Canadian Journal of Physiology and Pharmacology

Biased signalling in platelet G-‐coupled receptors.

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2020-0149.R2

Manuscript Type: Review

Date Submitted by the 03-Aug-2020 Author:

Complete List of Authors: Thibeault, Pierre; University of Western Ontario Schulich School of Medicine and Dentistry, Physiology and Pharmacology Ramachrandran, Rithwik; University of Western Ontario Schulich School of Medicine and Dentistry, Physiology and Pharmacology

Is the invited manuscript for consideration in a Special Not applicableDraft (regular submission) Issue:

Keyword: Platelet, GPCR, ADP, Thrombin, Prostanoid

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Biased signalling in platelet G protein‐coupled receptors.

Pierre E. Thibeault and Rithwik Ramachandran1 Department of Physiology and Pharmacology, University of Western Ontario, 1151 Richmond Street, London, ON, N6A5C1, Canada.

1To whom correspondence should be addressed: Rithwik Ramachandran, [email protected], Department of Physiology and Pharmacology, University of Western Ontario. London, ON, N6A 5C1. Tel: 519-661-2142

Draft

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Abstract Platelets are small megakaryocyte-derived, anucleate, disk-like structures that play an outsized role in human health and disease. Both a decrease in the number of platelets, as well as a variety of platelet function disorders, result in petechiae or bleeding which can be life threatening. Conversely, the inappropriate activation of platelets, within diseased blood vessels, remains the leading cause of death and morbidity through affecting heart attacks and stroke. The fine balance of the platelet state in healthy individuals is controlled by a number of receptor-mediated signalling pathways that allow the platelet to rapidly respond and maintain haemostasis. G-protein-coupled receptors (GPCRs) are particularly important regulators of platelet function. Here we focus on the major platelet-expressed GPCRs and discuss the roles of downstream signalling pathways (e.g. different G-protein subtypes or -arrestin) in regulating the different phases of the platelet activation. Further, we consider the potential for selectively targeting signalling pathways that may contribute to platelet responses in disease through development of biased agonists. Such selective targeting of GPCR-mediated signalling Draftpathways by drugs, often referred to as biased signalling, holds promise in delivering therapeutic interventions that do not present significant side-effects, especially in finely balanced physiological systems, such as platelet activation in haemostasis.

Keyword:

Platelet , GPCR , ADP , Thrombin , Prostanoid

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Platelet biogenesis and elimination Platelets were first described in the scientific literature in the mid to late 1800’s by a number of scientists, including William Osler and George Hayem, who recognized them as small anucleate disk-like structures in blood (Cooper, 2005). At this time, platelets were believed to be fragments of disintegrated leukocytes, clots of fibrin, or precursors to red blood cells. Giulio Bizzozero is widely credited with identifying platelets as a distinct morphological element in blood and for conducting the definitive studies that uncovered the critical role of platelets in haemostasis (Ribatti & Crivellato, 2007). Bizzozero, also credited with first describing bone marrow megakaryocytes, however failed to recognize them as the source of platelets, a finding attributed to James Wright who noted similarities between granule contents in megakaryocytes and platelets (Kaushansky, 2008). The production of platelets (thrombopoiesis) occurs primarily in the bone marrow from megakaryocytes. Megakaryocytes, formed by the differentiation of hematopoietic stem cells, home to the perivascular niche where they interact with sinusoidal bone marrow endothelial cells and form dynamic transendothelialDraft pseudopods which project into the lumen of bone marrow sinusoids. These extensions into the sinusoids elongate and are described as pro- platelets, which remain tethered to the megakaryocytes by thin cytoplasmic bridges. The release of the platelet into the blood occurs from the tip of the pro-platelets and represents the final step in the process of platelet biogenesis (Machlus & Italiano, 2013; Noetzli Leila J. et al., 2019). Platelets have a short lifespan and are eliminated within days (4-6 days in mice; 5-10 days in humans) (Leeksma & Cohen, 1955; Robinson et al., 2000) via seclusion in the spleen and other organs, or through deposition at sites of vascular injury. Even in the absence of such consumption, aged-platelets undergo apoptosis and are removed from circulation. A shift in the balance of the pro-apoptotic (BAX-BAK) and anti-apoptotic (BCL2) is believed to play an important role in this spontaneous apoptosis of ageing platelets (Mason et al., 2007). Thus, constant production and elimination of platelets serves to maintain normal platelet count in a healthy individual at around 150,000-300,000 cells per microliter.

The delicate balance of platelets in haemostasis and thrombosis Throughout their short lifespan, platelets must perform a delicate balancing act. They must remain poised for activation in response to a disruption of vascular integrity yet remain in their inactive state within intact vessels. Platelets are aided in performing this delicate act by the

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endothelial cells lining the blood vessels. The intact endothelial monolayer lining the inner surface of blood vessels acts to actively suppress platelet activation. Healthy vascular endothelia do so by physically separating platelets from the underlying platelet-activating matrix components, such as collagen, and by constantly producing and secreting factors that prevent platelet activation (e.g. thrombomodulin, ectonucleotides, prostaglandin I2 (PGI2), nitric oxide (NO)). In the event of vascular injury, platelet activation is rapid and is initially mediated by platelet glycoprotein (GP) Ib-V-IX interaction with von Willebrand factor (vWF) in the subendothelial matrix. This initial interaction slows the circulating platelets and enables further interaction between platelet adhesion receptors/integrins and their counter receptors; and/or adhesive matrix components (e.g. GPVI-Collagen or IIb3-Fibronectin). At the cellular level, these signals also trigger a cytoskeletal rearrangement in the platelets and cause platelet spreading which increases their attachment to subendothelial surfaces at the sites of injury. The initial activation of platelets further involves signalling mediated by factors released from the damaged cells in an injured blood vessel (e.g. ADP)Draft and produced during coagulation (e.g. thrombin). The inflammatory response accompanying the injury may also produce factors (e.g. platelet-activating factor) that further activate platelets. Crucially, these signals from the injured vascular cells are amplified dramatically by the release of platelet granule contents including thromboxane A2

