Blood Reviews 29 (2015) 153–162

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Blood Reviews

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REVIEW

Platelet secretion: From haemostasis to wound healing and beyond

Ewelina M. Golebiewska, Alastair W. Poole ⁎Medical Sciences Building, School of Physiology and Pharmacology, University of Bristol, University Walk, BS8 1TD Bristol, UK

⁎ Corresponding author at: School of Physiology & PhBuilding, University Walk, Bristol BS8 1TD, UK. Tel.: +4331 2288.

E-mail address: a.poole@bris.ac.uk (A.W. Poole).

http://dx.doi.org/10.1016/j.blre.2014.10.0030268-960X/© 2014 The Authors. Published by Elsevier Ltd

a b s t r a c t

a r t i c l e i n f o

Keywords:

PlateletsSecretionSNARE proteinsThrombosisHaemostasisCancer metastasisInflammation

Upon activation, platelets secretemore than 300 active substances from their intracellular granules. Platelet densegranule components, such as ADP and polyphosphates, contribute to haemostasis and coagulation, but also play arole in cancer metastasis. α-Granules contain multiple cytokines, mitogens, pro- and anti-inflammatory factorsand other bioactive molecules that are essential regulators in the complex microenvironment of the growingthrombus but also contribute to a number of disease processes. Our understanding of the molecular mechanismsof secretion and the genetic regulation of granule biogenesis still remains incomplete. In this reviewwe summariseour current understanding of the roles of platelet secretion in health and disease, and discuss some of the hypoth-eses that may explain how platelets may control the release of its many secreted components in a context-specificmanner, to allow platelets to play multiple roles in health and disease.

© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/3.0/).

1. Introduction

Platelets have been known to contribute to thrombosis andhaemostasis since their first identification by Bizzozero in the 1880s[1]. Recent evidence suggests that their functions extend beyondthe immediate environment of the thrombus and platelets have beenimplicated in a number of other physiological responses aimed atsafeguarding the integrity of the vessel. On the other hand, their prop-erties as haemostatic and inflammatory cells can result in disease statesunder certain conditions. Platelets can ‘communicate’ with each otherand with other cells via a range of bioactive substances secreted fromtheir intracellular granules. In this review, the contribution of plateletsecretion to those processes will be discussed.

2. Platelet activation cascade

Under physiological conditions, platelets circulate in close proximityto the vascular walls [2], but are protected from untimely activation bythe healthy endothelial monolayer which provides a natural ‘barrier’ tothrombosis, as well as by the release of inhibitory mediators such asnitric oxide and PGI2 from the intact endothelium. Platelets becomeactivated when the continuity of the endothelial layer is disrupted andthe underlying subendothelial matrix is exposed, or if inflammationperturbs the endothelium. Platelet receptors then interactwith collagenand von Willebrand Factor among others, which capture the platelets

armacology, Medical Sciences4 117 331 1435; fax: +44 117

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and induce activation signals. The initial events following plateletactivation are summarised in Fig. 1.

After the initial ‘platelet plug’ is formed at the site of injury, engage-ment of the coagulation cascade leads to fibrin mesh formation that en-capsulates and strengthens the thrombus. As well as serving as adhesionsites for coagulation factors (via surface exposure of phosphatidylserine,PS), platelets themselves are an important source of those factors (forexample Factors V [3] andXIII [4,5]), and other components that regulatecoagulation such as polyphosphates [6,7] and prothrombin [8].

Platelets can also become activated via G-protein coupled receptor(GPCR) signalling downstream from soluble agonists forming at the siteof thrombus. Those soluble agonists, and in particular ADP released fromdense granules of activated platelets, precipitate a number of positive feed-back cascades leading to rapid activation of large numbers of platelets. Ulti-mately, an orchestrated ‘effort’ of many other secreted mediators and cellsresults in restoration of vessel integrity. Arguably, secretion is the most farreaching result of platelet activation, and as such can also account for func-tions of platelets beyond the immediate environment of the thrombus.Therefore platelet secretion is at the heart of the control of vascular integri-ty, in health and disease, and is the focus of this review.

