P2Y12 receptor-mediated potentiation of thrombin-induced thromboxane A2 generation in platelets occurs through regulation of Erk1/2 activation

BACKGROUND: Thromboxane A2 (TXA2) is a positive feedback lipid mediator that is generated upon stimulation of platelets with various agonists. Aspirin works as an antithrombotic drug by blocking the generation of TXA2. The aim of this study was to evaluate the role of the purinergic P2Y receptors in thrombin-induced TXA2 generation. RESULTS: PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin, induced the activation of cytosolic phospholipase A2 (cPLA2), release of arachidonic acid (AA) from membrane-bound phospholipids, and subsequent TXA2 generation in human platelets. The actions of these agonists were significantly inhibited in the presence of the P2Y12 receptor antagonist, AR-C69931MX, but not the P2Y1 receptor antagonist, MRS2179. In addition, AYPGKF- and thrombin-induced TXA2 generation was significantly reduced in platelets from mice dosed with clopidogrel, confirming the results obtained with the human platelets. Also, Pearl mouse platelets that lack releasable nucleotides generated significantly less TXA2 when compared with the wild-type littermates in response to PAR stimulation. Inhibition of extracellular signal-regulated protein kinase 1/2 (Erk 1/2) activation using U0126, an inhibitor of MAP kinase kinase (MEK), suppressed PAR-mediated cPLA2 phosphorylation and TXA2 generation. Further, platelets that were pretreated with AR-C69931MX, as well as Pearl mouse platelets, displayed the reduced levels of Erk1/2 phosphorylation upon stimulation with the PAR agonists. CONCLUSIONS: Based on these findings, we conclude that thrombin-induced Erk1/2 activation is essential for PAR-mediated TXA2 generation, which is potentiated by the P2Y12 receptor-mediated signaling pathway but not the P2Y1 receptor-mediated signaling pathway. Finally, using selective inhibitors of Src kinases, we show that PAR-mediated Src activation precedes Erk1/2 activation.     

Different G protein-coupled signaling pathways are involved in alpha granule release from human platelets.

Alpha granule release plays an important role in propagating a hemostatic response upon platelet activation. We evaluated the ability of various agonists to cause alpha granule release in platelets. Alpha granule release was measured by determining P-selectin surface expression in aspirin-treated washed platelets. ADP-induced P-selectin expression was inhibited both by MRS 2179 (a P2Y1 selective antagonist) and AR-C69931MX (a P2Y12 selective antagonist), suggesting a role for both Galpha(q) and Galpha(i) pathways in ADP-mediated alpha granule release. Consistent with these observations, the combination of serotonin (a Galpha(q) pathway stimulator) and epinephrine (a Galpha(z) pathway stimulator) also caused alpha granule release. Furthermore, U46619-induced P-selectin expression was unaffected by MRS 2179 but was dramatically inhibited by AR-C69931, indicating a dominant role for P2Y12 in U46619-mediated alpha granule release. Additionally, the Galpha(12/13)-stimulating peptide YFLLRNP potentiated alpha granule secretion in combination with either ADP or serotonin/epinephrine costimulation but was unable to induce secretion by itself. Finally, costimulation of the Galpha(i) and Galpha(12/13) pathways resulted in a significant dose-dependent increase in alpha granule release. We conclude that ADP-induced alpha granule release in aspirin-treated platelets occurs through costimulation of Galpha(q) and Galpha(i) signaling pathways. The P2Y12 receptor plays an important role in thromboxane A(2)-mediated alpha granule release, and furthermore activation of Galpha(12/13) and Galpha(q) signaling pathway can cause alpha granule release.     

The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin α2β1

BACKGROUND: Activation of the platelet integrin alpha 2 beta 1 is closely regulated due to the high thrombogenicity of its ligand. As a beta 1 interacting kinase, ILK represents a candidate intracellular regulator of alpha 2 beta 1 in human platelets. OBJECTIVES: We investigated the regulation of ILK in human platelets and the role of ILK in regulating alpha 2 beta 1 activation in HEL cells, a megakaryocytic cell line. METHODS: An in-vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with beta 1-integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating alpha 2 beta 1 function assessed by overexpression studies in HEL cells. RESULTS: We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation-independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin-stimulated platelets and regulated by PI3K in collagen-stimulated cells. ILK associates with the beta 1-integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced alpha 2 beta 1-mediated adhesion whereas overexpression of kinase-dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of alpha 2 beta 1. CONCLUSIONS: Our findings that ILK regulates alpha 2 beta 1 in HEL cells, is activated in platelets and associates with beta 1-integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets.     

