Platelets are derived from larger progenitor cells called megakaryocytes that undergo fragmentation at their final stage of development. Hematopoietic stem cells (HSCs), which reside adjacent to the endosteal bone surface, generate progenitor cells that migrate to the vessels in the marrow cavity. A single daughter cell exits the bone after each division to proliferate and develop into numerous potential lineages, one of which is the megakaryocyte. The mechanical extension of proplatelet extensions into the sinusoidal blood vessels of the bone marrow is preceded by an elaborate intracellular program of nuclear amplification and protein production in maturing megakaryocytes. The released proplatelets continue to mature in the vasculature, eventually releasing individual platelets from the tips of the platelets [1,2]. Platelets are essential for forming clots by the formation of a thrombus and help in hemostasis. Platelets circulate in the bloodstream in a dormant discoid state but can be activated when they are exposed to damaged blood vessel walls [3].

Although platelets circulate in the blood in a quiescent state, when activated, they undergo major structural changes mediated by actin and myosin within the cytoplasm. The platelets are transformed from a discoid form to a compact sphere with dendritic extensions that allow the platelets to adhere to each other. These dendritic extensions are formed from filopodia which generate sheet-like lamellipodia that strengthen platelet’s contact with the area of injury [4]. This process of formation of filopodia and lamellipodia causes shape changes in platelets and aids in the adhesion of platelet to platelet for thrombus formation. Adhesion and aggregation of platelets are facilitated through a variety of membrane-bound receptors [5]. Platelets are not nucleated, but they do contain RNA, ribosomes, mitochondria, and other granules that are essential in enacting platelet function [6]. Platelet secretory organelles are classified into three types: α-granules, dense granules, and lysosomes. These granules include a variety of platelet-specific and non-specific proteins that aid in platelet activity [7-10].

As described above, platelets have a crucial role in thrombus formation and hemostasis. Following vascular injury, substances in the exposed extracellular matrix, including collagen and von Willebrand factor, can bind to the surface receptors of platelets causing the adhesion of platelets [3]. This adherence of platelets to the injury site leads to the activation of platelets. Upon activation, the platelets undergo a conformational change and begin secreting granular contents that promote aggregation and platelet clumping [4].

The signaling mechanism of platelet activation is complex and is mediated mainly via GPCRs and non-GPCRs. GPCRs are seven-transmembrane receptors and are the most numerous protein family encoded by the human genome that govern practically all physiological functions through G-protein signaling in various cells including platelets [11-15]. Non-GPCRs are essential receptors that activate platelets via the Glycoprotein VI (GPVI) receptors. While G protein-mediated signaling plays a minimal role in the initial adhesion of platelets to the vessel wall, mediators acting through G protein-coupled receptors are necessary for the subsequent recruitment of additional platelets into a growing platelet thrombus. In this review, we summarized the recent progress in understanding how platelet function is regulated by GPCR signaling and what are the molecular mechanisms underlying GPCR-mediated platelet activation.

GPCRs are seven-transmembrane receptors located on the cell membrane and are responsible for translating extracellular signals into important physiological effects [16]. Endogenous ligands of GPCRs include hormones, neurotransmitters, chemokines, and so on ranging from photons, amines, carbohydrates, lipids, and peptides to proteins [17].

GPCR-mediated platelet activation involves three primary G-protein-mediated pathways: G_q, G_12/13, and G_i. Heterotrimeric G-proteins are known to exist in their inactive state as stable complexes of G_α subunits and G_βγ dimers. G subunits cycle from a guanosine triphosphate (GDP)-bound inactive state to a GTP-bound active state, with guanosine diphosphate (GTP) hydrolysis limiting the active state’s lifespan. In vitro biochemical investigations have revealed that active G-protein heterotrimers break down into G_α-GTP and G_β/γ subunits [18]. The most soluble agonists produced by active cells, such as ADP, thromboxane A₂ (TxA₂), and thrombin activate platelets via GPCRs in the coagulation cascade [19-22]. Similarly, epinephrine and serotonin are involved in activating platelets through the GPCR pathway [23].