3. Secretion in primary haemostasis

3.1. Dense granules

Dense granules contain a range of small molecules [9] such as ADP,ATP, GDP, 5-HT, pyrophosphate, magnesium and calcium. Historicallyrelease from dense granules was described as ‘fast’ and indeed a recentstudy by Jonnalagadda et al. [10] showed that the release of [3H]-serotonin occurred more rapidly than PF-4 from α-granules or β-

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Fig. 1. Schematic of platelet activation cascade leading to the haemostatic plug formation. Platelet adhesion to ECM components via integrin or GPVI receptors or activation with solubleagonists via GPCR receptors leads to platelet activation. One of the hallmarks of platelet activation is secretion of bioactive molecules from dense and α-granules, which can then act toactivate further platelets, as well as in an autocrine manner to drive positive feedback cascade. ‘Inside-out’ signalling initiated by platelet activation also causes activation of integrinαIIbβ3. Platelets also undergo a dramatic shape change, increasing their surface area available for adhesion to ECM and to one another. Activated integrin αIIbβ3 and fibrin contributeto formation of the initial aggregate or platelet plug. Platelets also expose PS providing attachment sites for coagulation factors. The coagulation cascade contributes to the stabilisationof the thrombus.

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hexosaminidase from lysosomes, regardless of the agonist used tostimulate platelets [10].

Smallmolecules released fromdense granules togetherwith synthe-sised thromboxane A2 act back on circulating platelets and contribute tothe positive feedback signalling that sustains platelet aggregation. Thecentral role of the ADP–P2Y12 receptor axis in haemostasis is particular-ly well established. Platelet adhesion on vWF, inside-out activation ofintegrin αIIbβ3 and P-selectin expression are all defective in P2Y12

−/−

mice [11]. Recently, chemically mutagenised mice lacking Munc13-4(Unc13DJinxmice) that show a complete absence of platelet dense gran-ule secretion were generated. Those animals were also found to havesignificantly reduced aggregation and other markers of plateletactivation such as α-granule and lysosome secretion and integrin acti-vation [12], confirming that it is the secreted ADP rather than plasmaADP that contributes to those processes. They also failed to form throm-bi in the in vivo and in vitro models of thrombosis [13]. Co-stimulationwith exogenous ADP could partially rescue platelet function, underliningthe essential role of ADP in driving the positive feedback loop and thus theplatelet primary haemostatic response [13]. Interestingly, Munc13-4 dele-tion did not affect intracranial bleeding following stroke induction inmice, while at the same time they were protected from stroke progression[14]. This apparent paradox hints at different roles for ADP/P2Y12 signallingin haemostasis and thrombosis, possibly even in different disease scenarios.The role of ADP emerges as more important and complex than initiallythought and may account for some distinguishing features of thrombosisversus haemostasis. ADP/P2Y12 mediated signalling also enhances signal-ling towards procoagulant activity and thrombin activation [15].

3.1.1. ADP and the ‘core and shell’ model of thrombus structureDespite many different agonists shown to drive platelet activation

in vitro to completion, it is now known that in vivo platelet activationis non-homogeneous, possibly attributable to variations in shearunder physiological conditions [16,17]. α-Granule secretion and calci-um mobilization have also been shown to be heterogeneous [18].In a recent study, Stalker et al. suggested a new model of thrombusstructure, taking into account that non-uniform activation pattern.They showed that two distinct populations of platelets are present in a

growing thrombus in vivo: a ‘core’ of more stable P-selectin expressingplatelets, andamoreporous ‘shell’, containing less activated, P-selectinneg-ative platelets [19]. While the ‘core’ seemed to be more dependent uponthrombin and local contact-dependent activation, the recruitment of plate-lets to the ‘shell’was shown to be critically dependent uponADP signalling,with P2Y12 inhibition significantly decreasing the size of the ‘shell’ and notaffecting the ‘core’. They also show that permeability of the ‘core’ to plasma-borne molecules is limited, suggesting that efflux of platelet granule con-tents in that tightly packed region could be similarly limited [19,20].