A differential role of the platelet ADP receptors P2Y1 and P2Y12 in Rac activation

The dynamics of the actin cytoskeleton, largely controlled by the Rho family of small GTPases (Rho, Rac and Cdc42), is critical for the regulation of platelet responses such as shape change, adhesion, spreading and aggregation. Here, we investigated the role of adenosine diphosphate (ADP), a major co-activator of platelets, on the activation of Rac. ADP rapidly activated Rac in a dose-dependent manner and independently of GPIIb/IIIa and phosphoinositide 3-kinase. ADP alone, used as a primary agonist, activated Rac and its effector PAK via its P2Y1 receptor, through a G(q)-dependent pathway and independently of P2Y12. The P2Y12 receptor appeared unable to activate the GTPase per se as also observed for the adenosine triphosphate receptor P2X1. Conversely, secreted ADP strongly potentiated Rac activation induced by FcgammaRIIa clustering or TRAP via its P2Y12 receptor, the target of antithrombotic thienopyridines. Stimulation of the alpha(2A)-adrenergic receptor/G(z) pathway by epinephrine was able to replace the P2Y12/G(i)-mediated pathway to amplify Rac activation by FcgammaRIIa or by the thrombin receptor PAR-1. This co-activation appeared necessary to reach a full stimulation of Rac as well as PAK activation and actin polymerization and was blocked by a G-protein betagamma subunits scavenger peptide.     

Rap1b is critical for glycoprotein VI-mediated but not ADP receptor-mediated α2β1 activation

Summary. Background: The platelet α₂β₁ integrin functions as both an adhesion and signaling receptor upon exposure to collagen. Recent studies have indicated that α₂β₁ function can be activated via inside‐out signaling, similar to the prototypical platelet integrin α_IIbβ₃. However, signaling molecules that regulate α₂β₁ activation in platelets are not well defined. A strong candidate molecule is the small GTPase Rap1b, the dominant platelet isoform of Rap1, which regulates α_IIbβ₃ activation. Objectives: We hypothesized that Rap1b positively regulates α₂β₁ during agonist‐induced platelet activation. Methods: To test whether Rap1b activates α₂β₁ downstream of glycoprotein (GP)VI or other platelet receptors, we stimulated platelets purified from Rap1b^?/? or wild‐type mice with diverse agonists and measured α₂β₁ activation using fluorescein isothiocyanate‐labeled monomeric collagen. We also examined the role of Rap1b in outside‐in signaling pathways by analyzing adhesion and spreading of Rap1b^?/? or wild‐type platelets on monomeric, immobilized collagen. Finally, we monitored the activation status of related Rap GTPases to detect changes in signaling pathways potentially associated with Rap1b‐mediated events. Results: Rap1b^?/? platelets displayed comparable ADP‐induced or thrombin‐induced α₂β₁ activation as wild‐type platelets, but reduced convulxin‐dependent α₂β₁ activation. Rap1b^?/? platelets exhibited increased spreading on immobilized collagen but similar adhesion to immobilized collagen compared to wild‐type platelets. Rap1b^?/? platelets also showed Rap1a and Rap2 activation upon agonist stimulation, possibly revealing functional compensation among Rap family members. Conclusions: Rap1b is required for maximal GPVI‐induced but not ADP‐induced activation of α₂β₁ in murine platelets.     

The P2Y1 receptor plays an essential role in the platelet shape change induced by collagen when TxA2 formation is prevented.

