam did not inhibit hydrolysis:of 3H-PI by PI-PLC. BPLC caused phosphorylation of 47,000 Dalton protein (P47) in 32P-labelled platelets. Flurazepam did not inhibit phosphorylation of P47 in the first five minutes of incubation. However, flurazapam completely blocked phosphorylation of P47 by seven minutes. In Other experiments, flurazepam inhibited platelet aggregation induced by ionomycion, a calcium ionophore, in a concentration-dependent manner. These data lead us to suggest that flurazapam does not inhibit BPLC-ihduced platelet activation by inhibiting the action of BPLC or PI-PLC on platelet phospholipids or DAG production. However, the ability of flurazepam to inhibit ionomycin-induced platelet aggregation indicates that it may be blocking BPLC-induced platelet aggreagtion by interfering with the influx, of calcium ions into platelets. (Supported in part by the American Osteopathic Association, The Baker Award from Ohio University and the OUCOM).

In vitro Effects of Aprosulate sodium, a Novel Anticoagulant, On Platelet Activation: Possible Mechanism for Antiplatelet Action

1996 ◽  
Vol 76 (05) ◽  
pp. 786-790 ◽  
Author(s):  
Atsuhiro Sugidachi ◽  
Norbert Breiter ◽  
Taketoshi Ogawa ◽  
Fumitoshi Asai ◽  
Hiroyuki Koike
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Anticoagulant Activity ◽  
Acid Amide ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Free System ◽  
Induced Aggregation

SummaryAprosulate sodium, a bis-lactobionic acid amide derivative, is a novel synthetic polyanion with potent anticoagulant activities. In the present study, the effects of aprosulate on platelet aggregation were investigated in a plasma-free system. Aprosulate inhibited thrombin (0.03-0.3 U/ml)-induced aggregation in rat washed platelets in a concentration-dependent manner, with an IC50 value of 0.38 Μg/ml. In contrast, aprosulate, at up to 10 Μg/ml, did not affect collagen (1 Μg/ml) - or ADP (3 ΜM)-induced aggregation. In fura 2-loaded platelets, aprosulate (1-10 Μg/ml) inhibited intracellular Ca2+ mobilization induced by thrombin, but not that by ADP. Protamine, a highly basic protein, abolished aprosulate-mediated inhibition of thrombin-induced platelet aggregation, suggesting that the observed inhibition is primarily due to the negative charge contained on the aprosulate molecule. In human platelets, aprosulate inhibited thrombin-induced aggregation, but failed to inhibit platelet aggregation induced by SFLLRN, a synthetic tethered ligand of a thrombin receptor. Antiplatelet profiles of aprosulate were largely similar to those of heparin, although heparin inhibited both thrombin- and collagen-induced aggregation. These in vitro studies indicate that aprosulate is capable of inhibiting thrombin-induced platelet activation and that this effect is independent of its anticoagulant activity. These results suggest that the polyanionic feature of aprosulate plays an essential role in promoting its antiplatelet activities, and that a plausible mechanism to explain the observed inhibition conferred by this agent, would be one which involves blocking the platelet-thrombin interaction.

Evidence that Calcium Regulates Platelet Function

1978 ◽  
Vol 40 (02) ◽  
pp. 207-211 ◽  
Author(s):  
Thomas C Detwiler ◽  
Israel F Charo ◽  
Richard D Feinman
Keyword(s):  
Platelet Aggregation ◽  
Sarcoplasmic Reticulum ◽  
Platelet Activation ◽  
Platelet Function ◽  
Calcium Ions ◽  
Direct Evidence ◽  
Calcium Ionophore ◽  
Indirect Evidence ◽  
Ionophore A23187 ◽  
General Role