The most effective anti-thrombotic treatments currently usedspecifically target the ADP/P2Y12 positive feedback cascade, blockingthe P2Y12 receptors and thus limiting platelet activation and aggregationand the risks of pathological thrombosis, particularly in the managementof coronary heart disease [21]. However, the central role of the ADP/P2Y12axis in haemostasis means that abnormal bleeding often occurs in pa-tients treated with potent P2Y12 inhibitors such as clopidogrel, prasugrelor ticagrelor [22]. On the other hand, the new core and shell modelcould account for the ability of the same agents to reduce platelet accu-mulation without always causing bleeding, as they would not affect theinitial platelet adhesion or the size of the ‘core’ [19]. It is possible that inhumans, interindividual variations in platelet activity account for thosedifferences. Increasing our understanding of ADP/P2Y12 signalling mayhelp in discoveringmore selective platelet inhibitors that could specifical-ly limit thrombosis without causing bleeding.

3.1.2. Polyphosphate in coagulationDense granule secretion is also important in coagulation. Released

calcium for instance is required at several steps of the coagulationcascade and for activation of the prothrombinase pathway. Recentlymore insight was gained into the role of another dense granule cargomolecule, polyphosphate (polyP). PolyP is a highly anionic linear poly-mer that is synthesised from ATP and secreted by platelets after activa-tion [23]. Synthetic polyP could restore defective clot formation inplatelet-rich plasma from Hermansky–Pudlak patients who lack densegranules [24], suggesting a role in haemostasis. The putativemechanisminvolves direct polyP binding to Factor XII, thus triggering coagulationby the tissue factor-independent, contact activation pathway [24,25].

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On the other hand, humans and animals lacking Factor XII were shownto have no bleeding tendencies suggesting that the contact pathway isnot essential in primary haemostasis, contrary to the tissue factorpathway [26]. This would not support the idea of the polyP–Factor XIIpathway being essential for haemostasis. However, more recent studiesof mice deficient in hexakisphosphate 6 (IP6) kinase required forsynthesis of polyP showed that a 3-fold reduction in polyP levels ledto widespread platelet function and coagulation defects, again suggest-ing a role in haemostasis [6]. As well as its debatable role in Factor XIIactivation, polyP was also found to accelerate thrombin generation[27] and enhance fibrin clot stability [28]. Although the role of polyPand its potential in antithrombotic therapy remain largely unclear, thegeneral consensus is that this previously under-appreciated inorganicmolecule may represent an important player in the thrombotic–haemostatic environment [7]. Indeed, in 2012 two groups showed thatpharmacological inhibition of polyP could prevent thrombosis withoutincreasing surgical bleeding inmousemodels [29,30], again emphasisingthe multifaceted role of the platelet secretome in haemostasis.

3.2. α-Granules

A number of α-granule cargoes, such as vWF and fibrinogen, act topropagate activation and aggregation of platelets at the site of injury.In addition, an estimated one third to a half of total αIIbβ3 and onethird or more GPVI receptors reside in α-granules and are trafficked tothe surface of the platelet following platelet activation, further amplify-ing platelet aggregation [31].

3.2.1. Thrombospondin and CD36 in thrombus stabilisationIn addition, a recent study identified the thrombospondin (TSP1)/

CD36 axis as another pathway that may help differentiate physiologicalhaemostasis from thrombosis. TSP1 is one of the most highly expressedproteins in platelet α-granules and unlike vWF or fibrinogen is presentin plasma in very low concentrations. As shown by Kuijpers et al.,knockdown of the TSP1/CD36 axis in mouse platelets leads to a defectin thrombus stabilisation without affecting primary haemostasis [32].Initial platelet adhesion under shear in whole blood was not affectedin Tsp1−/− mice, but activation as measured by PS exposure was signif-icantly reduced. Similarly, they also showed that in the whole bloodperfusion model, Tsp1−/− or CD36−/− thrombi disintegrated faster,but the thrombus size was the same between genotypes. In vivo bothCD36−/− and Tsp1−/− mice showed longer time to occlusion andincreased embolization in thrombosis assays. The Tsp1/CD36 axis maytherefore be another contributor to the complex haemostasis/thrombosisbalance.