ADP and TxA2 are secondary agonists which play an important role as cofactors when platelets are activated by agonists such as collagen or thrombin. The aim of the present study was to characterize the role of the ADP receptor P2Y(1) in collagen-induced activation of washed platelets. Inhibition of P2Y(1) alone with the selective antagonist MRS2179 prolonged the lag phase preceding aggregation in response to low or high concentrations of fibrillar collagen, without affecting the maximum amplitude of aggregation or secretion. A combination of MRS2179 and aspirin resulted in complete inhibition of platelet shape change at low and high collagen concentrations, together with a profound decrease in aggregation and secretion. Scanning electron microscopy showed that these platelets had conserved the discoid morphology typical of the resting state. A lack of shape change was also observed in aspirin-treated P2Y(1)- and G(alphaq)-deficient mouse platelets and in delta-storage pool-deficient platelets from Fawn Hooded rats. In contrast, when the second ADP receptor P2Y(12) was inhibited with AR-C69931MX, aspirin-treated platelets were still able to change shape and displayed only a moderate decrease in aggregation and secretion. In conclusion, this study provides evidence that collagen requires not only the TxA2 receptor Tpalpha, but also P2Y(1), to induce platelet shape change.     

Platelet integrin αIIbβ3: activation mechanisms

Integrin alpha(IIb)beta(3) plays a critical role in platelet aggregation, a central response in hemostasis and thrombosis. This function of alpha(IIb)beta(3) depends upon a transition from a resting to an activated state such that it acquires the capacity to bind soluble ligands. Diverse platelet agonists alter the cytoplasmic domain of alpha(IIb)beta(3) and initiate a conformational change that traverses the transmembrane region and ultimately triggers rearrangements in the extracellular domain to permit ligand binding. The membrane-proximal regions of alpha(IIb) and beta(3) cytoplasmic tails, together with the transmembrane segments of the subunits, contact each other to form a complex which restrains the integrin in the resting state. It is unclasping of this complex that induces integrin activation. This clasping/unclasping process is influenced by multiple cytoplasmic tail binding partners. Among them, talin appears to be a critical trigger of alpha(IIb)beta(3) activation, but other binding partners, which function as activators or suppressors, are likely to act as co-regulators of integrin activation.     

A role for PKCθ in outside-in αIIbβ3 signaling

Fibrinogen binding to platelets triggers alpha(IIb)beta3-dependent outside-in signals that promote actin rearrangements and cell spreading. Studies with chemical inhibitors or activators have implicated protein kinase C (PKC) in alpha(IIb)beta3 function. However, the role of individual PKC isoforms is poorly understood. Biochemical and genetic approaches were used to determine whether PKCtheta is involved in alpha(IIb)beta3 signaling. PKCtheta was constitutively associated with alpha(IIb)beta3 in human and murine platelets. Fibrinogen binding to alpha(IIb)beta3 stimulated the association of PKCtheta with tyrosine kinases Btk and Syk, and tyrosine phosphorylation of PKCtheta, Btk and the actin regulator, Wiskott-Aldrich syndrome protein (WASP). Mouse platelets deficient in PKCtheta or Btk failed to spread on fibrinogen. Furthermore, PKCtheta was required for phosphorylation of WASP-interacting protein on Ser-488, an event that has been linked to WASP activation of the Arp2/3 complex and actin polymerization in lymphocytes. Neither PKCtheta nor Btk were required for agonist-induced inside-out signaling and fibrinogen binding to alpha(IIb)beta3. Thus, PKCtheta is a newly identified, essential member of a dynamic outside-in signaling complex that includes Btk and that couples alpha(IIb)beta3 to the actin cytoskeleton.     

Thrombin induces GPIb-IX-mediated fibrin binding to αIIbβ3 in a reconstituted Chinese hamster ovary cell model