SummaryIt is generally believed that calcium ions play a key role in regulation of platelet function.This is based on 3 types of evidence.1. Analogies with other cells. Calcium ions are known to trigger secretion and contraction in many cells, possibly reflecting a general role for calcium in all secretion and contraction.2. Indirect evidence. Platelet aggregation and secretion are induced by divalent cation ionophores. The response to the ionophore A23187 is identical to that induced by other potent stimuli.3. Direct evidence. Platelet activation can be blocked by drugs (e. g. certain local anesthetics) that block release of calcium ions from sarcoplasmic reticulum; the inhibition can be overcome by addition of extracellular calcium in the presence of a calcium ionophore. While this does not constitute definitive proof, the central role for calcium ions remains an attractive hypothesis that justifies attempts to further define calcium pools and fluxes in platelets.

scholarly journals Resolvin E1, an EPA-derived mediator in whole blood, selectively counterregulates leukocytes and platelets

Blood ◽  
2008 ◽  
Vol 112 (3) ◽  
pp. 848-855 ◽  
Author(s):  
Maria Dona ◽  
Gabrielle Fredman ◽  
Jan M. Schwab ◽  
Nan Chiang ◽  
Makoto Arita ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Lipid Mediator ◽  
Dependent Manner ◽  
Omega 3 ◽  
Concentration Dependent Manner ◽  
Chemokine Production ◽  
Thromboxane Receptor

Abstract Resolvin E1 (RvE1) is an omega-3 eicosapentaenoic acid (EPA)–derived lipid mediator generated during resolution of inflammation and in human vasculature via leukocyte-endothelial cell interactions. RvE1 possesses anti-inflammatory and proresolving actions. Here, we report that RvE1 in human whole blood rapidly regulates leukocyte expression of adhesion molecules. RvE1 in the 10- to 100-nM range stimulated L-selectin shedding, while reducing CD18 expression in both neutrophils and monocytes. When added to whole blood, RvE1 did not stimulate reactive oxygen species by either neutrophils or monocytes, nor did it directly stimulate cytokine/chemokine production in heparinized blood. Intravital microscopy (IVM) demonstrated that RvE1 rapidly reduced leukocyte rolling (∼ 40%) in venules of mice. In human platelet-rich plasma (PRP), RvE1 selectively blocked both ADP-stimulated and thromboxane receptor agonist U46619-stimulated platelet aggregation in a concentration-dependent manner. In contrast, Δ6,14-trans-RvE1 isomer was inactive. RvE1 did not block collagen-stimulated aggregation, and regulation of ADP-induced platelet aggregation was not further enhanced with aspirin treatment. These results indicate RvE1 is a potent modulator of leukocytes as well as selective platelet responses in blood and PRP, respectively. Moreover, the results demonstrate novel agonist-specific antiplatelet actions of RvE1 that are potent and may underlie some of the beneficial actions of EPA in humans.

scholarly journals Src family kinase–mediated and Erk-mediated thromboxane A2 generation are essential for VWF/GPIb-induced fibrinogen receptor activation in human platelets

Blood ◽  
2005 ◽  
Vol 106 (10) ◽  
pp. 3410-3414 ◽  
Author(s):  
Analia Garcia ◽  
Todd M. Quinton ◽  
Robert T. Dorsam ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Aggregation ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Kinase Inhibitor ◽  
Thromboxane A2 ◽  
Receptor Activation ◽  
Src Family Kinases ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Src Family Kinase

