Platelet calcium signaling by G-protein coupled and ITAM-linked receptors regulating anoctamin-6 and procoagulant activity

Abstract

Most agonists stimulate platelet Ca²⁺ rises via G-protein coupled receptors (GPCRs) or ITAM-linked receptors (ILRs). Well studied are the GPCRs stimulated by the soluble agonists thrombin (PAR1, PAR4), ADP (P2Y₁, P2Y₁₂), and thromboxane A₂ (TP), signaling via phospholipase (PLC)β isoforms. The platelet ILRs glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2), and FcγRIIa are stimulated by adhesive ligands or antibody complexes and signal via tyrosine protein kinases and PLCγ isoforms. Marked differences exist between the GPCR- and ILR-induced Ca²⁺ signaling in: (i) dependency of tyrosine phosphorylation; (ii) oscillatory versus continued Ca²⁺ rises by mobilization from the endoplasmic reticulum; and (iii) smaller or larger role of extracellular Ca²⁺ entry via STIM1/ORAI1. Co-stimulation of both types of receptors, especially by thrombin (PAR1/4) and collagen (GPVI), leads to a highly enforced Ca²⁺ rise, involving mitochondrial Ca²⁺ release, which activates the ion and phospholipid channel, anoctamin-6. This highly Ca²⁺-dependent process causes swelling, ballooning, and phosphatidylserine expression, establishing a unique platelet population swinging between vital and necrotic (procoagulant ‘zombie’ platelets). Additionally, the high Ca²⁺ status of procoagulant platelets induces a set of additional events: (i) Ca²⁺ dependent cleavage of signaling proteins and receptors via calpain and ADAM isoforms; (ii) microvesiculation; (iii) enhanced coagulation factor binding; and (iv) fibrin-coat formation involving transglutaminases. Given the additive roles of GPCR and ILR in Ca²⁺ signal generation, high-throughput screening of biomolecules or small molecules based on Ca²⁺ flux measurements provides a promising way to find new inhibitors interfering with prolonged high Ca²⁺, phosphatidylserine expression, and hence platelet procoagulant activity.

Keywords:

• Calcium channels
• glycoprotein VI
• GPCR
• thrombin
• TMEM16F

Introduction

Blood platelets respond to and become activated by multiple (patho)physiological agonists and chemically synthetized drugs. The majority of these compounds act by one out of two types of signaling pathways, i.e. via G-protein coupled receptors (GPCRs) or via ITAM-linked receptors (ILRs, immunoreceptor tyrosine-based activation motif-linked receptors) [1,2]. Typically, soluble agonists mostly trigger platelets via GPCRs, with as an example the coagulation product thrombin, acting on the protease-activated receptors (PAR)1 and PAR4. On the other hand, but not exclusively, vascular-bound agonists trigger platelets via ILRs, with as an example collagen, stimulating via the ILR glycoprotein VI (GPVI). Both thrombin and collagen are considered to be key agonists in (pathological) thrombus formation in man and rodents [3,4], and both trigger directly or indirectly a whole range of platelet responses [1]. Markedly, thrombin and collagen also directly steer the coagulation process [2]. Given this, it may not be surprising that also platelets have a potent regulatory role in blood clotting [5], a role that is concentrated in the population of ‘procoagulant platelets’ stimulated through the membrane channel protein anoctamin-6 (previously known as TMEM16F) [6].

In the present paper, we outline how platelet activation via GPCR and ILR signaling pathways, synergizing into cytosolic Ca²⁺ rises, induces anoctamin-6 activation, and causes procoagulant platelet formation. This also leads to a whole range of downstream responses in thrombosis and hemostasis, which culminate in the formation of a fibrin clot. Finally, we stipulate that high-throughput screening for molecules affecting the agonist-induced Ca²⁺ rises can provide a promising way to interfere in platelet-mediated blood clotting.