(TXA2), Adenosine diphosphate (ADP), and serotonin, which further amplify platelet activation and serve to recruit additional platelets to the site of a growing thrombus. The release of platelet granules involves cytoskeletal rearrangement, fusion of granules with the open-canalicular system, and release of granule contents (Flaumenhaft Robert, 2003; Selvadurai & Hamilton, 2018). Activated platelets also provide a surface on which additional thrombin is generated and fibrin can accumulate (Andersen et al., 1999). Many of the factors that contribute to platelet activation in response to injury are also engaged in the inappropriate activation of platelets, which occurs within blood vessels in disease. Endothelial dysfunction, resulting from diseases such as diabetes, leads to a loss of negative endothelial derived signals (PGI2 and NO) and is an important driver of platelet activation in disease (Deanfield et al., 2005; H. Ding & Triggle, 2010; Pober et al., 2009). Factors that are produced in vessel injury can also be generated in unhealthy blood vessels, as a result of inappropriate endothelial signalling. For example, (LPA), in atherosclerotic

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plaques, can trigger platelet activation that in turn causes the release of factors such as ADP and

TXA2 from platelets, which trigger inappropriate platelet aggregation and thrombosis. Many of these important mediators of platelet activation act on platelet cell surface G- protein coupled receptors (GPCRs) to mediate the various phases of platelet activation. In the following sections, we discuss the major receptor proximal signaling pathways engaged by GPCRs, provide a brief overview of the different GPCRs expressed in human platelets, and discuss whether selective targeting of signalling pathways downstream of these different platelet GPCRs might provide a route to safer and more efficacious anti-platelet agents.

GPCR signalling mechanisms GPCRs form a large family of cell surface receptors that respond to a diversity of activators (Lagerstrom & Schioth, 2008). Upon ligand binding, GPCRs trigger activation of intracellular second messenger signalling pathways by engaging one of four major class of guanine nucleotide binding proteins (G-proteins) – Gi/o/zDraft, Gs, Gq/11, and G12/13 (Cabrera-Vera et al., 2003; Wettschureck & Offermanns, 2005). In the inactive state, the heterotrimeric G-proteins consist of a guanosine diphosphate (GDP) bound  subunit that is associated with  subunits. Ligand bound GPCRs act as a guanine exchange factor (GEF) and trigger guanine nucleotide exchange at the G- protein  subunit (Gilman, 1987; Mahoney & Sunahara, 2016). The release of GDP and subsequent binding of GTP in G-protein  subunits leads to the release or conformational remodelling of the  subunits, and allows the G-protein  and  subunits to activate second messengers and propagate intracellular signals (Bunemann et al., 2003; Galés et al., 2006; Smrcka, 2008). G-protein signalling is terminated by the intrinsic GTPase activity in the G-protein  subunits which can be further accelerated by interactions with Regulators of G-protein Signalling (RGS) proteins (Berman & Gilman, 1998). The activated GPCR is rapidly phosphorylated by GPCR kinases (GRKs), often at serine and threonine residues on the receptor C-terminal tail, enabling binding with -arrestins (Gurevich & Gurevich, 2019). Initial docking of -arrestins on the phosphorylated GPCR C-terminal tail is followed by interactions with the intracellular core of the GPCR transmembrane bundle, which sterically blocks G interaction and terminates G- protein-mediated signalling (Cahill et al., 2017; Sente et al., 2018; Shukla et al., 2014). -arrestins also scaffold the activated receptors to cellular endocytic proteins and promote receptor

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internalization (Ferguson et al., 1996; Lefkowitz & Shenoy, 2005; Peterson & Luttrell, 2017). Depending on the strength of receptor interaction with -arrestins, receptors can either be recycled back to cell membrane (weak binders) or traffic to the lysosomes for degradation (strong binders) (Jean-Charles et al., 2017; Oakley et al., 2000; Thomsen et al., 2016; Tian et al., 2014). In addition to the classical role in terminating GPCR signalling, -arrestins are now recognized as mediators of G-protein-independent signalling from GPCRs by scaffolding other signalling mediators (Jean-Charles et al., 2017; Shenoy et al., 2006; van Gastel et al., 2018). For a number of different GPCRs it has been proposed that both G-protein-mediated and -arrestin mediated signalling lead to distinct cellular responses, some being beneficial and others being pathological (Dewire et al., 2007; Peterson & Luttrell, 2017). This extends the now well-accepted idea that a single receptor can and may couples to multiple G-protein subtypes, and further, adds -arrestins to the list of GPCR interacting signalling effectors. The ability of a single receptor to activate multiple signalling effectors has also provided the stimulus for seeking ligands that are able to stabilize different GPCR conformationsDraft – enabling the receptor to selectively engage different signalling pathways (Terry Kenakin, 2019; Rajagopal et al., 2011; Seyedabadi et al., 2019). This paradigm, variously termed as functional selectivity, biased agonism, or biased signalling, has opened up a novel strategy for therapeutically targeting GPCRs, that goes beyond simply blocking or activating receptor signalling.

Biased agonism in GPCRs Biased agonism describes the phenomenon where a ligand can preferentially stabilize a receptor conformation that favours interaction with one of many possible signalling effectors. It follows thus, that these ligands are able to preferentially-activate certain receptor-associated signalling pathways, but not others downstream of the same receptor, or simply activate certain pathways to different degrees of efficacy within a given system (Jarpe et al., 1998; T Kenakin, 1995; Kudlacek et al., 2002; Michel & Alewijnse, 2007; Rankovic et al., 2016). Across cell types, additional factors may influence signalling bias, including the expression levels of different signalling effectors and their dynamic regulation, for example by transcriptional regulation (Terry Kenakin & Christopoulos, 2013). Therapeutically, biased agonism opens the door to the exciting possibility of developing drugs that can selectively modulate a signalling pathway that is implicated in a disease setting, while effectively sparing (or promoting) the beneficial signalling