At the site of vascular injury, ADP is released from the cells that act as an autocrine and paracrine stimulation for the recruitment of extra platelets and stabilize the hemostatic plug [11]. In platelet, ADP is stored in the dense granules and is released upon platelet activation. Two different GPCRs for ADP designated P2Y₁ and P2Y₁₂, are expressed in both human and mouse platelets (Fig. 1) [24,25]. P2Y₁ and P2Y₁₂ receptors couple to G_q and G_i family members of G-protein, respectively [26-29]. For complete platelet activation, co-activation of P2Y₁ and P2Y₁₂ receptors is essential [30-33]. ADP-induced platelet aggregation is impaired and there is no shape change in mice lacking P2Y₁ receptors [34,35]. Additionally, mice lacking P2Y₁ have a slightly prolonged bleeding time and are also relatively resistant to ADP-induced thromboembolism [36,37]. Interestingly, mice platelet deficient in P2Y₁₂ have a normal shape change but impaired platelet aggregation in response to ADP [38,39]. In humans, deficiency of P2Y₁₂ has shown moderate hemorrhagia, however, mice with P2Y₁₂ deficiency do not have this trait [34,35,40,41]. Interestingly, platelet responses to low and intermediate concentrations of TxA₂ and thrombin were reduced in the absence of ADP, highlighting the importance of ADP as a positive feedback mediator required for sustained platelet activation [36-38,42]. Thienopyridines such as clopidogrel, currently used in the secondary prevention of cardiovascular events, irreversibly inhibit the P2Y₁₂ receptor [43,44].

Thrombin being one of the most potent activators of platelets, is the major effector protease of the coagulation system. After the disruption of the vascular endothelium, tissue factor is exposed to plasma coagulation factors, which causes thrombin production. Thrombin is formed on cellular surfaces, particularly those of activated platelets [45]. An important mechanism by which activated platelets stimulate coagulation is the local production of thrombin on the platelet surface. Thrombin induces platelet activation through the protease-activated receptor (PAR) family member of GPCRs (Fig. 1). Among the four members of the PAR family, three (PAR1, PAR3, and PAR4) are activated by thrombin. In human platelet, PAR1 and PAR4, and in mouse platelet PAR3 and PAR4 are expressed [46-48]. Synthetic peptides based on the sequence of the PAR1- and PAR4- tethered ligand domains can activate the receptors, imitating at least part of the activities of thrombin. PAR3 receptor in human platelet signal in response to the thrombin in transfected cells while in murine platelets it is mainly involved in facilitating PAR4 receptor cleavage rather than in signaling by its own [49]. Platelets can be activated by thrombin at concentrations as low as 0.1 nM. While various platelet agonists can trigger hydrolysis of phosphoinositide, none appear to couple to phospholipase C (PLC), an important enzyme that hydrolyzes the membrane-bound phosphatidylinositol 4,5-bisphosphate (PIP2) to produce secondary messenger as efficiently as thrombin [50]. PARs that couple to the G_q and G₁₂/G₁₃ family of heterotrimeric G proteins which are directly involved in the thrombin-induced platelet activation (Fig. 1) [51-53]. PAR1 antagonists or antibodies that prevent PAR1 or PAR4 activation have been used in studies to show that PAR1 causes human platelet activation at low thrombin concentrations, but PAR4 only contributes to thrombin-induced platelet activation at high thrombin concentrations [46,54-56]. It is assumed that the presence of PAR1 and PAR4 in human platelets is analogous to the PAR3 and PAR4 in murine platelets which is indicated by the study using PAR3-deficient mice which did not respond to any low and intermediate concentrations of thrombin but could be activated by high concentrations via PAR4 [46]. The greater potency of thrombin to activate PAR1 is most likely due to the presence of a hirudin-like sequence adjacent to the C-terminal thrombin cleavage site, which enhances thrombin binding and is missing in PAR4 [46,57]. However, experiments using heterologously produced receptors indicated that unless PAR4 was present, mouse PAR3, which has a hirudin-like region near the thrombin cleavage site, did not mediate thrombin-induced transmembrane signaling. This shows that at low thrombin concentrations, mouse PAR3 acts as a co-receptor for mouse PAR4 and enhances cleavage and activation of PAR4 [48]. Similarly, PAR4 knockout mice’s platelets are unresponsive to thrombin and are protected from thrombosis [58,59]. Thus, thrombin-induced platelet activation in mice is completely dependent on PAR4-mediated signaling and requires only PAR3 to facilitate PAR4 cleavage at low thrombin concentrations, whereas thrombin activates human platelets by cleaving and activating PAR1 and PAR4 at low and high concentrations, respectively. It is not clear that in mouse PAR3 and PAR4 forms stable heterodimers in which the former promotes the activation and the latter helps in signaling or whether thrombin binding to PAR3 only aids PAR4 cleavage [11].