Platelet Activation via GPCRs

As in their name, GPCRs couple to heterotrimeric G-proteins, comprising of α, β, and γ subunits, each of them present different isoforms. For all GPCRs, receptor binding causes a switch from the inactive (GDP binding) to the active (GTP binding) form of the Gα subunits. The most relevant Gα-subunits for platelet Ca²⁺ signaling and ensuing processes are: (i) Gαq (raising cytosolic Ca²⁺ via PLCβ isoforms) and (ii) Gαs (lowering cytosolic Ca²⁺ via cAMP production) (Figure 1). The other G-proteins, Gα12/13 (activating the monomeric GTPase, RhoA) and Gαi (suppressing cAMP production and activating phosphatidylinositol 3-kinases via βγ subunit), modulate the Ca²⁺ signal only indirectly, as detailed below [1,7]. Of the dozens of GPCRs expressed by platelets, most well studied in relation to Ca²⁺ signaling are the receptors for thrombin (PAR1, PAR4), ADP (P2Y₁, P2Y₁₂), and thromboxane A₂ (TP). Dysfunctional mutations of most of these receptors have been described [8,9], as detailed below.

Figure 1. Schematic overview of the different signaling pathways in terms of calcium signaling. Left part GPCRs: G-protein coupled receptors bearing the Gαq isoform activating subunit promote phospholipase C (PLC) β to generate phospholipase inositol 1,4,5-triphosphate (IP₃) and diacylglycerol (DAG). IP₃ triggers the release of Ca²⁺ from intracellular stores and DAG activates several proteins, such as different isoforms of protein kinase C (PKC). Other Gα isoforms (Gα12/13, Gαs, Gαi) trigger signaling pathways that synergize or negatively regulate Ca²⁺ increase, and modulate integrin activation, thromboxane generation and granule release. Right part ILRs: ITAM-linked-receptor activation results in the recruitment of several tyrosine kinase proteins to LAT. The formation of this signalosome results in the activation of PLCγ2, also eliciting IP₃ and DAG formation. Other abbreviations: AC: adenylyl cyclase, CLEC2: C-type lectin-like receptor 2, FcRγ: Fc receptor γ-chain, FcγRIIa: Fc γ receptor IIa, GPVI: glycoprotein VI, LAT: linker of activated T cells, MAPK: mitogen-activated protein kinase, PAR1/4: protease-activated receptor 1 and 4, PI3K: phosphatidylinositol 3-kinase, PKA: protein kinase A, PKC: protein kinase C, PLC: phospholipase C, P2Y₁ and P2Y₁₂: purinergic receptor 1 and 12, ROCK: Rho-associated protein kinase, SFK: Src family kinase, TP: thromboxane receptor, Syk: spleen tyrosine kinase, TxA₂: thromboxane A₂. This figure was created using BioRender.com

Protease-Activated Receptors (PARs)

Handbook knowledge is that the central coagulation protease thrombin cleaves and activates two GPCRs of human platelets, PAR1 and PAR4, both of which are directly coupled to Gαq. The isoform PAR1 is known to be more sensitive for thrombin and to prime for activation of PAR4 [7,10,11]. PAR1 induces an acute and rather short-lived rise in Ca²⁺ plus initial integrin activation, whereas PAR4 causes a steady and more sustained Ca²⁺ signal that also depends on autocrine P2Y₁₂-mediated events. Accordingly, in thrombin-stimulated platelets, the total amount of Ca²⁺ signal is the time-integrated sum of contributions of both receptors (Figure 2A) [12,16,17]. In mouse platelets, PAR3 as a non-cleaved receptor replaces PAR1 [7]. Regardless of the species, the more prolonged PAR4 signaling is considered to be needed for the platelet procoagulant response and for thrombus stabilization [18,19]. Part of the PAR-dependent signaling can occur independently of Ca²⁺ mobilization via a Gα12/13 pathway, which contributes to the platelet shape-change response [20].