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events occurring downstream of receptor activation. Thus, development of biased ligands for therapeutic targeting of GPCRs is of particular interest (Terry Kenakin & Christopoulos, 2013; Rankovic et al., 2016). One of the best examples of therapeutic harnessing of biased signalling comes from efforts targeting the opioid receptors. The opioid receptors are an attractive, yet challenging, pharmaceutical target since the effective analgesic effects of receptor agonists are accompanied by undesired side-effects including constipation, respiratory depression, risk of dependency, and development of tolerance. The potential of biased agonists to improve targeting of opioid receptors was initially highlighted by studies in -arrestin-2-knockout mice where morphine showed improved analgesic properties (Bohn et al., 2000) as well as decreased respiratory and GI depression, compared to the wildtype mice (Bohn et al., 2000; Raehal et al., 2005). This led to the hypothesis that G-protein-biased OR agonists, that lack the ability to activate -arrestins, would provide an analgesic with little to no side effects. Several G-protein-biased OR ligands have been generated and are in various stages of clinicalDraft development (DeWire et al., 2013; Manglik et al., 2016). TRV130 (oliceridine) is the most advanced compound and has shown fewer adverse effects than morphine in phase II trials (Singla et al., 2019; Viscusi et al., 2016, 2019), however, some safety concerns (e.g. instances of QT prolongation) have led regulators to seek additional clinical safety trials prior to drug approval. Similar strategies have been employed to design and test G-protein-biased agonists of the κ- (KOR) and δ-opioid receptors (DOR). KOR is well-studied to be involved in spinally-mediated peripheral and visceral pain analgesia, however, agonism of this receptor for therapeutic purposes leads to adverse side-effects including sedation, dysphoria, and hallucinations (Ho et al., 2018; Madariaga-Mazón et al., 2017). G-protein-biased agonists of the KOR have been associated with anti-nociception and decreased in associated side effects of KOR activation (Brust et al., 2016; Morgenweck et al., 2015; White et al., 2015; Zhou et al., 2013). DOR is an attractive therapeutic target for treatment of chronic pain, depression, and anxiety (Conibear et al., 2020; Pradhan et al., 2011). In a model of chronic pain, a G-protein-biased DOR agonist (PN6047) showed analgesic and antidepressant activity, without the commonly associated tolerance and dependency profile (Conibear et al., 2020). Thus, biased agonism is an important area of ongoing study and has the potential to improve therapeutic benefit while abrogating adverse outcomes and improving the safety profile of ligands targeting the opioid receptors (Conibear & Kelly, 2019; Madariaga-Mazón

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et al., 2017; Piekielna-Ciesielska & Janecka, 2020) and also a number of other GPCRs, including peptide and aminergic GPCRs (McCorvy et al., 2018; Urban et al., 2007; Vidal et al., 2018).

Platelet GPCRs Platelets express many GPCRs that respond to soluble mediators produced at the site of injury or in diseases to initiate the platelet aggregation sequalae. As such, GPCR signalling in platelets is an attractive therapeutic target that may allow some level of platelet activation while concurrently preventing the amplification that occurs through autocrine and paracrine signalling. Indeed, GPCRs have proven to be highly tractable drug targets and a number of anti-platelet agents currently in clinical use target platelet GPCRs (Dowal & Flaumenhaft, 2011). In the following sections, we outline the major platelet GPCRs and describe the currently available literature on their engagement with different G-protein-mediated and G-protein-independent signalling pathways and their contribution to different aspects of platelet activation. Prostanoid receptors. Prostanoids,Draft a subfamily of eicosanoids, are formed from arachidonic acid by the action of prostaglandin G/H synthase [cyclooxygenase (COX) 1 and 2, respectively] to form the common prostaglandin precursors PGG2 and PGH2, which are then acted upon by additional synthases to form prostaglandins (e.g., PGD2, PGE2, PGF2a), thromboxane A2

(TXA2), and prostacyclin (PGI2) (Figure 1). These prostanoids mediate cellular signalling through a family of -like GPCRs that include IP1 (PGI2), TP (TXA2), EP1-4 (PGE2), FP (PGF2) and DP1-2 (PGD2) receptors (Narumiya et al., 1999; Woodward et al., 2011). The DP1, EP2, EP4 and IP1 receptors couple to Gs and mediate production of cyclic adenosine monophosphate

(cAMP) (Figure 1). EP1, FP, and TP subfamilies couple to Gq/11 to initiate phospholipase C activation, leading to phosphatidylinositol 4,5, bisphosphate conversion to inositol 1,4, 5-

2+ trisphosphate (IP3) and 1,2,-diacylglycerol (DAG), which results in Ca mobilization from the endoplasmic reticulum and protein kinase C (PKC) activation (Figure 1). The EP3 receptor couples largely to the pertussis toxin sensitive Gi pathway resulting in a decrease in cAMP levels (Figure 1). It must also be stressed that the prostanoid receptors often couple to more than one G-protein to activate second messenger signalling cascades - for example the Gq/11-coupled EP1, FP and TP receptor families also couple to G12/13 to activate Rho-associated kinases (Offermanns et al.,

1994) (See Table 1; Figure 1). Generally, in platelets the activation of Gs-coupled prostanoid

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receptors result in inhibition of platelets, while Gi-, Gq/11-, and G12/13-coupled receptor activation results in platelet activation and aggregation (Table 1, Figure 1).

