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(Circulation Research. 2006;99:1293.)
© 2006 American Heart Association, Inc.

Reviews

Activation of Platelet Function Through G Protein–Coupled Receptors

Stefan Offermanns

From the Institute of Pharmacology, University of Heidelberg, Germany.

Correspondence to Stefan Offermanns, Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany. E-mail stefan.offermanns{at}pharma.uni-heidelberg.de

This Review is part of a thematic series on Mechanisms, Models, and In Vivo Imaging of Thrombus Formation, which includes the following articles:

Activation of Platelet Function Through G Protein–Coupled Receptors

Platelets As Immune Cells: Bridging Inflammation and Cardiovascular Disease

In Vivo Thrombus Formation

Platelet Adhesion

Platelet Inhibitors and Thrombus Formation
Bernhard Nieswandt and Ulrich Walter Guest Editors

	Abstract

Top Abstract Introduction Platelet Activators Working... Adenosine Diphosphate Thrombin Thromboxane A2 Other Platelet Stimuli Inhibitors of Platelets Acting... G Protein-Mediated Signaling... Platelet-Shape Change Platelet Aggregation Platelet Secretion and... Implications for Future... Conclusions References

 
Because of their ability to become rapidly activated at places of vascular injury, platelets are important players in primary hemostasis as well as in arterial thrombosis. In addition, they are also involved in chronic pathological processes including the atherosclerotic remodeling of the vascular system. Although primary adhesion of platelets to the vessel wall is largely independent of G protein–mediated signaling, the subsequent recruitment of additional platelets into a growing platelet thrombus requires mediators such as ADP, thromboxane A₂, or thrombin, which act through G protein–coupled receptors. Platelet activation via G protein–coupled receptors involves 3 major G protein–mediated signaling pathways that are initiated by the activation of the G proteins G_q, G₁₃, and G_i. This review summarizes recent progress in understanding the mechanisms underlying platelet activation and thrombus extension via G protein–mediated signaling pathways.

Key Words: platelet activation • heterotrimeric G proteins • GPCRs • thrombosis

	Introduction

Top Abstract Introduction Platelet Activators Working... Adenosine Diphosphate Thrombin Thromboxane A2 Other Platelet Stimuli Inhibitors of Platelets Acting... G Protein-Mediated Signaling... Platelet-Shape Change Platelet Aggregation Platelet Secretion and... Implications for Future... Conclusions References

 
Platelet adhesion and activation at sites of vascular wall injury is initiated by a multistep process involving the interaction of platelets with the subendothelial extracellular matrix which contains adhesive macromolecules including collagen and von Willebrand factor (vWF).^1,2 The initial interaction of platelets with the extracellular matrix under conditions of high sheer rates and the subsequent strengthening of this interaction involves the platelet vWF receptor GPIb/V/IX and the collagen receptor GPVI^2–4 (which will be covered in a forthcoming review in this series). GPVI is unable to mediate adhesion but, via activation of the FcR chain, induces intracellular signaling processes which promote the inside-out activation of integrins such as _IIbß₃ (GPIIb/IIIa) or ₂ß₁ (GPIa/IIa).^4,5 The interaction of activated integrins with the extracellular matrix then mediates the firm adhesion of platelets to the injured vessel wall, resulting in the formation of a platelet monolayer.

During the next stage of platelet activation, a platelet plug forms through the recruitment of additional platelets from the circulation and their integrin _IIbß₃-mediated aggregation (see Figure 1). The recruitment of additional platelets is mediated by a variety of locally accumulating mediators that are produced or released once platelet adhesion has been initiated and some level of platelet activation through platelet adhesion receptors has occurred. These mediators include ADP/ATP and thromboxane A₂ (TxA₂), which are secreted or released from activated platelets and thrombin, which is produced on the surface of activated platelets. These diffusible mediators have in common that they act via G protein–coupled receptors (GPCRs). Through the activation of G protein–mediated signaling pathways, they can further increase their own formation and release, thus acting as positive-feedback mediators that amplify the initial signals to ensure the rapid activation and recruitment of platelets into a growing thrombus (Figure 1).

View larger version (59K): [in this window] [in a new window]   Figure 1. Activation of platelets at sites of vascular injury. The initial contact of platelets with the subendothelial extracellular matrix is mediated by vWF and GPIb, followed by the activation of platelets by collagen via the collagen receptor GPVI. This results in the integrin-mediated adhesion of platelets and some cellular activation, which leads to the release and production of diffusible mediators such as ADP, TxA2, and thrombin acting via GPCRs. Activation of these receptors strengthens the adhesion of the first and second layers of adhering platelets, induces platelet-shape change, and further drives the formation and release of mediators. These diffusible mediators then act on platelets nearby and recruit them into the growing platelet plug. ECM indicates extracellular matrix; Fg, fibrinogen.

Thus, G proteins are centrally involved in the second phase of platelet-dependent thrombus formation. Pharmacological studies as well as work based on the analysis of mice lacking individual components of G protein–mediated signaling pathways have identified GPCRs, G proteins, and effector pathways regulating platelet activation (see Tables 1 and 2). This review describes the major GPCRs involved in platelet activation and summarizes recent data on the role of individual G proteins and their downstream effectors in the regulation of platelet functions such as integrin-mediated aggregation, degranulation, or platelet-shape change. Finally, the review discusses whether G protein–mediated signaling pathways could serve as targets for the development of new antiplatelet strategies.