Figure 2. Schematic representation of the different pattern of calcium responses in a single platelet stimulated by GPCRs or ILRs agonists. Fura2-labeled platelets were allowed to adhere to fibrinogen (A) or collagen (B, C) surfaces. Thrombin addition is indicated by an arrow. A) The GPCR agonist, thrombin, promotes oscillatory calcium traces in single platelets calcium signaling, while B) collagen, as an agonist of ILRs, promotes a sustained calcium rise. C) Procoagulant response, due to anoctamin-6 activation, was triggered by a continued high calcium rise after stimulation by collagen and thrombin. Schematic traces are based on published measurements in single platelets [12–15].[103]

Several human PAR4 variants (gene F2RL3) are known to link to alterations in platelet responses [8,21]. Furthermore, blocking of PAR4 or its hyperreactive forms was found to provide an antithrombotic potential [22,23]. Regarding the other receptor PAR1 (gene F2R), antagonists like vorapaxar are being used in some countries for secondary prevention of atherothrombotic disease [24]. Current judgment is that better understanding of the interindividual variation in the PARs’ activities is needed to come to a better personalized medication [25].

Purinergic Receptors P2Y₁ and P2Y₁₂

The two key purinergic GPCRs for ADP in platelets are P2Y₁ and P2Y₁₂ [26]. While ADP is considered to be a weak platelet agonist, it plays an important role in the amplification and stabilization of platelets responses to many other receptor agonists, including thrombin and thromboxane A₂ [27]. The low-level P2Y₁ receptors signal via Gαq- and PLCβ-mediated Ca²⁺ rises, whereas the more abundant P2Y₁₂ receptors are coupled to Gαi [26]. The relatively small Ca²⁺ signal evoked by P2Y₁ contributes to platelet shape change. On the other hand, the P2Y₁₂ pathway involves inhibition of adenylyl cyclase activity via the Gαi subunit and activation of phosphatidylinositol 3-kinases (PI3Ks) via the βγ subunits, the latter directing to integrin α_IIbβ₃ activation [1]. Markedly, P2Y₁₂ was also found to contribute to the thrombin-induced exposure of phosphatidylserine (PS) in isolated platelets [28].

The literature provides also ample evidence for synergic action between the P2Y₁ and P2Y₁₂ receptors. Recent work shows that in rat megakaryocytes P2Y₁₂ signaling can enhance the P2Y₁-induced Ca²⁺ oscillations, which then stimulates integrin α_IIbβ₃ activation and fibrinogen binding [29]. This adds well to earlier evidence that P2Y₁₂ contributes to platelet activation by potentiation of the P2Y₁-induced Ca²⁺ responses both via inhibition of adenylate cyclase and activation of PI3Ks with a complex additional role of Src kinase [30]. Critical in this interplay of Ca²⁺-dependent platelet activation via both P2Y receptors is also the guanine nucleotide exchange factor CalDAG-GEFI, which in a Ca²⁺-dependent way regulates integrin activation through Rap1b [31].

Dysfunctional (gain- or loss-of-function) variants of the genes for both P2Y₁ and P2Y₁₂ (P2RY1, P2YR12) have been identified, which associate with a bleeding or a prothrombotic propensity [32,33]. As an illustration of the relevance of the P2Y₁₂-PI3K-α_IIbβ₃ activation pathway, (pro)drugs inhibiting this receptor are widely used for the secondary prevention of arterial thrombosis, even as monotherapy. Mostly prescribed drugs are thienopyridines that irreversibly block the receptor (prodrugs: clopidogrel and prasugrel), and the compounds ticagrelor and cangrelor that reversibly bind to P2Y₁₂ [34].