TxA₂ release, similar to ADP secretion, enhances the initial stimulus for the activation of platelets and aids in the recruitment of more platelets. The short half-life of TxA₂ in solution is the limiting factor of this process which helps to restrict the spread of platelet activation to the initial site of injury. The activities of the TxA₂ are restricted to the local area only because of its half-life. TxA₂ is synthesized from arachidonic acid where cyclooxygenase-1 enzyme is used which targets the low-dose aspirin and thromboxane synthase. Thromboxane exerts its effect via the TxA₂ receptor (TP). This receptor is also known to activate through prostaglandin endoperoxides PGG₂ and PGH₂. TP receptor couples to the G_q and G₁₂/G₁₃ family protein of GPCR and exerts downstream signaling in platelet [52,60,61]. Studies using platelets from TP-deficient mice suggested that platelets do not respond to TxA₂, which indicate the role of TP as a TxA₂ receptor on platelets [62]. Additionally, TP-knockout mice show increased bleeding time and are not able to form stable thrombus [62]. It has been suggested that reduced activation of TP-deficient platelets may contribute to a reduction in injury-induced vascular proliferation and atherosclerotic progression [63,64].

Epinephrine, also called adrenaline is both a hormone and a neurotransmitter. In human platelets, epinephrine is a weak activator, compared to thrombin. However, in the case of humans, mild bleeding disorder is related to reduced epinephrine-induced aggregation. Epinephrine has the ability to enhance the effects of other agonists in both murine and human platelets. Epinephrine induces platelet aggregation mediated through α_2A-adrenergic receptors [23,65,66]. It is suggested that epinephrine induces platelet activation through its receptor coupled to G_i family members of GPCR (Fig. 1). Epinephrine couples to G_z which belongs to the G_i class of G-protein [67]. Knockout studies show that loss of G_zα expression abolishes adrenergic responses in mouse platelets, whereas loss of G_i does not. The study revealed that epinephrine responses in mouse platelets are nullified when G_zα expression is abolished, whereas G_i-knockout has no effect. The ability of adrenaline to activate Rap1b also appears to be mediated by G_z [68,69].

Platelets take up serotonin and are stored in dense granules. They are only released after activation of the platelet. The concentration of serotonin is higher in dense granules, however, platelets are unable to produce serotonin due to the lack of an enzyme (5-hydroxytryptophan decarboxylase) to cause decarboxylation [70,71]. Serotonin is able to induce its responses through the platelet surface 5-HT_2A receptor which is a member of GPCR (Fig. 1) [72-75]. Serotonin binding to 5-HT_2A initiates typical Gq-linked signal transduction events via the PLC pathway, promoting the production of inositol triphosphate (IP₃) and diacylglycerol (DAG), culminating in calcium mobilization [76-78].

Interestingly, it has been found that serotonin alone only induces shape change while epinephrine alone induces neither shape change nor aggregation in platelets [79]. It has been known that the co-stimulation of epinephrine and serotonin induces platelet aggregation by bypassing the P2Y₁ and P2Y₁₂ receptors and activates G_q-coupled 5HT_2A and G_z coupled α_2A adrenergic receptors respectively [80].

ADP, TxA₂, and thrombin are the principal diffusible platelet stimuli that attract platelets into a developing thrombus by activating numerous G protein-mediated signaling pathways that produce platelet-shape change, degranulation, and integrin αIIβ3-mediated aggregation. For the complete activation of platelets, agonists use different mechanisms mediated via G-protein. As described above, ADP induces platelet activation binding to P2Y₁ and P2Y₁₂ receptors and activates G_q and G_i respectively. Similarly, thromboxane binds to the TP receptor and thrombin binds to protease-activated receptors PAR1/PAR4 or PAR3/PAR4 which activates G₁₂/G₁₃ and G_q [24,25,46,47]. All these mediators increase the formation and release of ADP, TxA₂, and thrombin which ultimately amplifies the G-protein-mediated signaling pathway. This positive feedback mechanism is limiting the analysis of the role of individual G-protein-mediated signaling pathways involved in platelet activation. Studies with α subunits knockout platelets to analyze the G-protein mediated signaling pathway independent of agonist use and their receptors have shown that the G proteins G_q, G_12/13, and G_i2 (Table 1) play a crucial role in platelet activation [11].