View this table: [in this window] [in a new window]   Table 1. Main Platelet Stimuli Acting via GPCRs and Phenotypes Observed in Receptor-Deficient Mice

View this table: [in this window] [in a new window]   Table 2. Platelet Phenotypes of Mice Lacking G Protein Subunits

	Platelet Activators Working Through G Protein–Coupled Receptors

Top Abstract Introduction Platelet Activators Working... Adenosine Diphosphate Thrombin Thromboxane A2 Other Platelet Stimuli Inhibitors of Platelets Acting... G Protein-Mediated Signaling... Platelet-Shape Change Platelet Aggregation Platelet Secretion and... Implications for Future... Conclusions References

 
GPCRs constitute the largest family of proteins in the human genome.^6–8 They can be activated by a chemically very diverse group of ligands including amines, lipids, peptides, ions, nucleotides, or proteases.⁹ Because of their ability to specifically interact with various functionally different heterotrimeric guanine nucleotide-binding proteins (G proteins), agonist-activated GPCRs can induce different signaling pathways to change cellular functions.^10,11 The large versatility of the G protein–mediated signaling system may explain why it is the primary mediator of the second phase of platelet activation during thrombosis and hemostasis, which requires the coordinated and fast action of a variety of diffusible mediators to activate platelets and to recruit them into the growing thrombus.

	Adenosine Diphosphate

Top Abstract Introduction Platelet Activators Working... Adenosine Diphosphate Thrombin Thromboxane A2 Other Platelet Stimuli Inhibitors of Platelets Acting... G Protein-Mediated Signaling... Platelet-Shape Change Platelet Aggregation Platelet Secretion and... Implications for Future... Conclusions References

 
ADP is stored at high concentrations in dense granules of platelets and is released on platelet activation. Released ADP strongly activates platelets in an autocrine and paracrine fashion. It can also be released from damaged cells at places of vascular injury. Platelet activation by ADP is mediated by 2 G protein-coupled receptors, P2Y₁ and P2Y₁₂.^12,13 Whereas P2Y₁ couples to G_q,^14,15 P2Y₁₂ is coupled to G_i-type G proteins, in particular to G_i2.^16,17 Studies using receptor agonists had suggested that activation of both receptors is required for a full response of platelets to ADP.^18–20 Platelets from mice lacking P2Y₁ do not undergo shape change in response to ADP, and ADP-induced aggregation is severely impaired. In addition, mice lacking P2Y₁ have mildly increased bleeding times and a relative resistance to ADP-induced thromboembolism.^21,22 Absence of P2Y₁₂ in humans results in a mild form of hemorrhagia,^23,24 a phenotype not observed in mice lacking P2Y₁₂.^25,26 Platelets from mice deficient in P2Y₁₂ have a normal shape change but an impaired aggregation in response to ADP.^25,26 In addition, these animals have severely prolonged bleeding times and form smaller and unstable thrombi.²⁷ Both P2Y₁ and P2Y₁₂ are also involved in ADP-induced platelet procoagulant activity.²⁸ Interestingly, platelet responses to thrombin and TxA₂ at low and intermediate concentrations are reduced in the absence of ADP receptors,^21,22,25,27 underlining the important role of ADP as a positive-feedback mediator required for sustained platelet activation. The P2Y₁₂ receptor is irreversibly inhibited by thienopyridines such as clopidogrel, which is currently used for the secondary prevention of cardiovascular events.^29,30

	Thrombin

Top Abstract Introduction Platelet Activators Working... Adenosine Diphosphate Thrombin Thromboxane A2 Other Platelet Stimuli Inhibitors of Platelets Acting... G Protein-Mediated Signaling... Platelet-Shape Change Platelet Aggregation Platelet Secretion and... Implications for Future... Conclusions References

 
Thrombin is the main effector protease of the coagulation system and is among the most effective activators of platelets. Thrombin formation is initiated by the exposure of tissue factor to plasma coagulation factors after disruption of the vascular endothelium. Thrombin formation takes place on cellular surfaces including that of activated platelets.³¹ The local production of thrombin on the platelet surface represents an important mechanism by which activated platelets stimulate coagulatory processes. In addition, it may facilitate activation of platelets by thrombin, which is rapidly inactivated after its formation. Activation of platelets by thrombin is mediated by protease-activated receptors (PARs),³² which couple to G_q, G₁₂/G₁₃ and, in some cases, also to the G_i family of heterotrimeric G proteins.^33,34 Of the four protease-activated receptors, PAR1 and PAR4 are present on human platelets, whereas mouse platelets express PAR3 and PAR4.^35,36 Studies using PAR1 antagonists or antibodies blocking PAR1 or PAR4 activation have indicated that PAR1 mediates human platelet activation at low thrombin concentrations, whereas PAR4 contributes to thrombin-induced platelet activation only at high thrombin concentrations.^35,37–39 The higher potency of thrombin toward PAR1 activation is most likely attributable to the presence of a hirudin-like sequence close to the C-terminal thrombin cleavage site, which facilitates binding of thrombin and is absent in PAR4.^35,40 The presence of PAR3 and PAR4 in mouse platelets suggested that they function analogous to PAR1 and PAR4 in human platelets. This was initially suggested by studies using platelets from PAR3-deficient mice, which did not respond any more to low and intermediate concentrations of thrombin but could be activated by high concentrations via PAR4.³⁵ However, studies with heterologously expressed receptors revealed that mouse PAR3, which contains a hirudin-like sequence close to the thrombin cleavage site, did not mediate thrombin-induced transmembrane signaling unless PAR4 was present. This suggests that mouse PAR3 functions as a coreceptor for mouse PAR4 and facilitates cleavage and activation of PAR4 at low thrombin concentrations.⁴¹ Consistent with this, platelets from PAR4-deficient mice are unresponsive to thrombin and protected against thrombosis.^42,43 Thus, although thrombin activates human platelets by cleaving and activating PAR1 and PAR4 at low and high concentrations, respectively, thrombin-induced mouse platelet activation is completely dependent on PAR4-mediated signaling and requires PAR3 only to facilitate cleavage of PAR4 at low thrombin concentrations. It is not known whether mouse PAR3 and PAR4 form stable heterodimers in which PAR3 mediates activation and PAR4 signaling, or whether binding of thrombin to PAR3 simply facilitates cleavage of PAR4.