BACKGROUND: The interaction of thrombin with platelet glycoprotein (GP) Ib-IX-V has been recently suggested to induce fibrin-dependent platelet aggregation associated with signaling events. The approaches used to avoid the protease-activated receptor (PAR) thrombin receptors in platelets have provided controversial conclusions regarding the precise mechanism and molecules involved in the response. OBJECTIVES: In the present study, we developed a cellular model to investigate the functional consequences following the binding of thrombin to GPIb-IX. METHODS: We used Chinese hamster ovary (CHO) cells stably expressing human alpha(IIb)beta(3) and/or GPIb-IX complexes (CHO-alpha(IIb)beta(3)-IbIX cells) to analyze the effect of thrombin on the binding of polymerizing fibrin by using fluorescein isothiocyanate-fibrinogen as precursor. RESULTS: Thrombin induces, in a dose-dependent manner, the binding of polymerizing fibrin to CHO-alpha(IIb)beta(3)-IbIX cells. This response is not observed in cells expressing only one of the receptors, and it can be blocked by monoclonal antibodies against alpha(IIb)beta(3) and GPIbalpha. We show that the reaction is not due to simple cell trapping by the fibrin clot, and provide data supporting a role of a signaling pathway in which the 14-3-3zeta adaptor and calcium-calmodulin-dependent events are involved. CONCLUSIONS: The present data support a significant role of GPIb-IX and alpha(IIb)beta(3) receptors in an alternative fibrin-mediated pathway of platelet activation induced by thrombin.     

Inhibition of the platelet P2Y12 receptor for adenosine diphosphate potentiates the antiplatelet effect of prostacyclin

BACKGROUND: Activation of two receptors for adenosine diphosphate (ADP), P2Y(1) and P2Y(12), is necessary for ADP-induced platelet aggregation (PA). It is generally believed that the antithrombotic effects of drugs inhibiting P2Y(12), such as clopidogrel, are uniquely mediated by inhibition of P2Y(12)-dependent PA. However, as P2Y(12) is negatively coupled to adenylyl cyclase (AC), its inhibition may also exert antithrombotic effects through the potentiation of prostacyclin (PGI(2)), which inhibit PA by stimulating AC. OBJECTIVES: To test whether inhibition of P2Y(12) potentiates the antiplatelet effects of PGI(2). METHODS: We measured the effects of PGI(2) (0.01-10 microm) on PA of washed human platelets induced by thrombin (0.5 U mL(-1)) in the presence or absence of ARC69931MX (anti-P2Y(12)) or MRS2500 (anti-P2Y(1)). Results: PGI(2) inhibited PA in the presence of anti-P2Y(12), but not in the presence of anti-P2Y(1) or in the absence of inhibitors. In contrast, dibutyryl-cyclicAMP inhibited PA both in the presence and absence of anti-P2Y(1) or anti-P2Y(12). PGI(2) increased platelet cyclicAMP levels only in the absence of thrombin or in the presence of thrombin plus anti-P2Y(12). CONCLUSIONS: PGI(2) did not inhibit PA induced by thrombin, because its effect on AC was prevented by released ADP interacting with P2Y(12). Anti-P2Y(12) drugs, by rescuing AC activity, potentiate the antiplatelet effect of PGI(2), which may contribute to their antithrombotic effect.     

von Willebrand factor mediates platelet spreading through glycoprotein Ib and αIIbβ3 in the presence of botrocetin and ristocetin, respectively

BACKGROUND: von Willebrand factor (VWF) plays a critical role in the process of hemostasis by mediating flow-dependent adhesion and spreading of platelets on exposed extracellular matrix proteins following vascular injury. To accomplish this, VWF binds to two distinct platelet receptors: glycoprotein (GP)Ib-IX-V and integrin alpha(IIb)beta3. OBJECTIVE: To evaluate the ability of GPIb and alpha(IIb)beta3 to mediate platelet adhesion and lamellipodia formation on immobilized VWF in the presence of the biochemical modulators, ristocetin and botrocetin. RESULTS: In the presence of botrocetin and inhibitors of adenosine diphosphate (ADP) and thromboxane A2 (TxA2), VWF is able to support formation of lamellipodia through a GPIb-dependent mechanism that is independent of alpha(IIb)beta3 and PI3-kinase. Lamellipodia formation under these conditions is incomplete. In marked contrast, in the presence of ristocetin, VWF stimulates formation of fully spread lamellipodia through a pathway that is dependent upon alpha(IIb)beta3 and PI3-kinase. Furthermore, alpha(IIb)beta3 also supports platelet spreading on VWF alone, but only in the absence of inhibitors of ADP and TxA2. The localization of filamentous actin and the Arp2/3 complex in platelets on VWF in the presence of botrocetin and ristocetin are distinct, yielding disparate lamellipodium kinetic signatures. Interestingly, botrocetin significantly enhances platelet adhesion to VWF under flow in whole blood in an alpha(IIb)beta3-independent manner, while ristocetin augments washed platelet adhesion and spreading to VWF under flow in an alpha(IIb)beta3-dependent manner. CONCLUSIONS: These observations demonstrate that VWF is able to induce lamellipodia formation through distinct receptors, and has important consequences for investigation of the role of VWF-GPIb interactions in the context of platelet regulation.     