Thromboxane A₂ Receptor (TP)

The TP receptor for thromboxane A₂ is expressed on platelets and on vascular cells, and it couples to Gαq (regulating Ca²⁺ release) and Gα12 family proteins (regulating integrin activation) [7,35]. Aspirin, one of the hallmarks of current anti-platelet therapy, blocks the formation of thromboxane A₂, thus preventing its interaction with the TP receptor. Without this drug, the autocrine platelet-released thromboxane A₂ greatly enhances the responses of other platelet agonists, in particular ADP and collagen [2]. Patients with a mutation in the TBXA2R gene develop bleeding symptoms, and their platelets react less to thromboxane A₂, ADP, and other agonists [36,37]. In the past, several efforts have been made to develop specific TP antagonists. The extra advantage of such antagonists was questioned, given the high clinical importance of aspirin as a ‘gold-standard’ anti-platelet drug for the prevention of cardiovascular events. However, it is well possible that receptor antagonists have less bleeding side effects.

Other GPCRs

In the context of Ca²⁺ signal generation, another relevant GPCR on platelets is the IP receptor for endothelial-derived prostacyclin. This receptor couples to Gαs and raises the cAMP level, which downregulates Ca²⁺ rises via protein kinase A [38].

Platelet Activation via ILRs

The most widely studied ILR in terms of signaling is GPVI, while less attention has been paid to the podoplanin receptor C-type lectin-like receptor 2 (CLEC2) and the IgG receptor FcγRIIa. In platelets, signaling via these ILRs relies on a tyrosine phosphorylation cascade starting at the (hemi-) ITAM motif YxxI/L [39]. Tyrosine phosphorylation of this domain creates a docking site for SH2-containing proteins of the Src kinase family, which mediate Syk phosphorylation-ensuing activation of PLCγ and PI3K isoforms (Figure 1). Positive feedback loops mediated by a crosstalk between the small-GTPases Rap1b and Rac1 appear to be critical for a full ITAM-induced platelet activation [31].

As further detailed below, these ILRs, in particular, the collagen/fibrin receptor GPVI, have a well-established enhancing effect on the GPCR-induced responses by thrombin, ADP, and thromboxane A₂ [1]. Exemplary is the platelet procoagulant response mediated by PS exposure, requiring the combined collagen and thrombin stimulation [5,40,41].

Glycoprotein VI (GPVI)

GPVI is uniquely expressed by megakaryocytes and platelets and requires co-expression with the ITAM-bearing co-receptor Fc receptor γ-chain (FcRγ) [42]. It is the central signaling receptor of collagen, collagen-related peptides (CRP), and the snake venom convulxin [43,44]. The strength of the GPVI signal in response to collagen ligands is regulated by receptor clustering [45]. Recent overviews of mouse studies indicate that GPVI is one of the few platelet-signaling proteins that contribute to murine arterial thrombosis with only a minor role in hemostasis [46]. This feature makes GPVI an attractive target for the development of improved anti-platelet drugs to prevent thrombosis. Indeed, GPVI-deficient patients described so far present with no more than a minor bleeding tendency [43,47]. Interestingly, a genetic variant of GP6 was found to associate with an increased risk of venous thrombosis [48].

Other (hem)-ITAM-Linked Receptors

The podoplanin receptor CLEC2 is highly expressed as a homodimer in platelets and megakaryocytes, and it is present at low levels in inflammatory dendritic cells and myeloid cells [39,49]. Mouse thrombosis studies suggest a blood-borne ligand, but this has not been identified yet [50]. In comparison to the GPVI-linked FcRγ chain, the CLEC2 receptor contains a hemi-ITAM domain with a single YxxL sequence [51], but its signaling pathway shows considerable overlap with that of GPVI. Besides a role in arterial thrombosis, CLEC2 is also involved in the maintenance of lymphatic vascular integrity and inflammation [39,49]. When stimulated by the snake venom rhodocytin, CLEC2 appeared to have a no more than limited role in platelet procoagulant activity [52]. This is in agreement with the reported moderate Ca²⁺ rise induced by CLEC2 activation [53].