	Thromboxane A2

Top Abstract Introduction Platelet Activators Working... Adenosine Diphosphate Thrombin Thromboxane A2 Other Platelet Stimuli Inhibitors of Platelets Acting... G Protein-Mediated Signaling... Platelet-Shape Change Platelet Aggregation Platelet Secretion and... Implications for Future... Conclusions References

 
Like ADP, TxA₂ functions as a positive-feedback mediator during platelet activation. It is produced from arachidonic acid through conversion by cyclooxygenase-1, the target of low-dose aspirin, and thromboxane synthase. The action of TxA₂ is locally restricted because of its short half-life. The TxA₂ receptor (TP), which is also activated by the prostaglandin endoperoxides PGG₂ and PGH₂, couples to G_q and G₁₂/G₁₃.^33,44,45 The role of TP as the platelet TxA₂ receptor has been demonstrated in studies using platelets from TP-deficient mice, which become unresponsive to TxA₂.⁴⁶ TP-deficient mice have prolonged bleeding times and are unable to form stable thrombi.⁴⁶ A reduced activation of TP-deficient platelets has been suggested to contribute to a reduced injury-induced vascular proliferation as well as to a reduced progression of atherosclerosis observed in mice lacking TP.^47,48

	Other Platelet Stimuli

Top Abstract Introduction Platelet Activators Working... Adenosine Diphosphate Thrombin Thromboxane A2 Other Platelet Stimuli Inhibitors of Platelets Acting... G Protein-Mediated Signaling... Platelet-Shape Change Platelet Aggregation Platelet Secretion and... Implications for Future... Conclusions References

 
Several other stimuli have been identified that act through GPCRs. However, in contrast to ADP, thrombin, and TxA₂, they are only weak activators of platelets and appear to serve primarily as potentiators of platelet responses to other stimuli.

For instance, epinephrine alone is not able to activate platelets but potentiates the effect of other stimuli by acting through the _2A-adrenergic receptor. Interestingly, the _2A receptor in platelets preferentially couples to the G_i-type G protein G_z.⁴⁹ In mice lacking the _2A-adrenergic receptor, the potentiating effect of epinephrine on platelet activation was absent, bleeding time was moderately prolonged, and the formation of stable thrombi was impaired.⁵⁰

Prostaglandin E₂ can potentiate platelet activation via the EP3 receptor. The EP3 receptor form expressed on platelets is believed to be coupled to G_i-type G proteins. In mice lacking EP3, bleeding times are increased and the potentiating effects of prostaglandin E₂ are abrogated.^51,52

Serotonin is taken up by platelets, stored in dense granules, and released on platelet activation. Part of its action serves as a positive-feedback mechanism by activating 5-hydroxtryptamine 2A receptors on platelets, which are G_q-coupled.

Various chemokines such as platelet factor 4 (PF4/CXCL4), RANTES (CCL5), or CXCL5 are released from activated platelets. During recent years, it has been demonstrated that platelets express chemokine receptors such as CXCR4, the receptor of stromal cell-derived factors 1 (SDF-1/CXCL12), or CCR4, which is activated by macrophage-derived chemokine (MDC/CCL22) and by TARC (CCL17).^53–57 Also, the chemokine receptors CCR1, CCR2, and CX3CR1, which are activated by RANTES (CCL5), MCP-1 (CCL2), and fractalkine (CX3CL1), respectively, have been shown to be expressed by platelets.^56,58 Chemokine receptors are coupled to G_i-type G proteins and mediate a rather weak activation of platelets. However, they are able to potentiate the effects of other platelet stimuli. There is increasing evidence that the secretion and presentation of chemokines by platelets, as well as the activation of platelets by chemokines, play an important role in the development of atherosclerotic vascular disease.^59,60

Lysophospholipids such as lysophosphatidic acid (LPA) can activate platelets.⁶¹ There is evidence that platelets express LPA₁, LPA₂, and LPA₃ receptors.^62,63 LPA and related lysophospholipid species are found in mildly oxidized low-density lipoprotein, as well as in advanced atherosclerotic lesions, and may contribute to platelet activation at various stages of atherosclerosis.⁶⁴