3.2.2. α-Granules in coagulationFactors V, XI and XIII are all stored in α-granules, as is the throm-

bin precursor, prothrombin. Platelet derived Factor V is a potentprocoagulant, thanks to higher resistance to activated Protein C-mediated inactivation than its plasma counterpart and targeted releaseat the site of injury [33,34]. It was also shown to explain the paradoxi-cally mild bleeding diathesis in patients with congenital plasma FactorV deficiency, its residual secretion from platelets being able to rescuethrombin generation and thereby prevent bleeding [3]. Likewise,releasate from washed platelets, even from plasma Factor XI-deficientdonors, was able to correct the clotting defects observed in FactorXI-deficient plasma in vitro [35].

Importantly, platelet α granules also contain numerous anti-coagulation cargoes. Tissue factor pathway inhibitor (TFPI), protein S,protease nexin-2 (amyloid β-A4 protein), plasmin and its inactive pre-cursor plasminogen can all limit progression of coagulation by inhibitingor cleaving activated clotting factors or initiating fibrinolysis [31]. Theseapparently conflicting functions of platelets in the coagulation pathwayare essential to sustain haemostatic balance and to prevent pathologicalthrombosis under physiological conditions.

4. Importance of secretion in normal haemostasis: lessons frompatients

Since the primary function of platelets is haemostasis, or preventionof bleeding, it is no surprise that many patients presenting with abnor-mal bleeding are found to have defects in their platelet function and se-cretion. There are two classes of heritable bleeding disorders associatedwith poor platelet secretion: (i) those that result from defective plateletgranule formation, leading to low numbers of platelet granules andcargo and (ii) those that arise from defects in secretory machinery,where plateletsmay have normal numbers of granules, but have defectsin their ability to secrete them. Both of these classes of secretiondeficiency have helped us to understand the role and mechanisms ofplatelet secretion on different levels.

4.1. Platelet granule formation defects

Platelets derive from megakaryocytes (MKs) — large polyploid pro-genitor cells that reside primarily in the bone marrow. The synthesisand packaging of granules are thought to occur mainly at the earlymegakaryocyte stage [36], but little is known about exact mechanismsof granule sorting and trafficking to their final destination in platelets[37]. Limited evidence suggests that, unlike most of the other secretoryorganelles that bud from the Golgi apparatus, both α- and dense gran-ules may instead originate from the multivesicular bodies (MVBs)/lateendosomes in megakaryocytes [38]. Most inherited platelet storagedefects involve either α- or dense granules, but not both, suggesting theexistence of distinct granule biogenesis pathways for the two subtypes.Understanding of congenital platelet disorders has mademajor contribu-tions to our understanding of granule biogenesis as well as shed light onthe roles of platelet granules in haemostasis and thrombosis.

4.1.1. Dense granulesSince dense granules are lysosome-related organelles, the best de-

scribed dense granule formation defects are associated with systemiclysosome-like organelle biogenesis defects [39]. Therefore patientstend to manifest with other systemic lysosome-related organellessymptoms such as innate immunity defects [40], and in both humansand related mice genetic models of disease melanosome deficiency,resulting in defective eye, skin and hair or coat pigmentation, is oftenpresent [41].