The FcγRIIa receptor (also named CD32a) acts as a low-affinity receptor for the constant fragment of immunoglobulin G [54]. Upon dimerization it allows platelets to bind to IgG immune complexes, resulting in platelet activation via a similar tyrosine kinase cascade as GPVI and ensuing Ca²⁺ mobilization [55]. A role of the platelet FcγRIIa is known for a range of pathologies, such as immune thrombocytopenia, heparin-induced thrombocytopenia, bacterial infections, and cancer [54].

Synergistic Platelet Calcium Signaling via GPCRs and ILRs

Platelets respond in a context-dependent manner to the different stimuli to which they are exposed [2,56]. Accordingly, when these cells are stimulated through GPCR (e.g., with thrombin, autocrine ADP, thromboxane A₂) and through ILR (e.g., with collagen) this results in additive responses because of the complementarity of both types of signaling pathways [1]. A key quantifiable and integratory signal of GPCR plus ILR stimulation is the rise in cytosolic-free Ca²⁺, which then feeds forward to other responses such as pseudopod formation, integrin α_IIbβ₃ activation, granular secretion, procoagulant activity, and formation of contracting aggregates [1].

A notable mechanism of Ca²⁺ signal integration is provided by the different PLC isoforms that become activated through GPCRs (PLCβ2/3) and ILRs (PLCγ2) [57,58]. Both PLC types hydrolyze phosphatidylinositol 4,5-biphosphate into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP₃) [18,59]. The messenger IP₃ binds to its receptors in the endoplasmic reticulum and promotes the release of stored Ca²⁺ (Figure 1). Feedback is provided by concomitant signals, including protein kinase C isoforms and CalDAG-GEFI [31]. In a way not very well understood, the signaling route via PLCβ2/3 or PLCγ2 lead to a different shape of the IP₃-induced Ca²⁺ mobilization and the related entry of extracellular Ca²⁺, which hence differ between GPCR and ILR agonists.

In single platelets, a marked difference between GPCR and ILR stimulation is the IP₃-induced oscillatory, spiking Ca²⁺ rises in response to thrombin or ADP [13], and the continued Ca²⁺ rises in response to collagen [60] (Figure 2A and B). The reason for this difference is still not well understood, but it likely involves a higher activity of Ca²⁺-ATPases (SERCAs), pumping back the released Ca²⁺ into the stores after each spike [61], in the case of GPCR-stimulated platelets.

The agonist-induced regulation of Ca²⁺ entry channels in platelets is reviewed in detail elsewhere [62]. In brief, following Ca²⁺ store depletion, the endoplasmic reticulum Ca²⁺ sensor STIM1 interacts with the store-operated Ca²⁺ entry (SOCE) channel Orai1, causing influx of Ca²⁺ from the extracellular medium (Figure 3). Studies with genetically modified mice have shown that this SOCE pathway is more predominant upon ILR stimulation (collagen) than upon GPCR stimulation (thrombin) [62]. Yet, patients with mutations in STIM1 or ORAI1 were found to suffer from immune deficiency and bleeding symptoms, linked to defects in GPCR signals and thrombus formation [63]. An additional non-SOCE Ca²⁺ entry mechanism via the nonselective TRPC cation channels is stimulated by thrombin, with a significant role in PS exposure [64]. In particular, co-stimulation with collagen and thrombin activates these TRPC, allowing entry of Na⁺ and Ca²⁺ [64].