	Inhibitors of Platelets Acting Through GPCRs

Top Abstract Introduction Platelet Activators Working... Adenosine Diphosphate Thrombin Thromboxane A2 Other Platelet Stimuli Inhibitors of Platelets Acting... G Protein-Mediated Signaling... Platelet-Shape Change Platelet Aggregation Platelet Secretion and... Implications for Future... Conclusions References

 
The major endothelium-derived inhibitors of platelet activation are nitric oxide (NO) and prostacyclin (PGI₂), which raise the levels of the cyclic nucleotides cGMP and cAMP. Although NO directly activates guanylyl cyclase, PGI₂ acts through a G_s-coupled receptor, the IP receptor, to stimulate adenylyl cyclase. Recent evidence suggests that PGI₂-dependent platelet inhibition may play an important role in the protective effect of prostacyclin. In IP receptor–deficient mice, injury-induced vascular proliferation and platelet activation was enhanced. In mice lacking both IP and TP receptors, this augmented response was abolished, suggesting that PGI₂ and TxA₂, acting through their respective receptors, antagonistically regulate vascular and platelet functions.⁴⁷ This complex functional antagonism may also explain the adverse cardiovascular effects associated with selective cyclooxygenase-2 inhibitors, which inhibit the formation of PGI₂ but not of TxA₂.⁶⁵

Adenosine released during cell damage or by conversion of ADP by the endothelial ectonucleotidase CD39 inhibits platelet function by activating the G_s-coupled A_2A receptor.⁶⁶

	G Protein–Mediated Signaling Pathways Mediating Platelet Activation

Top Abstract Introduction Platelet Activators Working... Adenosine Diphosphate Thrombin Thromboxane A2 Other Platelet Stimuli Inhibitors of Platelets Acting... G Protein-Mediated Signaling... Platelet-Shape Change Platelet Aggregation Platelet Secretion and... Implications for Future... Conclusions References

 
The main diffusible platelet stimuli ADP, TxA₂, and thrombin recruit platelets into a growing thrombus by activating multiple G protein–mediated signaling pathways to induce platelet-shape change, degranulation, and integrin _IIbß₃-mediated aggregation. They use distinct mechanisms to induce full platelet activation. ADP activates G_q and G_i through its receptors P2Y₁ and P2Y₁₂, whereas TxA₂ and thrombin activate mainly G_q and G₁₂/G₁₃ via the TxA₂ receptor or the protease-activated receptors PAR1/PAR4 or PAR3/PAR4. Because all mediators can in turn increase the formation and release of thrombin, TxA₂, and ADP, their effects are amplified and eventually all major G protein–mediated signaling pathways are activated. These positive-feedback mechanisms have obscured the analysis of the roles individual G protein–mediated signaling pathways play in platelet activation. Platelets lacking the subunits of individual G-protein subtypes have been used to study the function of the main G protein–mediated signaling pathways in platelet activation independently of the mediators and their respective receptors involved. In the course of these studies the G proteins G_q, G₁₃, and G_i2 have been demonstrated to play important roles in platelet activation (see Table 2).

The G_q/G₁₁ family of G proteins couples receptors to ß isoforms of phospholipase C (PLC), of which, especially the ß₂ and the ß₃ isoforms, are present in platelets. Activation of PLC results in the formation of IP₃ and diacyl glycerol leading to an elevation of free cytoplasmic [Ca²⁺] and activation of protein kinase C (PKC), respectively. Although most cells in the mammalian organism express both G_q and G₁₁, platelets are an exception in that they only contain G_q.^67,68 So far, no physiological significance for the lack of G₁₁ in platelets has been reported. G₁₃, a member of the G₁₂/G₁₃ family, has been shown to regulate several signaling pathways of which the Rho/Rho-kinase–mediated pathway is the best established. Activated G₁₃ binds and activates a subgroup of Rho-specific guanine nucleotide exchange factors.^69,70 G_i2, the main member of the G_i family expressed on platelets couples receptors in an inhibitory fashion to adenylyl cyclase. In addition, G_i-type G proteins are a major source for ß complexes, which are released on G-protein activation and can regulate a variety of channels or enzymes including adenylyl cyclases or phosphatidylinositol 3-kinases (PI3Ks).⁷¹ The latter enzyme produces phosphatidylinositol-3,4,5-trisphosphate, which activates a variety of downstream effectors including the serine/threonine kinase Akt/protein kinase B (PKB).^72,73

	Platelet-Shape Change

Top Abstract Introduction Platelet Activators Working... Adenosine Diphosphate Thrombin Thromboxane A2 Other Platelet Stimuli Inhibitors of Platelets Acting... G Protein-Mediated Signaling... Platelet-Shape Change Platelet Aggregation Platelet Secretion and... Implications for Future... Conclusions References

 
A shape change is the initial response of platelets to activators such as thrombin, ADP, or TxA₂. It is an extremely rapid process based on the reorganization of the cytoskeleton.^74,75 During platelet-shape change, new actin filaments are formed, leading to the formation of a submembranous actin filament network and the extension of filopodia. In addition, actomyosin-based contractile processes are stimulated, resulting in the centralization of dense and granules. Finally, the circumferential microtubule coil depolymerizes, which allows the platelet to change from a discoid to a spherical shape. The platelet-shape change, which is induced by agonist concentrations lower than those required for degranulation and aggregation, is believed to be a prerequisite for efficient secretion of granule contents and to greatly facilitate adhesion of platelets to each other and to components of the extracellular matrix.