Prostacyclin (PGI2) is the major product of cyclooxygenase (COX) and prostaglandin I synthase-catalyzed metabolism of arachidonic acid in macrovascular endothelium (Bunting et al.,

1976; Moncada et al., 1976) and is a key endothelium-derived inhibitor of platelet activation. PGI2

stimulates the Gs-coupled IP receptor to activate adenylyl cyclase and elevate levels of cAMP. Nitric Oxide (NO), the other negative regulator of platelets produced by endothelial cells, acts directly on guanylyl cyclase to elevate levels of cyclic guanosine monophosphate (cGMP) (Radomski et al., 1987). cAMP, in turn, activates protein kinase A (PKA) which can blunt multiple aspects of platelet function including Ca2+ mobilisation, integrin activation, secretion (at least in part through inhibition of RhoA signalling), and myosin light chain phosphorylation (Aburima et al., 2017; Aburima et al., 2013). Further evidence for IP receptor-mediated signalling as an inhibitor of platelets comes from knockout mouse studies. IP-knockout mice do not develop spontaneous thrombosis, however, are moreDraft susceptible to thrombotic stimuli than their wild-type littermates (Murata et al., 1997). Like PGI2, TXA2 is produced by the cyclooxygenase pathway

and the action of thromboxane synthase on PGH2. TXA2 acts on the TP receptor on platelets and

is a potent activator of platelets. Platelets from TP-deficient mice are unresponsive to TXA2 and exhibit a mild bleeding tendency and are unable to form stable thrombi (Thomas et al., 1998). G-protein-mediated signalling, through agonist-activated GPCRs, is temporally restricted by second messenger or GPCR kinase-mediated (GRK) phosphorylation of the receptor and - arrestin recruitment. It is now well established that -arrestin recruitment can also serve as a scaffold that regulates GPCR-mediated signalling, independent of G-protein activation. It is challenging to directly examine a role for -arrestins in the regulation of GPCR mediated platelet function because of embryonic lethality in -arrestin-1 and -2 double knockout mice and the significant redundancy in function of the two arrestin isoforms. Platelets are also not amenable to other genetic methods such as siRNA and CRISPR/Cas9 mediated target methods that are frequently employed to study -arrestin regulation of GPCRs. Thus, the role of -arrestins in

regulating prostanoid receptor in platelets has not been extensively studied. In other cell types, EP2

and EP4 receptor interaction with -arrestins form scaffolds with Src-Kinase, which results in transactivation of the epidermal growth factor receptor (EGFR) and AKT activation (Buchanan et al., 2006; Chu et al., 2015; Chun et al., 2010; J. I. Kim et al., 2010). AKT signalling can play

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important roles in platelets (D. S. Woulfe, 2010) and whether such -arrestin-mediated AKT activation downstream of prostanoid receptors is important in platelets remains to be established. As described above, prostanoid receptor-mediated signalling that results in increased cAMP production is associated with platelet inhibition; while a decrease in cAMP, promotes platelet aggregation induced by calcium mobilization (Table 1). This balance is reflected in the cardioprotective effects seen with aspirin, which is in large-part, attributed to its irreversible inactivation of platelet COX enzymes - resulting in a reduction in the pro-aggregating effects of

TXA2. The inherent lack of significant transcriptional activity, in the anuclear platelets, results in a long-lasting inhibition of TXA2 production for the duration of the platelet lifespan. Aspirin and other traditional non-steroidal anti-inflammatory drugs (NSAIDS) also inhibit endothelial production of PGI2 which is largely derived from endothelial cell COX-2 enzymes. The development of the COX-2-selective inhibitors (Coxibs), which was driven by a quest for NSAIDS with greater gastro-intestinal safety, unfortunately resulted in an increase in myocardial infarctions resultant from the incorrect assumptionDraft that PGI2 was derived from the constitutively expressed COX-1 and endothelial specific expression of PGI synthase, while platelet expression of thromboxane synthase was believed to underlie the predominant production of thromboxane by platelets (Needleman, Minkes, et al., 1976; Needleman, Moncada, et al., 1976). The levels of PGI2 in the healthy endothelial is now established to be produced by COX-2 which is induced by shear forces generated by normal blood flow (Caughey et al., 2001; FitzGerald, 2004; McAdam et al., 1999; Yu et al., 2012). Overall, synthetic prostacyclins, thromboxane synthase inhibitors, and antagonists are likely to be useful agents for inhibiting platelet function, often though they are thought to be mechanistically too similar to their inexpensive counterpart, aspirin, limiting their development and use except in cases where aspirin is contraindicated (Ritter, 2011). Proteinase Activated Receptors. The action of thrombin as a coagulation cascade-derived enzyme, that converts fibrinogen to fibrin, has been recognized as far back as the late 1800’s, when it was described by Schmidt and others as fibrinferment (Halliburton, 1888; Marcum, 1998). However, it was soon realized that thrombin could mediate a diversity of actions including the ability to activate platelets (Schmid et al., 1962; Shermer et al., 1961), stimulate mitogenesis (Carney & Cunningham, 1978; L. B. Chen & Buchanan, 1975), regulate vascular tone (De Mey et al., 1982; Haver & Namm, 1984), and affect neuronal cell function (Gurwitz & Cunningham,

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1990). The search for a , which mediated the diverse actions of this enzyme, initially employed ligand binding approaches with radiolabelled thrombin and were unsuccessful (Carney & Cunningham, 1978; Van Obberghen-Schilling & Pouysségur, 1985). Ultimately, it was an expression cloning approach that led to the successful identification of the thrombin receptor (a GPCR now known as Proteinase Activated Receptor-1) (Rasmussen et al., 1991; Vu et al., 1991). The discovery of the thrombin receptor and the necessity for thrombin’s proteolytic activity for the receptor-mediated mitogenic action of thrombin also led to the discovery of the remarkable mechanisms of activation of PAR1. Activation of PAR1 involves the proteolytic unmasking of a N-terminal receptor activating sequence of amino acids, the so-called tethered ligand of the receptor (Coughlin, 1999). Additional receptors (PAR2, PAR3 and PAR4) were soon identified that all bear the hallmark proteolytic cleavage-dependent tethered ligand mechanism of receptor activation (Coughlin, 1994; Hollenberg & Compton, 2002). The initial discovery of PAR1 also made the notable discovery that the PAR receptors could be activated using synthetic peptides that mimic the sequence that is revealed by Draftreceptor proteolysis. These synthetic peptides have been widely used as surrogate activators of these receptors to study their mechanisms of activation and role in physiology (Ramachandran et al., 2012). Human platelets express two thrombin-activated PARs, PAR1 and PAR4, while murine platelets express PAR3 and PAR4 (Coughlin, 2005a). Since thrombin is one of the strongest stimuli for platelet activation, considerable effort has focused on the study of the platelet-expressed thrombin receptors to understand the mechanisms underlying their activation of platelets. PAR1 is the high-affinity thrombin receptor and is engaged at very low concentrations of thrombin. This high-affinity is based on the presence of a thombin-exosite I-binding hirudin-like motif in the N-terminus of PAR1 (Jacques et al., 2000). This enables thrombin to bind the receptor and subsequently cleave it at the Arg41/Ser42 site to reveal the tethered ligand and activate the receptor. PAR4, the other thrombin receptor that is expressed on human platelets, activation typically requires much higher concentrations of thrombin to be present. Higher concentrations of thrombin are believed to be required for the activation of PAR4 because it lacks the thrombin-binding site. Instead, PAR4 interactions with thrombin occur through weaker interactions with an anionic motif that slows the dissociation of PAR4 from the cationic thrombin (Jacques & Kuliopulos, 2003). Thrombin cleaves PAR4 at the Arg47/Gly48 site to reveal the receptor-activating tethered ligand. Both PAR1 and PAR4 activation can stimulate multiple G-protein-dependent signalling cascades. PAR1- and PAR4-coupling to