A tripeptide mimetic of von Willebrand factor residues 981–983 enhances platelet adhesion to fibrinogen by signaling through integrin βIIbβ3

BACKGROUND: RGD is a major recognition sequence for ligands of platelet alpha(IIb)beta3. OBJECTIVE AND METHODS: To identify potential binding sites for alpha(IIb)beta3 apart from RGD, we screened phage display libraries by blocking the enrichment of RGD-containing phages with a GRGDS peptide and identified a novel integrin recognition tripeptide sequence, VPW. RESULTS: Platelets adhered to an immobilized cyclic VPW containing peptide in a alpha(IIb)beta3-dependent manner; platelets and alpha(IIb)beta3-expressing CHO cells adhered faster to immobilized alpha(IIb)beta3-ligands in the presence of soluble VPW. In platelets adhering to fibrinogen, VPW accelerated the activation of the tyrosine kinase Syk which controls cytoskeletal rearrangements. In alpha(IIb)beta3-expressing CHO cells, VPW induced a faster formation of stress fibers. Sequence alignment positioned VPW to V980-P981-W982 in the von Willebrand factor (vWf) A-3 domain. In blood from a vWf-deficient individual, VPW increased platelet adhesion to fibrinogen but not to collagen under flow and rescued the impaired adhesion to vWf deficient in A-3. CONCLUSION: These data reveal a VPW sequence that contributes to alpha(IIb)beta3 activation in in vitro experiments. Whether the V980-P981-W982 sequence in vWf shows similar properties under in vivo conditions remains to be established.     

Prevention of arterial thrombosis by intravenously administered platelet P2T receptor antagonist AR-C69931MX in a canine model

P2Y1, P2X1, and P2T receptors mediate ADP-induced platelet aggregation. The antithrombotic effects of AR-C69931MX (N6-[2-methylthio)ethyl]-2-[3,3,3-trifluoropropylthio]-5'-adenylic acid, monoanhydride with dichloromethylenebiphosphonic acid), a selective P2T platelet receptor antagonist, was assessed in a canine model of arterial thrombosis. Placebo or AR-C69931MX (4.0 microg/kg/min for 6 h) pretreatment was administered as an intravenous infusion beginning 15 min before inducing vessel wall injury. A 300-microA anodal current was applied to the intima of the carotid artery for 180 min or discontinued 30 min after cessation of blood flow due to thrombus formation. Each of five control animals developed occlusive thrombi within 3 h after induction of vessel wall injury. In contrast, carotid artery blood flow in five of six AR-C69931MX-treated animals was maintained for the duration of the protocol. Ex vivo platelet aggregation in response to adenosine diphosphate was inhibited at the first measurement time point of 75 min after the start of drug infusion and remained inhibited during drug administration. Bleeding time values were increased in the drug-treated group. Values for both the ex vivo platelet aggregation and the bleeding times returned to control values shortly after discontinuation of AR-C69931MX. The results indicate that AR-C69931MX antagonizes the ex vivo and in vivo aggregatory actions of ADP, and displays a rapid onset and offset of action with the ability to prevent occlusive arterial thrombus formation. AR-C69931MX may be suitable for the management of patients who require short-term modulation of platelet function.     