Platelet agonists can induce shape change under conditions in which they do not stimulate PLC or induce an increase in [Ca²⁺]_i, suggesting that an elevation of [Ca²⁺]_i alone is not sufficient to induce platelet-shape change.^76–79 Consistent with that, thrombin and TxA₂ can still induce platelet-shape change in the absence of G_q-mediated PLC activation.⁶⁸ This suggests that other G proteins activated by thrombin and TxA₂ receptors such as G₁₂ and G₁₃ are involved in the induction of platelet-shape change.³⁴ In platelets lacking G₁₃ but not G₁₂, low and intermediate concentrations of thrombin and TxA₂ were not able to induce a shape change.⁸⁰ At high agonist concentrations, TxA₂ and thrombin activated platelet-shape change also in the absence of G₁₃. However, in the absence of both G₁₃ and G_q, platelet-shape change could not be induced even at maximal agonist concentrations.⁸¹ This clearly indicates that platelet-shape change can be induced through G_q as well as through G₁₃. Stimuli that are able to activate both G proteins via their respective receptors such as TxA₂ and thrombin preferentially use G₁₃ to induce platelet-shape change. Stimuli such as ADP, which activates G_q-mediated signaling pathways but not signaling via G₁₃, induce platelet-shape change solely via G_q.^68,81 Activation of G_i does not appear to be required for the induction of platelet-shape change (see Figure 2).

View larger version (62K): [in this window] [in a new window]   Figure 2. Signaling pathways induced via GPCRs in platelets. Shown are some of the upstream signaling mechanisms linking the activation of GPCRs by ADP, TxA2, and thrombin via the G proteins G13, Gq, and Gi to the induction of platelet-shape change, inside-out activation of integrins, and degranulation. RhoGEF indicates Rho-guanine nucleotide exchange factor; PIP5K, phosphatidylinositol 4-phosphate 5-kinase; MPase, myosin phosphatase; MLCK, MLC kinase; DAG, diacyl glycerol; CalDAG-GEF, calcium and diacyl glycerol-regulated guanine nucleotide exchange factor; PIP3, phosphatidylinositol-3,4,5-trisphosphate.

The regulation of myosin light chain (MLC) phosphorylation has been suggested to be involved in the induction of platelet-shape change.^82,83 MLC phosphorylation can be controlled through a Ca²⁺/calmodulin-dependent regulation of MLC kinase and through a Rho/Rho-kinase–mediated regulation of myosin phosphatase.⁸⁴ In G_q-deficient platelets the agonist-induced platelet-shape change could be blocked by inactivation of Rho and inhibition of Rho-kinase,³⁴ and MLC phosphorylation and shape change in response to TxA₂ and low concentrations of thrombin were strongly dependent on the Rho/Rho-kinase pathway in wild-type platelets.^85,86 The central role of the Rho/Rho-kinase–mediated regulation of MLC phosphorylation and platelet-shape change is supported by the strong correlation between the ability of stimuli to induce platelet-shape change and their ability to stimulate RhoA activation and MLC phosphorylation. In platelets lacking G_q, TxA₂ and thrombin were still able to induce shape change, RhoA activation, and MLC phosphorylation.^68,80,81 High concentrations of TxA₂ and thrombin as well as ADP are able to induce platelet-shape change as well as RhoA activation and MLC phosphorylation also in the absence of G₁₃. However, in the absence of both G_q and G₁₃, none of the stimuli can induce platelet-shape change or activation of RhoA or MLC phosphorylation.⁸¹ This indicates that activation of the Rho/Rho-kinase–mediated signaling pathway is required for agonist-induced MLC phosphorylation in platelets. When the G₁₃-mediated signaling pathway resulting in Rho/Rho-kinase activation is blocked, G_q-mediated MLC phosphorylation can be induced only under sufficiently high agonist concentrations which result in RhoA activation through G_q. G_q has been shown to be able to mediate RhoA activation in a PLC-independent manner via Rho guanine nucleotide exchange factors.^87–89 Stimulation of Rho/Rho-kinase and MLC phosphorylation increases actomyosin contractility, and activation of Rho-kinase has been suggested to be required for the dynamic regulation of microtubule coils during platelet-shape change.⁹⁰

In addition to MLC phosphorylation, several other signaling processes may be involved in agonist-induced platelet-shape change. This includes tyrosine kinases such as pp60c-src or pp72syk.^34,78,91,92 Actin assembly during platelet-shape change has also been shown to be controlled by polyphosphoinositides such as phosphatidylinositol 4,5-bisphosphate. Formation of phosphatidylinositol 4,5-bisphosphate by phosphatidylinositol 4-phosphate 5-kinase can be regulated by the small GTP-binding protein Rac,^75,93,94 as well as by RhoA.⁹⁵ Rac becomes activated in platelets on receptor activation⁹⁶ and can also induce actin polymerization through the activation of the Arp2/3 complex mediated by WAVE proteins.⁹⁷ However, in G_q-deficient platelets, activation of Rac by various agonists is abrogated while they are still able to induce platelet-shape change,⁹⁸ indicating that Rac activation is not required for agonist-induced platelet-shape change. Under these conditions, actin assembly and polymerization may be induced through other RhoA-dependent pathways such as the activation of 4-phosphate 5-kinase⁹⁹ or of formins, which promote linear elongation of actin filaments.¹⁰⁰