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Gq/11, Gi, and G12/13 is fairly well established (Hung et al., 1992; McCoy et al., 2010; Voss et al., 2007), though in some instances, coupling to Gi may require crosstalk with certain purinergic receptors (S. Kim et al., 2002; Nylander et al., 2003) (Table 1; Figure 2). Aside from thrombin, enzymes such as matrix metalloproteinases (MMPs) and leukocyte granule enzymes (Elastase, Proteinase 3 and Cathepsin-G) can also activate platelet PAR1 and PAR4 receptors (Austin et al., 2013; Mihara et al., 2013; Molino et al., 1995; Trivedi et al., 2009). In some instances, cleavage by these non-canonical activators of PAR1 and PAR4 result in signalling events that are different from those triggered by thrombin. -arrestin-mediated scaffolding of signalling effectors can also occur downstream of both PAR1 (Kanki et al., 2019; Soh & Trejo, 2011) and PAR4 (Ramachandran et al., 2017; Thibeault et al., 2019a), though a role for PAR1 coupling to -arrestin in platelets has not yet been established. PAR4 activation of -arrestins is involved in activation of the Akt signalling pathway and may synergise with Gq/11-mediated signalling to activate platelets (Ramachandran et al.,

2017). Association of PAR4 with the purinergicDraft receptor is also proposed to support such -arrestin-mediated sustained signalling to Akt (Khan et al., 2011, 2014).

Purinergic Receptors. ADP is stored in platelet dense granules and released upon platelet activation. ADP is also released from damaged cells, at sites of vascular injury, acting in both an autocrine and paracrine manner for recruiting platelets and stabilizing the hemostatic plug. (Born,

1962; Born & Cross, 1963). Platelets express two receptors for ADP, the Gq/11-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor (Table 1; Figure 3) (Hollopeter et al., 2001; J. Jin et al., 1998). Activation of both of these receptors is required for ADP to exert its maximal effect on platelets (Jianguo Jin & Kunapuli, 1998) however, P2Y1 deficient mice do not show significant increases in bleeding tendency or protection from thromboembolic mortality and platelets from these mice still aggregate in response to ADP. Conversely, P2Y12-deficient mouse platelets do not aggregate in response to ADP stimulation (Foster et al., 2001; D. Woulfe et al., 2001), suggesting that ADP-meditated activation of the Gi signalling pathway is the predominant stimulus from

ADP, synergizing with Gq/11 activation by P2Y1 and other Gq/11- or G12/13-coupled GPCRs in platelets (Dorsam & Kunapuli, 2004; S. Kim et al., 2002).

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As with the other receptors described above, few studies have directly studied -arrestin mediated signalling downstream of P2Y receptors in platelets. A role for GRKs and second

messenger kinases in regulating P2Y1 and P2Y12 receptor desensitization has been examined (Hardy et al., 2005). This study showed that platelets express GRK2 and GRK6 and overexpression of these kinases (in non-platelet cells) was able to increase desensitization of the P2Y12 receptor.

Similarly GRK6 deletion resulted in increased signalling through P2Y12 (and PAR1/4 receptors)

while this deletion was without effect on TxA2 responses (X. Chen et al., 2020). GRK and second messenger mediated phosphorylation of the P2Y receptors would enable -arrestin recruitment and these desensitization responses can indirectly infer -arrestin interaction with the respective receptors. Whether -arrestin interaction with these receptors mediates signalling that is relevant to platelet function remains unclear. As described above it has been proposed that PAR4

stabilization of platelet thrombi is dependent on P2Y12 signalling and formation of PAR4/P2Y12 complex with -arrestin, allowing the scaffolding of Lyn and PI3K to phosphorylate and activate AKT. Further studies are necessary, but Draftlack of pharmacological modulators of -arrestins remains a limitation. Other platelet GPCRs. As described above the prostanoid receptors, thrombin receptors and purinergic receptors are major activators of platelets with profound effects on platelet responsiveness seen when these receptors are genetically or pharmacologically blocked. While these are primarily the targets of interest in platelet studies and for the purpose of therapeutic design, proteomic and functional studies have established the expression of a number of other GPCRs on platelets that have more modest effects on platelet function (Table 1). While these receptors do not act by themselves to affect full platelet activation, they interact with the three major platelet activating receptor families to influence platelet reactivity. Broadly these include

the adenosine receptors (A2A>A2B) (Johnston-Cox & Ravid, 2011), 2A-

(Newman et al., 1978), lysophosphatidic acid (LPA) receptors (LPA1-3) (Khandoga et al., 2008), serotonin receptors (Lopez-Vilchez et al., 2009; Vanags et al., 1992), cysteinyl leukotriene

receptors (Hasegawa et al., 2010) and others (Table 1). Functional studies with the 2A-adrenergic

and serotonin receptors which couple to Gi or Gq/11 show synergy with the strong platelet activating receptors to enhance platelet activation (Vanags et al., 1992) while activation of the Gs

coupled A2A receptor inhibits platelet aggregation (Johnston-Cox & Ravid, 2011). Overall the