AbstractThe binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein Ib-IX (GPIb-IX) results in platelet activation. In this study, we sought to clarify previous conflicting reports and to elucidate the mechanism of activation and the precise role of extracellular signal-regulated kinase (Erk) in VWF-induced platelet activation. Erk2 is activated in platelets on stimulation with VWF/ristocetin in a time-dependent manner. VWF-induced Erk2 phosphorylation and thromboxane A2 (TXA2) release were completely blocked by PP2, an Src family kinase inhibitor, suggesting that Erk is downstream of Src family kinases. U73122, a phospholipase C inhibitor, also abolished TXA2 generation and Erk phosphorylation. Although VWF fostered the agglutination of platelets regardless of any additional treatment, the inhibition of mitogen-activated protein kinase kinase (MEK) with U0126 abolished VWF-induced platelet aggregation and thromboxane production in non–aspirin-treated washed platelets. However, in platelets treated with aspirin, VWF failed to cause any aggregation. Thus, we conclude that VWF stimulation of platelets results in phospholipase A2 activation through Erk stimulation and that Src family kinases and phospholipase C play essential roles in this event. We further conclude that VWF-induced platelet aggregation does not directly depend on Erk activation but has an absolute requirement for Src/Erk-mediated TXA2 generation.

scholarly journals The phospholipase C/protein kinase C pathway is involved in cathepsin G-induced human platelet activation: comparison with thrombin

1996 ◽  
Vol 313 (2) ◽  
pp. 401-408 ◽  
Author(s):  
Mustapha SI-TAHAR ◽  
Patricia RENESTO ◽  
Hervé FALET ◽  
Francine RENDU ◽  
Michel CHIGNARD
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Inositol Phosphates ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Functional Responses ◽  
Kinase C

Cathepsin G, an enzyme released by stimulated polymorphonuclear neutrophils, and thrombin are two human proteinases which potently trigger platelet activation. Unlike thrombin, the mechanisms by which cathepsin G initiates platelet activation have yet to be elucidated. The involvement of the phospholipase C (PLC)/protein kinase C (PKC) pathway in cathepsin G-induced activation was investigated and compared with stimulation by thrombin. Exposure of 5-[14C]hydroxytryptamine-labelled platelets to cathepsin G, in the presence of acetylsalicylic acid and phosphocreatine/creatine kinase, induced platelet aggregation and degranulation in a concentration-dependent manner (0.1-3.0 μM). Time-course studies (0-180 s) comparing equivalent concentrations of cathepsin G (3 μM) and thrombin (0.5 unit/ml) resulted in very similar transient hydrolysis of phosphatidylinositol 4,5-bisphosphate and steady accumulation of phosphatidic acid. In addition cathepsin G, like thrombin, initiated the production of inositol phosphates. The neutrophil-derived proteinase also induced phosphorylation of both the myosin light chain and pleckstrin, a substrate for PKC, to levels similar to those observed in platelets challenged with thrombin. Inhibition of PKC by GF 109203X, a specific inhibitor, suppressed platelet aggregation and degranulation to the same extent for both proteinases. Using fura 2-loaded platelets, the rise in the cytosolic free Ca2+ concentration induced by cathepsin G was shown to result, as for thrombin, from both mobilization of internal stores and Ca2+ entry across the plasma membrane. These findings provide evidence that cathepsin G stimulates the PLC/PKC pathway as potently as does thrombin, independently of thromboxane A2 formation and ADP release, and that this pathway is required for platelet functional responses.

Resveratrol Attenuates Adenosine Diphosphate-Induced Platelet Activation by Reducing Protein Kinase C Activity

2008 ◽  
Vol 36 (03) ◽  
pp. 603-613 ◽  
Author(s):  
Yu-Min Yang ◽  
Xing-Xiang Wang ◽  
Jun-Zhu Chen ◽  
Shi-Jun Wang ◽  
Hu Hu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Adenosine Diphosphate ◽  
Dependent Manner ◽  
Chinese Medicinal Herb ◽  
Concentration Dependent Manner ◽  
Kinase C