Impaired activation of murine platelets lacking G alpha(i2)

The intracellular signaling pathways by which G protein-coupled receptors on the platelet surface initiate aggregation, a critical process for hemostasis and thrombosis, are not well understood. In particular, the contribution of the G(i) pathway has not been directly addressed. We have investigated the activation of platelets from mice in which the gene for the predominant platelet G alpha(i) subtype, G alpha(i2), has been disrupted. In intact platelets from G alpha(i2)-deficient mice, the inhibition of adenylyl cyclase by ADP was found to be partially impaired compared with wild-type platelets. Moreover, both ADP-dependent platelet aggregation and the activation of the integrin alpha IIb beta 3 (GPIIb-IIIa) were strongly reduced in platelets from G alpha(i2)-deficient mice. In addition, G alpha(i2)-deficient platelets displayed impaired activation at low thrombin concentrations. This defect was mimicked by blocking the adenylyl cyclase--coupled platelet ADP receptor (P2Y(12)) on wild-type platelets with a selective antagonist. These observations suggest that G alpha(i2) is involved in the inhibition of platelet adenylyl cyclase in vivo and is a critical component of the signaling pathway for integrin activation by ADP, resulting in platelet aggregation. In addition, thrombin-dependent activation of mouse platelets is mediated, at least in part, by secreted ADP acting on the G alpha(i2)-linked ADP receptor.     

Protease-activated receptor-induced Akt activation – regulation and possible function

BACKGROUND: Thrombin induces the activation of the platelet serine/threonine kinase Akt. Akt activation is dependent on its phosphorylation at Thr308 and Ser473. The mechanism by which thrombin induces Akt phosphorylation is controversial, as is the role of Akt in platelet function. OBJECTIVES: To investigate how protease-activated receptors (PARs) stimulate Akt and the role that Akt plays in human platelet function. METHODS: Platelets were stimulated through PAR1 or PAR4. Specific inhibitors were used to evaluate, by Western blotting, signaling pathways regulating Akt phosphorylation, and the role of activated Akt was evaluated by aggregometry and flow cytometry. RESULTS: Phospholipase C (PLC) controls Akt phosphorylation elicited by PARs. Stimulation of PAR1 or PAR4 resulted in rapid Akt phosphorylation, independently of secreted ADP and phosphatidylinositol-3-kinase (PI3K) activation. Akt phosphorylation approximately 60 s after PAR1 stimulation became entirely dependent on the purinergic receptor P2Y(12) and the activation of PI3K. In contrast, PAR4 partially sustained Akt phosphorylation independently of P2Y(12) and PI3K for up to 300 s. Pharmacologic inhibition of Akt reduced P-selectin expression and fibrinogen binding in platelets stimulated through PAR1, and delayed platelet aggregation in response to submaximal PAR1 or PAR4 stimulation, although aggregation at 300 s was unaffected. CONCLUSIONS: Platelet PAR stimulation causes rapid Akt phosphorylation downstream of PLC, whereas with continuous stimulation, ADP and PI3K are required for maintaining Akt phosphorylation. Activated Akt regulates platelet function by modulating secretion and alpha(IIb)beta(3) activation.     

Megakaryocytes derived from human embryonic stem cells: a genetically tractable system to study megakaryocytopoiesis and integrin function

BACKGROUND: The platelet fibrinogen receptor, a heterodimer consisting of integrin subunits alpha(IIb) and beta(3), is required for platelet aggregation, spreading, and hemostasis. Platelet agonists such as thrombin and adenosine diphosphate (ADP) lead to the activation of alpha(IIb)beta(3), thereby enhancing its affinity and avidity for binding fibrinogen (inside-out signaling). Furthermore, fibrinogen binding to alpha(IIb)beta(3) triggers cytoskeletal changes and granule release (outside-in signaling). AIM: Genetic approaches to characterize the molecular pathways involved in alpha(IIb)beta(3) signaling are not possible with anucleate blood platelets. Therefore, we have established an OP9 stromal cell co-culture system to generate megakaryocytes from human embryonic stem cells (hESCs). RESULTS: alpha(IIb)beta(3) activation, measured by soluble fibrinogen binding to hESC-derived megakaryocytes, /GPIbalpha(+) cells, is readily detectable following stimulation with known platelet agonists. Dose-response curves for peptide agonists specific for the two platelet thrombin receptors, protease-activated receptor 1 (PAR1) and PAR4, show a relative responsiveness that mirrors that of human platelets, and sub-maximal ADP responses are augmented by epinephrine. Moreover, hESC-derived megakaryocytes undergo lamellipodia formation, actin filament assembly, and vinculin localization at focal adhesions when plated on a fibrinogen-coated surface, characteristic of alpha(IIb)beta(3) outside-in signaling. Undifferentiated hESCs genetically modified by lentiviral infection can be cloned and maintained in an undifferentiated state and then differentiated into megakaryocytes capable of alpha(IIb)beta(3) activation. CONCLUSION: Using hESCs, we have developed a renewable source of human megakaryocytes, and a genetically tractable system for studying megakaryocytopoiesis and alpha(IIb)beta(3) signaling in the native cellular environment.     