	Platelet Aggregation

Top Abstract Introduction Platelet Activators Working... Adenosine Diphosphate Thrombin Thromboxane A2 Other Platelet Stimuli Inhibitors of Platelets Acting... G Protein-Mediated Signaling... Platelet-Shape Change Platelet Aggregation Platelet Secretion and... Implications for Future... Conclusions References

 
The accumulation of platelets into a hemostatic thrombus is based on the formation of multiple platelet/platelet interactions (platelet aggregation). Platelet aggregation is mediated by integrin _IIbß₃, which binds various extracellular macromolecular ligands including fibrinogen and vWF. The dimeric structure of fibrinogen and the multimeric structure of vWF allow these ligands to crossbridge platelets and to generate a platelet aggregate. In unactivated, resting platelets, _IIbß₃ is in an "off" state, in which ligand binding affinities are low and no signaling occurs. Platelet activation induces intracellular signaling processes that influence the cytoplasmic part of _IIbß₃, rapidly converting _IIbß₃ into an active conformation (inside-out signaling), which results in fibrinogen/vWF-mediated platelet aggregation.^101–106

Although the exact signaling mechanisms that link receptors of platelet activators to the cytoplasmic domains of _IIbß₃ are incompletely understood, the role of different G-protein subfamilies and the role of some of their immediate effector pathways have been described during recent years. There is good evidence that activation of PLCß through G_q, which results in the formation of IP₃ and diacyl glycerol, plays an important role in mediating _IIbß₃ activation. IP₃-mediated increases in cytosolic-free Ca²⁺ appears to be required for integrin activation, but Ca²⁺ alone is obviously not sufficient to induce inside-out signaling.¹⁰⁷ Activation of PKC by diacyl glycerol or phorbol esters leads to _IIbß₃ activation.¹⁰⁷ It is, however, not clear which of the various PKC isoforms are involved in G protein–mediated inside-out activation and which are the relevant PKC substrates in this process. The requirement of G_q-mediated signaling for agonist-induced _IIbß₃ activation has also been demonstrated by the phenotype of G_q-deficient platelets, which fail to aggregate and to secrete in response to thrombin, ADP, and TxA₂ because of a lack of agonist-induced PLC activation.⁶⁸ There is, however, clear evidence for additional G_q-independent signaling processes which are involved in _IIbß₃ activation. This has first been suggested based on several observations made when the mechanisms of ADP-induced integrin _IIbß₃ activation were studied. Platelets lacking the G_i-coupled P2Y₁₂ receptor or in which P2Y₁₂ was blocked did not aggregate in response to ADP unless the G_i-mediated pathway was activated by alternative mechanisms.^14,25,86 A role for G_i-mediated signaling in integrin _IIbß₃ activation by platelet stimuli is also indicated by the fact that platelets lacking G_i2 or the related G_z show reduced aggregation in response to ADP, thrombin or adrenaline.^{17,49,108,109} Thus, G_q and G_i appear to synergize to induce platelet aggregation.

How G_i contributes to integrin _IIbß₃ activation in platelets is currently not clear. It is conceivable that the G_i-mediated decrease in cAMP levels counteracts the antiaggregatory effects of endothelial mediators such as PGI₂, which increases platelet cAMP levels via its G_s-coupled receptor. However, under in vitro conditions, a decrease in cAMP levels alone does not induce aggregation of platelets.^19,110,111 Activation of G_i results in the release of reasonable amounts of G-protein ß subunits, which, as dimers themselves, regulate various effectors and may contribute to _IIbß₃ activation and platelet aggregation. The 2 PI3K isoforms activated by ß complexes, p110ß (PI3Kß) and p110 (PI3K), are both expressed in platelets.^112,113 Activation of PI3K results in the formation of 3-phosphorylated phosphoinositides, which can activate a variety of effectors including various isoforms of PKC or PKB/Akt.^72,114 A role of PI3K in the activation of integrin _IIbß₃ is also supported by observations in PI3K-deficient mice, which show reduced aggregation responses to ADP,^115,116 as well as by studies using specific inhibitors of PI3Kß, which suggests that this enzyme plays an important role in sustaining platelet aggregation in response to low concentrations of platelet activators.¹¹⁷ Consistent with that, platelets lacking the downstream effectors of PI3K, Akt1, and/or Akt2 show reduced aggregation.^118,119

Recently, the small GTPase Rap1 has attracted interest as a potential mediator of integrin _IIbß₃ activation by various platelet activators. Rap1, which has been implicated in the activation of inside-out signaling for a variety of integrins including _IIbß₃,^120,121 is rapidly activated on platelet activation obviously through G_q- and G_i-mediated signaling pathways.^122,123 Mice lacking Rap1b show defects in the activation of integrin _IIbß₃ in response to various platelet stimuli and are protected from arterial thrombosis.¹²⁴ Similarly, mice lacking CalDAG-GEFI, a guanine nucleotide exchange factor mediating agonist-induced Rap1 activation in platelets, show decreased platelet activation and thrombus formation and have severely prolonged bleeding times.¹²⁵ These data clearly indicate that Rap1 is involved in at least 1 of several mechanisms mediating the integrin _IIbß₃ activation by various platelet activators.