Upon agonist stimulation, PLCβ is produced by the G_q family of GPCR. This family protein is essential for the granular secretion of platelets and integrin activation which results in platelet aggregation [81]. The downstream signaling of the G_q initiates with PLCβ activation which in turn leads to the formation of IP₃ and DAG, which increase cytosolic free calcium [Ca²⁺] and activate protein kinase C (PKC) [81,82]. In addition, there is also involvement of βγ-complex which interacts with phosphatidylinositol 3-kinase (PI3K), a downstream effector to induce platelet activation (Fig. 1) [83]. Although the majority of the mammalian cells express both G_q and G₁₁, interestingly platelet is only the exception which contains only G_q [81,82]. To date there is no physiological significance has been reported for the absence of G₁₁ in blood platelets. On platelet activation with several agonists including ADP, thrombin, and TxA₂ analog U46619 in G_q deficient mice, there is a decrease in granular secretion and aggregation [81]. Furthermore, in ADP-triggered shape change in platelets, G_q plays a crucial role most likely by stimulating calcium/calmodulin-dependent and/or RhoA-dependent signaling mechanism [84]. Platelet activation requires an increase in intracellular calcium. Increased calcium has a variety of consequences, including platelet shape alteration and activation of small GTPases, which increase platelet granule exocytosis [85,86]. It is worth noting that, in comparison to the degree of calcium mobilization elicited by ADP or thrombin activation, serotonin through the 5-HT_2A results in a lesser calcium influx [26,30]. This is in line with the research that describes serotonin as a helper agonist that stimulates platelet aggregation along with others and cannot induce sufficient platelet aggregation on its own but can potentiate the aggregation combined with other stimuli [26,87].

The G₁₂/G₁₃ protein family is abundantly expressed in platelets which show the Rho/Rho-kinase–mediated signaling pathway in platelet activation [52]. G_12/13 is bound to GTP which interacts and activates guanine nucleotide exchange factors (GEFs) for the p115Rho-GEF protein. p115RhoGEF protein helps to convert GDP-bound RhoA to GTP-bound RhoA [88-90]. Similarly, Rho kinases are phosphorylated after activation by RhoA and inhibit myosin light chain phosphatase [53]. As a result, phosphorylation of the myosin light chain as well as contraction via myosin light chain are enhanced. Therefore, G_12/13 promotes shape change in platelets along with granular secretion [53]. However, platelet aggregation in response to low doses of thrombin and TxA₂ analog U46619 in G₁₃-knockout platelets is reduced whereas granular secretion is reduced in response to U46619 but not when induced by thrombin [91]. G₁₃-knockout platelets show a reduction in the shape change when induced by the agonists. Interestingly, G_12/13 plays a critical role in integrin outside-in signaling by binding to the cytoplasmic domain of integrin [92].

In the G_i family protein, G_i2 is abundantly expressed in platelets, upon agonist couple to the receptor, the G_i2-α subunit inhibits the adenylyl cyclase enzyme and terminates the further downstream signaling [93]. Furthermore, G_i-βγ complexes, which are generated upon G-protein activation and have the ability to control a range of channels or enzymes such as PI3Ks and adenylyl cyclases [93]. The enzyme PI3Ks triggers a number of downstream effectors such as AKT/protein kinase B (PKB), serine/threonine kinase, ERK, Rap1b, and Src family kinases (Fig. 1). In platelet, ADP-activated receptor P2Y₁₂-coupled to Gi protein plays a key role in PI3K activation, specifically, βγ-subunit activated PI3K, which blocks RASA3 and causes persistently active Rap1b [94-96]. Activation of P2Y₁₂ results in prolonged active Rap1b because it inhibits adenylate cyclase, which lowers cAMP, and activates PI3K, which inhibits RASA3 [97].

Orphan GPCRs are receptors that lack endogenous ligands [98]. Currently, there are more than 140 GPCRs that have unidentified endogenous ligands which create doubt in the actual function of discovered GPCRs. These orphan GPCRs can have great potential for therapeutic targets [99].

Recently, GPR31 has been known to show the signaling through the Gi protein family which ultimately enhances the PAR-4 mediated platelet activation in human and mouse. Additionally, it has been known that the involvement of GPR31 also results in Rap1 and p38 activation and little calcium flux, however, it fails to induce platelet aggregation [100]. Similarly, GPR56 is also known to cause activation of G₁₃ signaling leading to platelet shape change. Additionally, in GPR56 knockout mice it has been found that there is prolonged bleeding and impaired platelet plug formation [101]. GPR55, another orphan receptor, is also known to induce platelet aggregation in humans but the mechanism is still unknown [102-104]. Further research is essential to fully understand how these orphan receptors affect platelet signaling and whether the knowledge can be translated from bench to clinics.