CHO cells expressing the high affinity αIIbβ3T562N integrin demonstrate enhanced adhesion under shear

BACKGROUND: Alpha(IIb)beta3-mediated platelet adhesive interactions in the vasculature, which are dependent on the functional state of this receptor, may be sensitive to shear forces. OBJECTIVES: To evaluate the influence of the alpha(IIb)beta3 affinity state on cell attachment under flow, we compared Chinese hamster ovary cells expressing the low affinity alpha(IIb)beta3 wild-type (wt) receptor to those expressing the high affinity alpha(IIb)beta3 T562N receptor. MATERIALS AND METHODS: We designed a real-time videomicroscopy adhesion assay for von Willebrand factor (VWF) or fibrinogen under flow conditions. RESULTS: At 50 s(-1), alpha(IIb)beta3 T562N supported higher cell adhesion to fibrinogen (63.3 +/- 2.9 cells/field) than alpha(IIb)beta3 wt (38.7 +/- 2.4 cells/field, P < 0.0001). At 100 s(-1), alpha(IIb)beta3 T562N mediated cell adhesion (40.5 +/- 3.8 cells/field), while alpha(IIb)beta3 wt did not (5.3 +/- 1.4 cells/field, P < 0.001), allowing to discriminate the efficiency of each receptor. Similar findings were observed for adhesion to VWF. Complete inhibition of cell adhesion to fibrinogen was achieved with 800 microM fibrinogen gamma-chain dodecapeptide [HHLGGAKQAGDV (H12)], while Arg-Gly-Asp-Ser (RGDS) peptide (10-1000 microM) induced a dose-dependent cell detachment. These results suggest that the H12 motif allows initial attachment, in contrast to the RGDS site, which strengthens the stability of adhesion. Interestingly, compared with wt, a 10-fold lower concentration of RGDS was required to reach a similar reduction of cell adhesion mediated by alpha(IIb)beta3 T562N. CONCLUSIONS: Our data show that alpha(IIb)beta3 activation is associated with a stabilization of integrin binding to fibrinogen or VWF under shear.     

Effect of glycoprotein IIb/IIIa inhibitors on CD62p expression, platelet aggregates, and microparticles in vitro

Flow cytometry can detect platelet activation (CD62p), aggregate formation, microparticle formation, and glycoprotein IIb/IIIa (GP IIb/IIIa) receptor occupancy in one sample at the level of single particles. We studied the effect of GP IIb/IIIa inhibitors on platelet activation with flow cytometry in vitro. Citrated whole blood was incubated with increasing concentrations of three different GP IIb/IIIa inhibitors (c7E3, DMP728, XJ757), then thrombin or adenosine diphosphate (ADP) was added, and after 1 minute the sample was fixed. Samples with thrombin but without c7E3 had a decrease in platelet count, from a mean of 260,000 platelets/microl to 56,000 platelets/microL, and aggregates increased. Samples with concentrations of c7E3 that resulted in 80% or more receptor blockade had no decrease in platelet count, and no aggregates were formed, but the number of CD62p-positive single platelets increased from 1200 to 7400 platelets/microL. The two other inhibitors (DMP 725, XJ757) or ADP instead of thrombin gave similar results. Microparticle formation did not change with platelet activation in the presence of a GP IIb/IIIa inhibitor. With small inhibitor doses resulting in <80% receptor blockade, the number of aggregates did not change or was even higher than that in samples without inhibitor. GP IIb/IIIa inhibitors do prevent aggregate formation but they do not prevent activation of platelets. With GP IIb/IIIa inhibition, more activated single platelets remain in the blood. One may expect an increasing number of circulating, activated platelets with the use of GP IIb/IIIa inhibitors.     