There is also clear evidence that G₁₃-mediated signaling contributes to efficient _IIbß₃ activation. This has first been suggested on the basis of the observation that some level of integrin _IIbß₃ activation and platelet aggregation can also be induced in the absence of G_q-mediated signaling when G_i- and G₁₂/G₁₃-dependent signaling pathways are concomitantly activated.^126,127 A role of G₁₃ in platelet integrin activation could be confirmed in studies with platelets lacking G₁₃. In the absence of G₁₃, agonist-induced _IIbß₃ activation was reduced. In addition, G₁₃-deficient animals had prolonged bleeding times and were protected against arterial thrombosis.⁸⁰ A role of the G₁₃-mediated activation of the Rho/Rho-kinase pathway is consistent with findings that indicate a role of RhoA in platelet aggregation under high-shear conditions and in the irreversible aggregation of platelets in suspension.^128,129

Although the precise signaling mechanisms linking GPCRs to integrin _IIbß₃–mediated platelet aggregation are only partially understood, it has become clear in recent years that the rapid platelet aggregation with high efficiency requires the activation of at least 3 signaling pathways mediated by the heterotrimeric G proteins G_q, G₁₃, and G_i. In the absence of 1 of the 3 pathways, activation of _IIbß₃ still occurs, although with lower efficacy.

	Platelet Secretion and Procoagulant Activity

Top Abstract Introduction Platelet Activators Working... Adenosine Diphosphate Thrombin Thromboxane A2 Other Platelet Stimuli Inhibitors of Platelets Acting... G Protein-Mediated Signaling... Platelet-Shape Change Platelet Aggregation Platelet Secretion and... Implications for Future... Conclusions References

 
Secretion from platelets is an important mechanism that amplifies platelet activation and results in the release of mediators that act on the vessel wall as well as on other blood cells. It occurs in 2 waves, the first consists of the release of dense core granules and -granules followed by the release of lysosomes. Dense granules contain small molecules such as nucleotides (ADP, ATP) or serotonin, whereas granules contain various proteins including growth factors, chemokines, adhesive molecules, and coagulation factors. There is good evidence that platelet secretion functions analogous to other systems of regulated secretion, and platelets have been shown to contain the necessary components of the secretory machinery.^130,131 Although a detailed model of the mechanisms linking platelet surface receptors to the activation of platelet secretion is still missing, some of the upstream regulatory processes have been elucidated. It is well established that an increase in the free cytosolic [Ca²⁺] and an activation of PKC are required for platelet activation.^76,132 Platelet secretion can be inhibited by various inhibitors of PKC isoforms.^133–136 However, the identity of the PKC isoforms involved in stimulus-induced platelet secretion is not clear. PKC has been suggested to be involved.¹³⁷ Several targets of Ca²⁺ and PKC have been suggested including Munc-18, MARCKS proteins, or phosphatidylinositol-5-phosphate 4-kinase.¹³¹ Although elevation of cytosolic [Ca²⁺] and activation of PKC are able to induce some platelet degranulation, full secretory responses of platelets to various stimuli require the synergistic activation of Ca²⁺ and PKC-mediated processes.^{136,138–140} The central role of the G_q/PLC-ß pathway in agonist-induced platelet granule secretion is supported by the finding that various platelet activators failed to induce secretion in platelets lacking G_q⁶⁸ and by the fact that secretion in response to TxA₂ and low concentrations of thrombin are reduced in PLCß–deficient platelets.¹¹⁶

Platelet-shape change does not appear to be required for platelet aggregation.¹⁴¹ Platelet-shape change, however, precedes platelet secretion, and contractile forces generated during platelet-shape change lead to the centralization of platelet granules. Centralization of platelet granules is believed to be required for fusion of granules with each other as well as with the open canalicular system and the plasma membrane. This implies that the cytoskeletal reorganization during platelet-shape change contributes to the secretion of platelet granules. Consistent with this, signaling pathways that are involved in the induction of platelet-shape change such as the Ca²⁺-dependent and the RhoA/Rho-kinase–mediated stimulation of MLC phosphorylation have been shown to be involved in platelet secretion,^142,143 and platelet secretion in response to thrombin and TxA₂ is severely impaired in platelets lacking G₁₃.⁸⁰

It is well established that the activation of platelets and the activation of the coagulation cascade are complementary processes that influence each other. Platelet secretion contributes to the procoagulant activity of activated platelets by providing additional coagulation factors including factor V, factor VIII, or fibrinogen.¹⁴⁴ In addition, strong platelet activation results in the exposure of phosphatidylserine at the outer surface of the plasma membrane as well as in the formation of membrane blebs and microvesicles. The exposure of phosphatidylserine supports the formation of thrombin by facilitating the assembly of the prothrombinase and tenase complexes on the surface of activated platelets.^31,145 The shedding of membrane blebs into the circulation is suggested to provide procoagulant microvesicles. There is good evidence that the procoagulant activity of platelets is induced by very strong and prolonged increases in intracellular [Ca²⁺]. The most important stimulus appears to be fibrillar collagen, which acts through primarily G protein–independent processes. The transient [Ca²⁺] increases induced via G_q-coupled receptors are obviously only sufficient to induce a small degree of phosphatidylserine exposure and procoagulant activity.³¹ Nevertheless, the autocrine activation of platelets by ADP and subsequent activation of P2Y₁ and P2Y₁₂ receptors contributes to the activation of platelet-procoagulant activity in vitro and in vivo.^28,146,147