Inappropriate platelet activation is the key point of thrombogenesis. The aim of the present study was to investigate the effects of resveratrol (RESV), a compound extracted from the Chinese medicinal herb Polygonum cuspidatum sieb et Zucc, on the platelet activation induced by adenosine diphosphate (ADP) and its possible mechanism. The percentage of platelet aggregation and surface P-selectin-positive platelets, and the activity of protein kinase C (PKC) of platelet were observed with platelet aggregometer, flow cytometry and phosphorimaging system, respectively. RESV at 25, 50 and 100 μM showed anti-platelet aggregation and inhibition of surface P-selectin-positive platelets in a concentration-dependent manner. RESV (50 μM) inhibited the activity of PKC in the membrane fraction of platelets and decreased the percentage of membrane associated PKC activity in total PKC activity. Moreover, DL-erythro-1,3-Dihydroxy-2-aminooctadecane, an elective protein kinase C inhibitor (PKCI), and RESV had additive effects of inhibiting the percentage of platelet aggregation and surface P-selectin-positive platelets. It is suggested that RESV may inhibit platelet aggregation, the percentage of surface P-selectin-positive platelets and subsequent thrombus formation. The mechanisms may be partly relative to the decrease of the activity of PKC of platelets.

Specific Pharmacologic Targeting of Rho GTPases Rac1, Cdc42 and RhoA Reveals Their Differential and Critical Roles In Regulation of Platelet Activation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2019-2019 ◽  
Author(s):  
Huzoor Akbar ◽  
Xun Shang ◽  
Rehana Perveen ◽  
Kevin Funk ◽  
Mark Berryman ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Rho Gtpase ◽  
Conflicts Of Interest ◽  
Small Gtpases ◽  
Signaling Cascades ◽  
Dependent Manner ◽  
Exchange Reactions ◽  
Concentration Dependent Manner ◽  
Induced Aggregation

Abstract Abstract 2019 Rac1, Cdc42 and RhoA, members of the Rho family of small GTPases, play critical roles in reorganization of actin cytoskeleton and aggregation in platelets. Although they have been implicated in regulation of platelet activation, the unique and redundant roles of each of the Rho GTPase in various signaling cascades and the resulting functional outcomes have yet to be clearly defined. In this study we compared their roles in several aspects of platelet activation by utilizing three small molecule inhibitors, NSC23766, CASIN, and GO4, that specifically suppress endogenous Rac1, Cdc42, and RhoA activities, respectively. These novel pharmacological inhibitors are active in direct binding to their specific GTPase substrates, i.e. NSC23766 to Rac1, CASIN to Cdc42, and G04 to RhoA, and in interfering with the GTP loading exchange reactions of each Rho GTPase catalyzed by respective guanine nucleotide exchange factors at 5–50 uM concentration range. First, effector-domain pull down assays confirmed that treatment of platelets with NSC23766 (30 uM), CASIN (10 uM) or GO4 (30-50 uM) specifically blocked collagen induced Rac1-GTP, Cdc42-GTP, and RhoA-GTP formations, respectively. Incubation of platelets with NSC23766 (30 uM) or CASIN (10 uM) effectively inhibited collagen-induced phosphorylation of the Rac/Cdc42 effector, PAK1. Addition of GO4 (30 uM) to platelets prior to stimulation with thrombin blocked RhoA/ROCK mediated phosphorylation of myosin light chain (MLC). Second, incubation of aspirin treated platelets containing apyrase (3 U/ml) with CASIN (10 uM), but not NSC23766 (30 uM) or GO4 (30 uM), inhibited filopodia formation on immobilized fibrinogen or collagen-related peptide (CRP), a GPVI agonist. On the other hand, treatment of platelets with CASIN (10 uM) or GO4 (30 uM), but not with NSC23766 (30 uM), inhibited spreading of platelets on immobilized fibrinogen in the presence of aspirin and apyrase. Third, NSC23766 (3-30 uM), CASIN (3-10 uM), and GO4 (5-50 uM) all inhibited secretion from platelet granules and secretion-dependent aggregation induced by threshold concentration of ADP, collagen, CRP, or thrombin in a concentration-dependent manner. However, while CASIN (10 uM) or GO4 (30 uM) completely blocked collagen or CRP induced aggregation in aspirin treated platelets containing apyrase, NSC23766 (30 uM) showed no effect. Fourth, while pre-incubation of platelets with 5 uM CASIN or 10 uM G04 alone only partially (15%) inhibited CRP induced platelet aggregation in aspirin treated samples, CASIN at 10 uM or a combination of 5 uM CASIN and 5 uM G04 were able to inhibit platelet aggregation by 90%. Fifth, GO4 (30 uM) but not CASIN (10 uM) inhibited thrombin stimulated phosphorylation of p38-MAPK (137%) in aspirin treated platelets in the presence of apyrase. Addition of GO4 (30 uM) or CASIN (10 uM) to aspirin treated platelets containing apyrase inhibited CRP induced phosphorylation of ERK1/2 by 94% and 53% respectively, However, in the absence of aspirin and apyrase GO4 (30 uM), but not CASIN (10 uM), completely inhibited CRP induced phosphorylation of ERK1/2. Finally, although both GO4 (30 uM) and CASIN (10 uM) completely inhibited CRP induced phosphorylation of MLC in aspirin treated platelets containing apyrase, GO4 (30 uM) maximally (94%) while CASIN (10 uM) partially (36%) inhibited phosphorylation of MLC in the absence of aspirin and apyrase. Taken together, these data suggest that: (a) Cdc42 is involved in integrin alphaIIbbeta3 and GPVI mediated filopodia formation, RhoA is involved in regulation of integrin alphaIIbbeta3 induced platelet spreading, whereas Rac1 is critical in secondary mediators (ADP/TXA2) mediated lamellipodia formation; (b) Cdc42 and RhoA regulate platelet aggregation in parallel pathways, possibly by affecting the RhoA/ROCK-MAPK-dependent and -independent phosphorylation of MLC; and (c) the crosstalk among Cdc42, Rac1 and RhoA plays an important role in signaling cascades involved in platelet activation. Disclosures: No relevant conflicts of interest to declare.