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involvement of these receptors in platelet regulation is likely context specific (tissue, pathology, etc.) and considering these signals may be important in understanding platelet function in conditions such as stress, inflammation or atherosclerosis (García-Sevilla et al., 2004; Wolfgang Siess et al., 1999; Wang Jong-Shyan & Cheng Lee-Ju, 1999). Further, the implications of alterations to signalling from these receptors on platelet haemostasis, which may be accomplished through therapeutic interventions or in pathology, remains poorly understood and warrant prioritization in order to understanding the full interplay of platelet-expressed GPCRs. Thus, GPCRs are important regulators of platelet function. GPCRs also couple to multiple receptor proximal signalling effectors to effect platelet activation. A deeper understanding of specific pathways engaged by each GPCR in the context of a specific platelet response is critical to determining the pathways that have to be selectively targeted in order to prevent inappropriate platelet activation in disease.

Biased signalling in platelets. Draft Anti-platelet therapy is the cornerstone of current cardiovascular disease management and has significantly reduced mortality and morbidity due to coronary artery thrombosis (Michelson, 2010). Yet significant limitations exist in current therapies, be it due to weak inhibition of platelet aggregation (e.g. with aspirin), lack of efficacy due to inter-patient pharmacogenomic differences (e.g. clopidogrel) or due to side effects such as clinically significant bleeding (e.g. PAR1 inhibition with voropaxar). While all of these limitations cannot be resolved by biased ligands that target selected platelet pathways, in some instances the side effect profile can likely be improved with such strategies. It is now accepted that activated GPCRs seldom couple to a single downstream signalling pathway. Depending on the ligand interacting with a given receptor, GPCRs can stabilize to multiple active conformations, each of which may favour interaction with one or more intracellular effectors – translating into different cellular responses (Galandrin et al., 2007; Terry Kenakin, 2011; Lefkowitz & Shenoy, 2005; Wootten et al., 2018). The ability of different ligands to stabilize distinct receptor conformations has been experimentally demonstrated with a number of techniques revealing that structural basis for such differences stems from favourable orientation of different amino acids that are critical for engaging different G-proteins or -arrestins (Manglik et al., 2015; Wingler et al., 2020). Most often, biased signalling is examined by monitoring the

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engagement of different G-protein subtypes and the recruitment of -arrestins as the major receptor proximal effectors. As described above, platelet aggregation involves integration of signalling inputs from multiple receptors, which are activated by multiple agonists (some of which act on more than one receptor), and frequently can couple to multiple intracellular signalling effectors (which can be triggered downstream of multiple receptors). Layered on top of this, the variation in abundance of and temporal release of agonists adds further complexity. This staggering array of interactions, which has gradually become evident over the last decades, presents an extremely challenging framework for the study of individual signalling pathways. Knocking out or inhibiting specific effectors has provided some indication of how biased signalling may be exploited for therapeutic targeting, however, deletion of any given effector has an impact of multiple signalling pathways. Disruption of multiple signalling pathways through receptor knockout further complicates the interpretation of findings and obscures analysis of the roles individual GPCR-coupled G protein- mediated and -arrestin-mediated signallingDraft pathways play in platelet activation. Further dissection of individual signalling pathways downstream of receptor activation requires the development of biased ligands that not only retain specificity to individual GPCRs, but also selectively engage specific signalling effectors subtypes. Development of such agents is ongoing for a number of GPCRs (Wacker et al., 2017) but studies focused on platelet GPCRs are sparse. In the case of the major prostanoid and purinergic receptors discussed above, we were unable to identify studies that have systematically tested the efficacy of ligands at individual G- protein- and -arrestin-mediated pathways in the platelets. It could however be hypothesized that

ligands biasing prostanoid receptors towards the Gs pathway and away from the Gi pathway are

likely to be beneficial. The TP receptor couples to both Gq/11 and G12/13 signalling. While TP

dependent Gq activation is necessary for triggering platelet aggregation by this receptor, TP

mediated G12/13 activation induces shape change (Klages et al., 1999). Thus, a drug that prevents

TP coupling to Gq might be sufficient to provide anti-platelet action. Similarly, in the case of the

thrombin receptor PAR1, which can couple to Gq/11, G12/13 and Gi, it could be postulated that

compounds that stabilize this receptor in a conformation that prevents activation of the Gq/11 pathway in the platelets would be desirable as anti-platelet agents. In the case of PAR1, additional

inhibition of the G12/13 pathway may also be desirable since activation of this pathway by

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thrombin leads to endothelial barrier disruption (Carbajal et al., 2000; Vouret-Craviari et al., 1998),

while sparing Gi (Bae et al., 2007) and -arrestin (Soh & Trejo, 2011) pathways will be desirable since these signals protect the endothelial barrier (Ramachandran, 2012). Our own recent work has focussed on developing such tools for the study of the thrombin-

activated PAR4 receptor in order to examine whether specific activation of the Gq/11 pathway or -arrestin recruitment is capable of activating platelet aggregation. Such studies have been challenging and perfectly biased compounds for each individual pathway, have been hard to come

by. Nevertheless, we were able to conclude that Gq/11 activation through PAR4 was necessary for PAR4-mediated platelet activation, while agents that showed increased efficacy for -arrestin recruitment synergised with the Gq/11-mediated signalling to cause a greater platelet aggregation response (Ramachandran et al., 2017; Thibeault et al., 2019b). Further development of compounds that are able to selectively engage Gq/11, G12/13, Gi, or -arrestin recruitment to PAR1 and

Gq/11, G12/13 or -arrestin recruitment to PAR4 is ongoing and will help to further clarify the relative contribution of each of these pathwaysDraft in thrombin receptor-mediated platelet aggregation. The variety of G-proteins that can be engaged by PAR receptors and the significant cross-reactivity observed with the endogenous ligands for the platelet-expressed prostanoid receptor subtypes receptors poses a significant challenge in dissecting signalling bias. signalling might be more tractable given that P2Y1 and P2Y12 receptor-coupling to G-protein pathways is more selective and seeking G-protein vs -arrestin activating ligands may be informative.