	Implications for Future Development of Antiplatelet Agents

Top Abstract Introduction Platelet Activators Working... Adenosine Diphosphate Thrombin Thromboxane A2 Other Platelet Stimuli Inhibitors of Platelets Acting... G Protein-Mediated Signaling... Platelet-Shape Change Platelet Aggregation Platelet Secretion and... Implications for Future... Conclusions References

 
Platelet adhesion at sites of atherosclerotic plaque rupture and subsequent formation of platelet aggregates is a central mechanism underlying the formation of arterial thrombi, resulting in acute coronary syndromes, stroke, and peripheral artery disease. In addition, platelet activation appears to be already involved in earlier stages of atherosclerosis development.^60,148 Platelets are therefore prime targets for therapeutic approaches to treat or prevent cardiovascular diseases.^30,149 Although considerable progress has been made in the development of antiplatelet agents as well as in the clinical evaluation of their effects, available antiplatelet therapies are still not optimal. This topic will be covered further in a forthcoming review in this series.

The currently most widely used antiplatelet drugs, aspirin, and clopidogrel have been shown to prevent cardiovascular diseases and are relatively well tolerated by treated patients. However, despite the proven beneficial effects, both drugs can reduce the risk for serious vascular events such as myocardial infarction or stroke in high-risk patients only by approximately one-quarter.^150,151 Thus, the efficacy of aspirin and clopidogrel is limited. This is probably attributable to the fact that both drugs inhibit the action of only 1 of several positive-feedback mediators acting via GPCRs. A third class of antiplatelet drug, _IIbß₃ (GPIIb/IIIa) antagonists, block the final convergence point of platelet activation. Blockers of _IIbß₃ have been shown in clinical trials to be very efficacious when used in patients undergoing coronary interventions. However, the increased efficacy is accompanied by a considerable reduction in safety, which restricts the use of _IIbß₃ antagonists to the treatment of acute clinical conditions or to the treatment of patients undergoing coronary interventions.^152,153 Thus, especially for the prevention of cardiovascular diseases, there is still a clinical need for antiplatelet drugs with higher antithrombotic efficacy than aspirin and clopidogrel but with safety profiles that allow for a preventive long-term administration.

Because G_q, G₁₃, and G_i integrate the effects of various platelet stimuli acting through their GPCRs, interference with signaling pathways downstream of GPCRs appears to be a promising strategy to develop antiplatelet agents with higher efficacy than those that block only individual platelet stimuli or their individual receptors. Because all 3 G protein–mediated signaling pathways show some level of redundancy, inhibition of 1 of the pathways should still be safer than blocking the common end point of platelet activation. Based on the fact that platelets lack G₁₁, which can compensate G_q deficiency in almost all tissues except platelets and the nervous system,^68,154 G_q has been suggested as a target for antiplatelet agents.⁶⁸ To circumvent potential side effects in the central nervous system, a G_q inhibitor should not be able to pass the blood/brain barrier. It has, however, turned out to be extremely difficult to selectively target the function of G_q because of its close structural similarity to G₁₁.¹⁵⁵ However, a G_q-specific approach would be required, because inhibition of both G_q and G₁₁ is likely to have deleterious effects in multiple organs.^156–158 Alternatively, signaling pathways downstream of G_q, G₁₃, or G_i such as the Rho/Rho-kinase or the PI3K-mediated signaling pathways represent attractive targets for antiplatelet drugs.^159,160 Because most components of G protein–mediated downstream signaling pathways are present in a variety of cells, potential inhibitors should act in a somewhat platelet-selective manner to reduce the risk of side effects in other cells and tissues. Depending on their pharmacokinetic and pharmacodynamic properties, drugs acting on widely expressed protein targets can have remarkable platelet selectivity, as demonstrated by the well-established antiplatelet activity of low-dose aspirin.¹⁶¹

	Conclusions

Top Abstract Introduction Platelet Activators Working... Adenosine Diphosphate Thrombin Thromboxane A2 Other Platelet Stimuli Inhibitors of Platelets Acting... G Protein-Mediated Signaling... Platelet-Shape Change Platelet Aggregation Platelet Secretion and... Implications for Future... Conclusions References

 
After the initial interaction of platelets with the altered vessel wall and their adhesion, the subsequent activation and formation of a platelet thrombus require the action of various diffusible mediators that act through GPCRs. In recent years, the major GPCRs involved in platelet activation under physiological and pathological conditions have been identified, and it has become clear that platelet activation requires the parallel signaling through several heterotrimeric G proteins. Although, in the absence of G_q-, G_i-, or G₁₃-mediated signaling, some platelet activation can occur, efficient activation of platelets in vitro and in vivo requires all 3 G protein–mediated signaling pathways. Mouse lines lacking individual G-protein subunits or other downstream signaling components of these pathways have provided the first models not only to evaluate the physiological role of individual signaling pathways but also to make predictions regarding their potential role as targets for new antiplatelet agents.

	Acknowledgments

 
The secretarial assistance of Rose LeFaucheur is gratefully acknowledged.

Sources of Funding

Work by the author has been supported by grants from the Deutsche Forschungsgemeinschaft.

Disclosures

None.

	Footnotes

 
Original received August 14, 2006; revision received October 2, 2006; accepted October 20, 2006.