Figure 3. Schematic overview of the Ca²⁺ entry pathways and PS exposure. Left part GPCRs: Ca²⁺ depletion from the intracellular stores by GPCRs is mainly triggered through PLCβ-induced generation of IP₃. The DAG mediates transient receptor potential channel 6 (TRPC6) opening, allowing extracellular Ca²⁺ entry. The rise in cytosolic Ca²⁺ results in activation of integrin α_IIbβ₃ via CalDAG-GEFI and Rap1b. Right part ILRs: ILR-induced Ca²⁺ depletion from the intracellular stores most strongly promotes STIM1-Orai1 channel coupling and activation by pumping Ca²⁺ ions inside the cytosol (store-operated calcium entry, SOCE). Mitochondrial depolarization and generation of mitochondrial permeability transition pore (MPTP) contributes to the increase of cytoplasmic Ca²⁺ levels. This high and sustained Ca²⁺ levels mediate anoctamin-6 (Ano6) scramblase activation and PS exposure, which also promotes ion channel opening and Na⁺, Cl⁻ and water entry. Among other processes, calpains are activated resulting in the cleavage of several proteins and receptors. In both pathways, high intracellular Ca²⁺ levels activate sarco/endoplasmic reticulum Ca²⁺ ATPases (SERCA), which pumps Ca²⁺ back into the intracellular stores. Several channels in the plasma membrane such as plasma membrane Ca²⁺ ATPases (PMCA) and Na⁺/Ca²⁺ exchanger (NCX) also reducing the cytoplasmic Ca²⁺ levels (not shown). ROCK: Rho-associated protein kinase. For other abbreviations, see Figure 1. This figure was created using BioRender.com

Furthermore, mitochondrial-stored Ca²⁺ can play a role upon dual platelet stimulation. Current understanding is that signals including elevated Ca²⁺ trigger the mitochondrial Ca²⁺ release via mitochondrial permeability transition pores, which results in the ‘supramaximal Ca²⁺ signals’ that are required for platelet PS exposure and procoagulant activity [65,66].

Another platelet ion channel highly selective for Ca²⁺ is provided by the P2X₁ receptor for ATP, which also plays a regulatory role in thrombus formation [67,68]. P2X₁ contributes to Ca²⁺ signaling and platelet function upon low-level stimulation by GPCRs and ILRs [69]. The contribution of the P2X₁ signal to PS exposure is less clear. Regarding secondary lowering of the Ca²⁺ signal, next to SERCA isoforms, also plasma membrane Ca²⁺-ATPases and Na⁺/Ca²⁺ exchangers play a role in this [62], although it is unclear if these roles differ between platelet agonists.

Overall, it appears that the magnitude and duration of the Ca²⁺ signal in platelets is a function of their exposure to agonists, in which GPCR and ILR agonists together evoke the platelet procoagulant response. A differential exposure to agonists can also explain, on top of differences between platelets, why the platelets in a thrombus are heterogeneous in activation stages [70,71]. Herein, the population of most strongly activated platelets is the one showing PS exposure [41,56]. Taken together, the physiological consequences of procoagulant platelet formation can be summarized as: (i) limiting the thrombus growth by reducing the adhesive function of platelets, (ii) enhancing the coagulation by thrombin and fibrin formation, and (iii) a thrombin-induced stabilization and contraction of the platelet plug.

Anoctamin-6 and the Platelet Procoagulant Response

The channel anoctamin-6 is a multi-spanning integral membrane protein, coded by the gene ANO6 (TMEM16F). It is highly expressed in platelets and serves as a Ca²⁺-dependent ion channel as well as a channel for negatively charged phospholipids like PS [6,72]. Whereas first papers considered anoctamin-6 as a channel permeable to both monovalent and divalent cations, including Cl⁻, Na⁺ and Ca²⁺ [73], later reports emphasized the high Ca²⁺-dependent Cl⁻ conductance [6,74]. Interestingly, recent structural and inter-species analyses suggest that the ion permeation occurs independently of phospholipid scrambling, i.e. PS exposure [74,75]. However, the ion channel activity is required for the anoctamin-6-dependent ballooning of platelets that accompanies PS exposure [76].

Dysfunctional mutations in ANO6 are causative for the Scott syndrome (Table I), resulting in a mild bleeding phenotype in mouse and man [82–84]. To date, only six Scott patients have been reported, who only bleed after major trauma (surgery) without easy bruising from superficial cuts [77].