Conclusions In this brief review, we have highlighted the diversity of GPCR-mediated signalling pathways that are triggered in a coordinated manner to result in full platelet activation. Different GPCRs and their associated G-protein- and -arrestin-mediated signals are required for platelet activation. These parallel signalling pathways make it challenging to tease out roles of individual receptor effector pairs, but also provide an opportunity for the development of biased ligands, that can subtly perturb platelet GPCR signalling for therapeutic benefit. Ultimately such biased ligands will increase our understanding of platelet signalling pathways, allow study of temporal hierarchy of different GPCR signalling events, and enable evaluation of the physiological roles of individual GPCR-stimulated signalling pathways in platelet activation. Development of such biased ligands

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also holds promise as a path towards novel therapeutic interventions with improved efficacy and safety compared to currently available anti-platelet drugs.

Draft

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Table 1. Summary of platelet expressed G-protein-coupled receptors identified by functional and/or proteomic approaches. Where possible, the primary endogenous agonist, G-protein subtypes, known interaction with -Arrestins (known interaction denoted by "Yes"; known not to couple denoted by "No"; unknown denoted by "-"), and known role in human platelets is described.

Coupling to Role in Endogenous G-protein /Signalling Receptor human Agonists effectors through platelets -Arrestins Shape change, G , G , G Thrombin q/11 12/13 i/o aggregation (Macfarlane et al., (Coughlin et al., (Coughlin, 2001; McCoy et Yes (Paing PAR1 1992), APC, 2005b; Kahn et al., 2010; et al., 2002) MMP1, MMP13 al., 1999; Traynelis & Trejo, (Austin et al., 2013) Shapiro et al., 2007) 2000) Aggregation (Sambrano et al., 2001), Draft stabilization (Ramachandran Gq/11, G12/13 Yes (Khan et al., 2017), (Offermanns et al., 2014; shape change, Thrombin, Stefan, 2006; Smith et al., granuale PAR4 cathepsin G, serine Rwibasira 2017; secretion proteases Rudinga et al., Thibeault et (Sambrano et 2018) al., 2019b) al., 2001) (Coughlin, 2005b; Kahn et al., 1999; Shapiro et al., 2000) Shape change G (Z. Ding et q/11 (J. Jin et al., al., 2005; Béatrice Yes (Reiner 1998), P2Y ADP, ATP Hechler & Gachet, 1 et al., 2009) aggregation (B. 2011; Waldo & Hechler et al., Harden, 2004) 1998) Aggregation, stabilization, G (Bodor et al., Yes (Khan i2 granule 2003; Béatrice et al., 2014; P2Y ADP secretion 12 Hechler & Gachet, Smith et al., (Gachet, 2008; 2011) 2017) Kahner et al., 2008;

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Schoenwaelder et al., 2007) - (Amisten et al., Sphingosine-1 P2Y 2008; Murakami - - 10 phosphate, LPA et al., 2008) Inhibition of aggregation G , G (Schwaner (Armstrong et IP PGI s i/o - 1 2 et al., 1995) al., 1989; S. M. Seiler et al., 1997) Inhibition of aggregation G (H. Sugimoto et DP PGD s - (Alvarez et al., 1 2 al., 2003) 1991; S. Seiler et al., 1990) Aggregation (Gresele et al., TP TBXA2 G , G - q/11 12/13 1987; Mais et Draft al., 1988) Gq/11, Gi/o (Abramovitz et al., EP1 PGE2 2000; Katoh et al., - Aggregation 1995; Schwaner et al., 1995) Inhibition of aggregation G (Schwaner et Yes (Chu et (Kuriyama et EP2 PGE s 2 al., 1995) al., 2015) al., 2010; Petrucci et al., 2011) Aggregation G , G i/o q/11 (Fox et al., (Amisten et al., 2013; Petrucci 2008; An et al., Yes (Bilson EP3 PGE et al., 2011; 2 1994; Satoh et al., et al., 2004) Schober et al., 1999; Yamaoka et 2011; Singh et al., 2009) al., 2010) Inhibition of aggregation G (Y. Sugimoto & Yes (Leduc (Kuriyama et EP4 PGE s 2 Narumiya, 2007) et al., 2009) al., 2010; Philipose Sonia et al., 2010)

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Gi/o, Gs, Gq/11 (Amisten et al., Yes A1 Adenosine 2008; Cordeaux et (Mundell & - al., 2004; Jockers Kelly, 2011) et al., 1994) Inhibition of G , G , G Yes z s q/11 aggregation A Adenosine (Fresco et al., (Mundell & 2A (Varani et al., 2004; Olah, 1997) Kelly, 2011) 1999, 2000) G , G (Fresco et Inhibition of s q/11 Yes al., 2004; Olah, aggregation A Adenosine (Mundell & 2B 1997) (Yang et al., Kelly, 2011) 2010) Gi/o, Gs (Bylund & Ray-Prenger, No Aggregation  - Epinephrine, 1989; Eason et al., (DeGraff et 2A (W Siess & adrenergic norepinephrine 1992; Jones et al., al., 1999, Lapetina, 1989) 1991; Limbird, 2002) 1988) Aggregation G (Amisten et Draftq/11 Yes (Hasan et al., Arginine al., 2008; V (Terrillon et 2006; 1A vasopressin Schöneberg et al., al., 2004) Serradeil-Le 1998) Gal et al., 1993) Gq/11, G12/13, Gi/o Yes (Amisten et al., (Alcántara- Shape change 2008; Fukushima Hernández LPA LPA (Khandoga et 1 et al., 1998; et al., 2015; al., 2008) Khandoga et al., Urs et al., 2008) 2005) farnesyl Yes diphosphate, Shape change G , G , G (Alcántara- LPA farnesyl i/o q/11 12/13 (Khandoga et 2 (Ishii et al., 2000) Hernández monophosphate, al., 2008) et al., 2015) LPA farnesyl G (Amisten et Yes diphosphate, q/11 Shape change al., 2008; (Alcántara- LPA farnesyl (Khandoga et 3 Khandoga et al., Hernández monophosphate, al., 2008) 2008) et al., 2015) LPA Inhibition of G , G , G s q/11 i/o aggregation (Amisten et al., LPA farnesyl Yes (H. Yin (Khandoga et 4 2008; Khandoga et (GPR23) diphosphate, LPA et al., 2009) al., 2008; al., 2008; Lee et Pamuklar et al., al., 2007, p. 23; 2008)