Lipid rafts are required in Gαi signaling downstream of the P2Y12 receptor during ADP-mediated platelet activation

ADP is important in propagating hemostasis upon its secretion from activated platelets in response to other agonists. Lipid rafts are microdomains within the plasma membrane that are rich in cholesterol and sphingolipids, and have been implicated in the stimulatory mechanisms of platelet agonists. We sought to determine the importance of lipid rafts in ADP-mediated platelet activation via the G protein-coupled P2Y1 and P2Y12 receptors using lipid raft disruption by cholesterol depletion with methyl-beta-cyclodextrin. Stimulation of cholesterol-depleted platelets with ADP resulted in a reduction in the extent of aggregation but no difference in the extent of shape change or intracellular calcium release. Furthermore, repletion of cholesterol to previously depleted membranes restored ADP-mediated platelet aggregation. In addition, P2Y12-mediated inhibition of cAMP formation was significantly decreased upon cholesterol depletion from platelets. Stimulation of cholesterol-depleted platelets with agonists that depend upon Galpha(i) activation for full activation displayed significant loss of aggregation and secretion, but showed restoration when simultaneously stimulated with the Galpha(z)-coupled agonist epinephrine. Finally, Galpha(i) preferentially localizes to lipid rafts as determined by sucrose density centrifugation. We conclude that Galpha(i) signaling downstream of P2Y12 activation, but not Galpha(q) or Galpha(z) signaling downstream of P2Y1 or alpha2A activation, respectively, has a requirement for lipid rafts that is necessary for its function in ADP-mediated platelet activation.     

Effect of cellular and receptor activation on the extent of integrin αIIbβ3 internalization

Summary. Previous studies by our laboratory demonstrated that internalization of fibrinogen‐bound α_IIbβ₃ correlated with both a loss of aggregation and a loss of bound fibrinogen from the platelet surface. However, these studies do not address whether cellular activation, receptor activation and/or receptor occupancy are responsible for the observed internalization of α_IIbβ₃. The present studies were designed to evaluate the roles of cellular and receptor activation states on the α_IIbβ₃ internalization process. In these studies, washed platelets were allowed to bind FITC‐D57, an antiα_IIb monoclonal antibody, and were subsequently treated with ADP, thrombin receptor activation peptide (TRAP) or antiLIBS6 monoclonal antibody. Following flow cytometric analyses for log green fluorescence, rabbit antifluorescein was added, and the samples were re‐analyzed for residual/unquenched fluorescence. Because access of the quenching antibody is limited to extracellular/surface‐associated fluorescein, protection from quenching by antifluorescein is taken as evidence of internalization. Stimulation of platelets with ADP or TRAP resulted in a significant increase in the percent internalization of α_IIbβ₃ compared to control (8.7% and 12.8% vs. 2.9%). Addition of cytochalasin E prior to stimulation resulted in a greater than 90% inhibition of both TRAP and ADP‐induced internalization, suggesting that activation‐dependent internalization is mediated by the actin cytoskeleton. To investigate whether receptor activation increases the extent of α_IIbβ₃ internalization, platelets were treated with anti‐LIBS6, which directly activates α_IIbβ₃. Stimulation with anti‐LIBS6 caused an approximate 8‐fold increase in the extent of α_IIbβ₃ internalization. To evaluate whether the activated pool of α_IIbβ₃ is preferentially internalized, platelets were incubated with PAC‐1, an antibody specific for activated α_IIbβ₃. Platelets stimulated with TRAP, demonstrated a dose‐dependent internalization of PAC‐1. However, approximately 29% of total PAC‐1 binding was internalized, irrespective of TRAP concentration, suggesting that a constant proportion of activated α_IIbβ₃ is selectively internalized in platelets. Collectively, these data suggest that α_IIbβ₃ is internalized to a greater extent in activated platelets in a cytoskeleton‐dependent manner. Furthermore, the active conformer of α_IIbβ₃ is preferentially internalized which may act as a mechanism for downregulating adhesiveness of activated platelets in the circulation.