Inhibition of Human Platelet Functions by Verapamil

1981 ◽  
Vol 45 (02) ◽  
pp. 158-161 ◽  
Author(s):  
Y Ikeda ◽  
M Kikuchi ◽  
K Toyama ◽  
K Watanabe ◽  
Y Ando
Keyword(s):  
Platelet Aggregation ◽  
Calcium Ionophore ◽  
Serotonin Release ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Dependent Inhibition ◽  
Platelet Functions

SummaryThe effects of verapamil, a coronary vasodilator, on platelet functions were studied.Platelet aggregation induced by ADP, epinephrine or collagen was inhibited by verapamil in vitro. Calcium ionophore A23187-induced platelet aggregation was also inhibited by verapamil in a concentration dependent manner. In washed platelets, verapamil caused a dose-dependent inhibition of serotonin release induced either by thrombin or A23187 in the absence of extracellular calcium. Addition of 1 mM CaCl2 with A23187 or thrombin partially overcame this inhibition. Addition of 1 mM CaCl2 in the absence of verapamil had no effect on thrombin- or A23187-induced secretion. When verapamil was administered to the healthy volunteers at the dosage commonly used, inhibition of platelet aggregation was observed 2 hrs after the drug ingestion. It is of great interest that verapamil potentiated the anti-aggregating activity of prostacyclin in vitro.Our results may suggest a potential role for verapamil in the treatment of thrombotic disorders.

Abstract 54: PSI Domain of ß3 Integrin Has Endogenous Thiol Isomerase Function and is a Novel Antithrombotic Therapeutic Target

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Emily C Reddy ◽  
Guangheng Zhu ◽  
Pingguo Chen ◽  
Adili Reheman ◽  
Xi Lei ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Cell Biology ◽  
Human Platelet ◽  
Critical Role ◽  
Rnase A ◽  
Dependent Manner ◽  
Integrin Activation ◽  
Free System ◽  
Integrin Β3 ◽  
Β3 Integrin