Table I. Genetic pathologies altering platelet PS exposure. Abbreviations: ΔCRAC, combined immunodeficiency due to CRAC channel dysfunction; EV, extracellular vesicle; WAS, Wiskott–Aldrich syndrome

Disorder	OMIM	Gene	Genetic transmission	Patient characteristics	Platelet defect	Ref.
Scott	262890	ANO6	autosomal recessive	mild bleeding	defected PS exposure and EV release	[77,78]
Stormorken	185070	STIM1	autosomal dominant (gain-of-function)	thrombocytopenia, asplenia, myopathy, mild bleeding	increased PS exposure, secondary loss of platelet function	[79]
WAS	301000	WASP	X-linked	infections, thrombocytopenia, bleeding	increased Ca^2+ rise and PS exposure	[80]
ΔCRAC	612782	ORAI1	autosomal recessive	immune defect, myopathy	reduced Ca^2+ rise and PS exposure	[63,81]
	612783	STIM1	autosomal recessive (loss-of-function)	immune defect, hypotonia	reduced Ca^2+ rise and PS exposure

Studies with human and mouse platelets have confirmed the role of anoctamin-6 in the procoagulant responses upon sustained intracellular Ca²⁺ rises, such as induced by the co-stimulation of collagen and thrombin (Figure 2C) or Ca²⁺ ionophores. Such platelets swell due to anoctamin-6-mediated influx of Cl⁻ and (in)directly Na⁺, forming balloon-like structures, and simultaneously expose procoagulant PS due to phospholipid scramblase activity. Both types of responses are annulled in platelets from Scott syndrome patients and whole-body or platelet-specific Ano6^−/− mice [76,83,84].

In platelets from at least two Scott patients, a reduced collagen/thrombin-induced activity of the Ca²⁺-dependent protease calpain was found, which can explain lower microvesicle formation and lower intracellular cleavage of integrin α_IIbβ₃ and talin in these platelets [14,83,85]. For the Scott_UK patient, this could be linked to a decreased expression of one of the calpain isoforms [86]. Of relevance, in platelet-specific Ano6^−/−- mice, the hemostatic and thrombotic reactions were reduced, thus confirming that platelet PS exposure is a regulatory factor in blood clotting in vivo [84].

As indicated before, a condition par excellence that induces anoctamin-6 mediated PS exposure and procoagulant activity is the activation of platelets by collagen plus thrombin (next to Ca²⁺ ionophore), resulting in ‘supramaximal’ high Ca²⁺ rises [5,87]. These high-Ca²⁺ PS-exposing platelets are furthermore characterized by a calpain-mediated cleavage and inactivation of integrin α_IIbβ₃ [88], the extracellular cleavage of GPVI and GPIbα [89], and a greatly enhanced binding ability of Gla-domain containing coagulation factors [18,90]. This assembly of coagulation factors causes a magnitude increase in the formation of factor Xa (tenase complex) and thrombin (prothrombinase complex) at the platelet surface, thereby strongly enhancing thrombin generation and the clotting process [1,52,91]. Since the ballooned PS-exposing platelets can collect a surrounding protein (fibrin) coat, they were also designated as COAT platelets (COllagen And Thrombin) [92,93]. Given the major morphological changes of the ballooned platelets, it is not a surprise that anoctamin-6 has a strong regulatory effect on the platelet neo-N-terminal and phospho-proteomes upon collagen/thrombin activation [86]. With the risk of popularizing, for these non-dead platelets, the term ‘zombie’ might be more appropriate than ‘necrotic.’