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Noguchi et al., 2003) farnesyl G , G diphosphate, q/11 12/13 Shape change, (Amisten et al., LPA farnesyl Yes (H. Yin activation 5 2008; Khandoga et (GPR92) monophosphate, et al., 2009) (Khandoga et al., 2008; Lee et LPA, N- al., 2008) al., 2006) arachidonoylglycine Modulator of ADP and thrombin induced Yes (Gray G , G aggregation q/11 i/o & Roth, 5HT Serotonin (Garnovskaya et (Dale et al., 2A 2001; Roth al., 1995) 2002; Li et al., et al., 2008) 1997; Markovitz Jerome H. et al., 1999) Gi/o (Adham et al., 5HT1F Serotonin Draft1993; Amisten et - - al., 2008) Gs, G12/13 (Amisten et al., Yes 2008; Ouadid et 5HT Serotonin (Barthet et - 4 al., 1992; al., 2009) Ponimaskin et al., 2002) Gi/o (Amisten et Yes mGlu3 L-glutamate al., 2008; Zhao et (Iacovelli et - al., 2001) al., 2009) Gi/o, Gq/11 (Amisten et al., mGlu L-glutamate - - 4 2008; S. Yin et al., 2013) G , G q/11 i/o Yes (Amisten et al., OT Oxytocin (Grotegut et - 2008; Strakova et al., 2011) al., 1998) G (Amisten et Yes Aggregation Succinate i/o Succinate al., 2008; Högberg (Geubelle et (Högberg et al., receptor 1 et al., 2011) al., 2017) 2011) Gi/o (Amisten et Yes CX3CR1 Fractalkine al., 2008; Oh et al., (Cederblad - 2002) et al., 2016;

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Wakita et al., 2017) - (Amisten et al., 2008; CMKOR1 Adrenomedullin, Balabanian Yes (CXCR7, CXCL11, et al., (Gustavsson - ACKR3) CXCL12a 2005; et al., 2017) Kapas & Clark, 1995) G , G q/11 i/o Yes Secretion Cysteinyl (Amisten et al., CysLT (Parhamifar (Hasegawa et 1 leukotrienes 2008; Capra et al., et al., 2010) al., 2010) 2003) Gq/11, Gi/o (Capra et al., 2003; Heise Cysteinyl Yes (Ni et CysLT et al., 2000; - 2 leukotrienes al., 2011) Mellor et al., 2003) Epstein- Draft Barr virus Gi/o (Amisten et Yes induced Oxysterols al., 2008; Liu et (Daugvilaite - gene 2 al., 2011) et al., 2017) (EIB2) - (Amisten et al., Yes (De GPR1 Chemerin 2008; Henau et al., - Barnea et 2016) al., 2008) Gs (Amisten et al., 2008; Bohnekamp GPR133 Unknown - - & Schöneberg, 2011)

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Draft

Figure 1. Synthesis of prostanoids (top) and their known receptor targets and G-protein- coupling effectors expressed in human platelets (bottom). Synthesis of prostanoids occurs

through biochemical conversion of arachidonic acid (AA) by cyclooxygenase 1 (COX-1) to PGH2 which then can be acted on by prostanoid synthases to generate active prostanoid receptor ligands. Prostanoid biosynthesis occurs in both the endothelium as well as through synthesis and release

following of platelet activation. Arrows indicate which cytosolic G-protein subtypes (Gαi/o - green,

Gαs - pink, Gαq/11 - blue, or Gα12/13 – purple) each prostanoid GPCR engages following receptor activation. Made in ©BioRender - biorender.com

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Figure 2. Schematic of Proteinase Activated Receptor (PAR) signalling in the human platelet. At the site of vascular injury tissue derived and coagulation cascade generated thrombin can act on two platelet receptors, PAR1 and PAR4. Thrombin, produced in low concentrations locally at the site of injury, activates the high-affinity PAR, PAR1 enabling signalling through Gαq/11 and

Gα12/13, and subsequent activation of platelets. As the thrombin concentration increases locally on the growing platelet thrombus, the lower affinity thrombin receptor, PAR4, is cleaved and able to signal through the same G-protein subtypes as PAR1. Importantly, while both PAR1 and PAR4 share common signalling pathways, studies have clearly demonstrated unique kinetics and signalling profiles through these common pathways, that enable PAR1 and PAR4 signalling to contribute differently to platelet activation and aggregation. Arrows indicate which cytosolic G-

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protein subtypes (Gq/11 - blue or G12/13 – purple) PAR1 and PAR4 engage following receptor activation. Made in ©BioRender - biorender.com

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Figure 3. Purinergic receptor signalling following activation by ATP (P2Y1), ADP (P2Y1 and

P2Y12), and lipids (sphingosine-1 phosphate, LPA; P2Y10). ADP and ATP are released by the injured endothelium but importantly serve as a major feed forward mechanism in platelet activation when they are released from platelet dense granules following initial priming of platelet by other stimuli. Activation of P2Y1 by ATP or ADP leads to Gαq/11-mediated shape change and platelet aggregation while activation of P2Y12 by ADP engages Gαi signalling and inhibition of cAMP. Full platelet activation of platelets by ADP is thought to require signalling from both receptors. Role of the P2Y10 receptor in platelets remains unclear. Arrows indicate which cytosolic

G-protein subtypes (Gi/o – green or Gq/11 - blue) each of the purinergic GPCRs engage following receptor activation. Made in ©BioRender - biorender.com

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