In recent years, it has been shown that platelets are not only activated but also desensitized through GPCRs. Several mechanisms prevent the hyperactivation of GPCR signaling in various cells, among which G Protein-Coupled Receptor Kinases (GRKs) and arrestins play an important role and these are therefore gaining popularity among researchers [105,106]. When GPCR is activated by ligand binding, G protein coupling, and signal transduction occur, GRK enters the picture and phosphorylates the active GPCR’s cytoplasmic serine and threonine residues, opening the door for arrestin recruitment. Desensitization is the process by which the recruited arrestin attaches to the phosphorylated GPCR, prevents further G-protein coupling, and stops the downstream signaling [107-110]. There are 7 isoforms for GRKs among which GRK2, GRK3 GRK5, and GRK6, and 4 isoforms of β-arrestin among which β-arrestin 1 and β-arrestin 2 are known to present abundantly in platelet [79,111-116]. There is a dearth of knowledge on the regulation and mechanisms of GPCR signaling and GPCR desensitization by GRKs in platelets, despite the crucial roles played by these cells in GPCR activities. We and others have just lately shown the intriguing aspects of the arrestin and GRK system in platelet GPCR signaling. We demonstrated using GRK6 knockout mouse platelets that GPCR agonists, such as 2-MeSADP, U46619 (TxA₂ analog), AYPGKF, and thrombin, dramatically increase platelet aggregation and dense granule production in comparison to wildtype platelets [79]. GRK6-deficient platelets do not, however, exhibit GPVI agonist collagen-related peptide (CRP)-induced platelet aggregation or dense granule secretion. This suggests that GRK6 regulates P2Y₁, P2Y₁₂, TPα, and PARs-mediated signaling in platelets, but not non-GPCR-mediated platelet activation. Recent research revealed that GRK6 contributes to ADP-induced P2Y₁₂ receptor desensitization in platelets, but not P2Y₁ receptor desensitization [115]. While co-stimulation of serotonin and epinephrine mimics the ADP-induced P2Y₁ and P2Y₁₂ receptor-mediated platelet activation pathway, it is noteworthy that GRK6 is not involved in serotonin-induced G_q-coupled 5-HT_2A receptor and epinephrine-induced G_z-coupled α_2A adrenergic receptor-mediated platelet activation, in contrast to ADP-induced aggregation. This suggests that the kinase activity of GRKs varies with different ligands, receptors, and proteins. According to reports, GRK5 is involved in the desensitization of GPCR receptors and controls thrombin signaling in platelets via the PAR-1 receptor [117,118]. Similarly, we recently assessed the functional significance of arrestin isoforms and their molecular basis of modulation of GPCR desensitization in platelets using β-arrestin1 and β-arrestin2 -/- mice. Our findings demonstrated that β-arrestin2 -/- but not β-arrestin1 -/- potentiates the aggregation and secretion of platelets triggered by agonists, including thrombin, ADP, AYPGKF, and U46619. In platelets lacking β-arrestin2, co-stimulation of serotonin and adrenaline enhanced platelet aggregation, indicating that β-arrestin2 is crucial for controlling overall GPCR signaling. Furthermore, we demonstrated that β-arrestin2 controls the desensitization of ADP and PAR4 receptors, which in turn controls the G_q- and G_i-mediated signaling in platelets. β-arrestin2 was additionally involved in thrombus formation in vivo. It is yet unclear, nevertheless, whether or not platelet GPCRs are phosphorylated by GRK isoforms other than GRK6 and GRK5, and whether or not this phosphorylation alters the receptor’s function. Furthermore, whereas arrestins play a crucial role in GPCR-mediated signaling, the process by which arrestins desensitize GPCRs in platelets remains unclear. We can find a new way to develop drugs in the field of vascular pathobiology by evaluating the functional significance of individual GRK and β-arrestin isoforms in platelets and characterizing the novel mechanisms involved in GPCR desensitization and trafficking in platelets using knockout mice for each isoform.

GPCRs are the largest and most diverse class of signaling proteins that affect an incredible array of physiological processes throughout the human body. Platelets operate mainly through the GPCR-linked G-protein signaling mechanisms after the stimulation with different agonists. Based on the diverse platelet physical, biochemical, and functional variability, the dynamic nature of platelet properties in health and disease varies. Nevertheless, there are significant gaps in our understanding of platelet function in these other disorders that prohibit us from reaching a thorough mechanistic understanding. Studying platelets and platelet GPCRs can provide insights into identifying several GPCR-targeted therapies in platelet-associated diseases in the future.