On the other hand, PS exposure of platelets can also be induced by a different pathway, acting independently of anoctamin-6 and elevated Ca²⁺ [94,95]. The latter pathway is triggered by the apoptotic stimulus ABT-737, relies on caspase activation and is accompanied by a different set of intracellular proteolytic events [86]. The responsible PS-exposing enzyme most likely is the phospholipid ‘scramblase’ XKR8 [96], the Ca²⁺ dependence of which is unclear. As summarized elsewhere, mouse studies have shown that many other signaling proteins can modulate collagen/thrombin-induced PS exposure [97]. However, in the vast majority of these are proteins that primarily alter the Ca²⁺ signal. As shown in Table I, the clinical relevance of the altered PS exposure in patients with Stormorken, Wiskott-Aldrich or Calcium Release-Activated Channel (CRAC) syndromes lies in bleeding symptoms and thrombocytopenia as a result of alterations in Ca²⁺ signaling proteins.

High-Throughput Screening to Find New Inhibitors Affecting Platelet Calcium Signaling and Procoagulant Activity

High-throughput screening (HTS) of small compounds or biomolecules is an established technique for the discovery of lead molecules to produce commercialized agents after several rounds of drug development [98]. So far, only a few high-throughput approaches have been used to search for platelet-related Ca²⁺ signal inhibitors (Table II). A first example is a well-plate based high-throughput approach employing Drosophila cell lines to identify novel inhibitors of the Orai1 CRAC channels [99]. Other examples are the use of HEK cells for identification of the novel PAR4 antagonist BMS-986120 [23], and for the search for a small molecule inhibitor of the nonselective Ca²⁺ influx channel TRPC6 [100]. In the platelet field, pharmaceutical screening approaches discriminating between GPCR- and ILR-induced Ca²⁺ signaling are still absent. In the reported studies of Table II, platelets were used only after initial screening.

Table II. Used approaches of high-throughput screening for platelet-related Ca²⁺ research. Abbreviations: HEK, human embryonic kidney; PP1cα, protein phosphatase 1catalytic subunit; TG, thrombin generation

Target protein	Approach	Secondary assay	Ca^2+/PS	Outcome	Ref.
Orai1 TRPC6	Drosophila cell line overexpressing HEK293 cells (hTRPC6-YPF)	platelets platelets	yes/no yes/no	CRAC channel small molecule	[94][95]
PP1cα	yeast two-hybrid interactions (PP1cα vector)	platelets	no/no	Gβ1 subunit	[100]
PAR1	platelet secretion, luciferase-based assay	platelets	no/no	small molecule	[101]
PAR4	overexpressing HEK293 cells	platelets (TG)	yes/no	BMS-986120	[20]
Factor Xa	chromogenic substrate assay, prothrombin time (plasma)	platelets (plasma)	no/yes	Rivaroxaban	[102]

In this context, it is of interest to make use of the high and sustained Ca²⁺ rise that triggers PS exposure for a high-throughput screening setting of platelets to find novel molecules interfering with procoagulant activity. Finding inhibitors that block the appearance of procoagulant platelets may reduce the risk of thrombotic events, while preserving other populations of adhesive, secreting and aggregating platelets, thereby limiting the risk of bleeding. However, a fine-tuning inhibitory dosing would be needed to minimize the bleeding complications, such as seen in Scott syndrome patients. Besides this, it will also be interesting to find new procoagulant molecules to strengthen hemostasis in conditions, where this is needed.

Conclusion

Several pathological conditions in the spectrum of cardiovascular disease present with hyperreactive platelets and increased levels of procoagulant platelets [38, Baaten, 104104104104104104104 #53, 53]. However, useful inhibitors – with a clinical potential – targeting the high Ca²⁺ levels and consequent PS exposure in platelets stimulated by GPCR plus ILR agonists have not yet been reported. Therefore, studies on the role of procoagulant platelets in pathological conditions are needed to find novel targets that help in the development of inhibitors that prevent procoagulant platelet formation.

Disclosure of Interest

The authors declare no conflicts of interest.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website

Additional information

Funding

DIF is supported by the H2020 Marie Skłodowska-Curie Actions agreement No. 766118 and is enrolled in a joint PhD program of the Universities of Maastricht (The Netherlands) and Santiago de Compostela (Spain).