Integrin αIIbβ3 plays a critical role in platelet aggregation and adhesion, key events in hemostasis and thrombosis. Integrin activation involves complex signalling events that lead to conformational changes exposing ligand-binding sites; however, mechanisms underlying integrin activation remain poorly understood. The β subunit contains a PSI domain that is highly conserved across integrins and species, though its function is unknown. Integrin β subunits are cysteine-rich and endogenous thiol isomerase activity in integrin β3 has been reported. The PSI domain contains two CXXC sequences, the active site motif of protein disulfide isomerase (PDI). Based on this observation and the location of this domain at the knee region of the integrin, we hypothesized that integrin PSI domain has endogenous thiol isomerase function, which plays a key regulatory role in integrin conformation and function. Targeting the PSI domain may have therapeutic potential. Using reduced, denatured RNase, a recombinant murine integrin β3 PSI domain demonstrated endogenous PDI-like activity. This PDI-like activity was dose-dependently inhibited by the PDI inhibitor, bacitracin. Mutation of either CXXC motif within the integrin β3 PSI domain reduced PDI-like activity, while removal of both CXXC motifs completely abolished this activity. We developed unique mouse anti-mouse/anti-human β3 PSI domain monoclonal antibodies (anti-PSI mAbs) that inhibited the PDI-like activity of both the murine recombinant integrin β3 PSI domain and purified human platelet β3 integrin, in a dose-dependent manner. Interestingly, the anti-PSI mAbs blocked fibrinogen to human platelet β3 integrin in a cell free system. Furthermore, anti-PSI mAbs inhibited murine and human platelet aggregation in vitro and ex vivo and inhibited murine thrombus formation in vivo without significantly changing bleeding time or platelet count. In conclusion, we identified that the PSI domain has PDI function, is a fundamental regulator of platelet β3 integrin activation, and is a potential novel target for anti-thrombotic therapies. Since PSI domain is conserved in all integrin β subunits, our discovery may have broad implications for the role of integrins in cell biology of many human diseases.

scholarly journals Insulin-like growth factor-1 regulates platelet activation through PI3-Kα isoform

Blood ◽  
2007 ◽  
Vol 110 (13) ◽  
pp. 4206-4213 ◽  
Author(s):  
Soochong Kim ◽  
Analia Garcia ◽  
Shaun P. Jackson ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Aggregation ◽  
Growth Factor ◽  
Platelet Activation ◽  
Shape Change ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Insulin Like Growth Factor ◽  
Concentration Dependent Manner ◽  
Induce Platelet Aggregation

Platelets release insulin-like growth factor-1 (IGF-1) from α granules upon activation. We have investigated the regulation of IGF-1 in Gi-dependent pathways leading to Akt activation and the role of IGF-1 in platelet activation. IGF-1 alone failed to induce platelet aggregation, but IGF-1 potentiated 2-MeSADP–induced platelet aggregation in a concentration-dependent manner. IGF-1 triggered platelet aggregation in combination with selective P2Y1 receptor activation. IGF-1 also caused platelet aggregation without shape change when combined with selective Gz stimulation by epinephrine, suggesting the role of IGF-1 in platelet aggregation by supplementing Gi pathways. The potentiating effect of IGF-1 was not affected by intracellular calcium chelation. Importantly, IGF-1 was unable to potentiate platelet aggregation by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, suggesting a critical regulation by PI3-K. Moreover, the potentiating effect of IGF-1 was abolished by the presence of PI3-K p110α inhibitor PIK-75. Stimulation of platelets with IGF-1 resulted in phosphorylation of Akt, a downstream effector of PI3-K, which was completely inhibited by wortmannin. IGF-1-induced Akt phosphorylation was abolished by PIK-75 suggesting the contribution of PI3-K p110α for activation of Akt by IGF-1. These results demonstrate that IGF-1 plays a role in potentiating platelet aggregation by complementing Gi- but not Gq-signaling pathways via PI3-K p110α.

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