Hermansky–Pudlak Syndrome (HPS) was first described in 1976[42], but it is only recently that advances in genomic approachesallowed for identification of the genes responsible for this diseasewhich in turn increased our understanding of granule biogenesis inplatelets. The lack of a secondary aggregation response of platelets toexogenous stimuli in HPS patients means that bleeding complicationsare common, although bleeding is not the most common cause ofdeath among HPS patients. Bleeding manifestations include spontane-ous bruising, epistaxis,menorrhagia, and prolonged oozing after traumaor minor surgery such as a tooth extraction [43]. Distinct subtypes ofHPS are caused by defects in one of at least eight genes (HPS-1 throughHPS-8) encoding the HPS proteins which interact with each other incomplexes termed BLOCS 1–3 (biogenesis of lysosome-related organ-elles complexes 1–3) [44].

Another dense granule storage defect with much poorer prognosisthan HPS is Chediak–Higashi syndrome (CHS) [45]. Again, patientslack dense granules and present with bleeding diathesis and decreasedpigmentation, accompanied with severe immunologic defects and neu-rological dysfunction [46]. The CHS gene has been cloned and a series ofmutations described, but the function of the affected protein (lysosomaltrafficking regulator, LYST) remains unknown [46]. Its distinct structuraldomains including the BEACH (Beige and Chediak–Higashi) domain[47] suggest a function in membrane trafficking and organelle biogene-sis, similar to genes affected in HPS.

Fig. 2. Currently accepted model of platelet secretion. Three SNARE proteins: trans-membrane VAMP8, with some additional VAMP2 and VAMP3 contribution (blue),and membrane-anchored STX11 (green) and SNAP23 (red) form the core SNAREcomplex in platelet secretion. MUNC13-4 and MUNC18-2 (purple) are also essentialfor secretion, although their exact mechanism of action is not fully understood.RAB27b, along with other small GTPases (orange) is also implicated.

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Isolated dense storage pool disorders not associated with systemicdefects in lysosomal-related organelles are quite common and can re-sult inmild to severe secretion defectswith orwithout affecting second-ary aggregation, and thus presenting with varying degrees of bleedingdiathesis [48]. Identification of the genes involved in those defectscould potentially help understand the differences between haemostasisand thrombosis for clinical benefit (see later sections in this review).

4.1.2. α-Granulesα-Granules are the storage site for many proteins including those

synthesised inMK or endocytosed fromplasma,withmultiple functionsin haemostasis and other processes described later in this article.Mechanisms of α-granule formation are less well understood. Defectsof protein packaging and α-granule biogenesis in MK result in a set ofheterogeneous disorders collectively known as Grey Platelet Syndrome,resulting in a usually mild to moderate bleeding disorder which canhowever on occasions be life-threatening [49]. In 2011, next generationDNA and RNA sequencing technologies were used by 3 groups indepen-dently to identify mutations in the NBEAL2 (neurobeachin-like 2) genein a large number of GPS cases [50–52]. NBEAL2 belongs to the samegene family as LYST responsible for CHS, and appears to be directly im-plicated in α-granule biogenesis in MKs. Nbeal2−/− mice show defectsin primary thrombus formation and are protected from inflammatorybrain infarction following focal cerebral ischaemia [53,54]. They alsohave defects in tissue repair after injury, reflecting the importance ofplatelet α-granule secretome beyond the haemostasis/thrombosis con-text [53]. Another α-granule biogenesis disorder, arthrogryposis, renaldysfunction, cholestasis syndrome (ARC) is a severe multisystem child-hood disorder caused by mutations in a novel Sec1/Munc18 familymember VPS33B [55]. VPS33B is known to be involved in intracellulartrafficking in yeast, but its role in α-granule biogenesis remains to beelucidated. Quebec syndrome on the other hand is not associated withgranule biogenesis defects as such, but with degradation of α-granulecargoes due to marked increase in urokinase plasminogen activator atthe megakaryopoietic stage, resulting in production of profibrinolyticplatelets [56,57].More research is needed to fully understand the geneticbasis forα-granule formation and defectswhich could perhaps enable ustomodify them for clinical benefits associatedwith their multifunctionalcargoes.

4.2. Defects in secretory machinery

Relatively recently another family of congenital disorderswas linkedwith its genetic correlates to help us understand how secretion in plate-lets is regulated. Familial haemophagocytic lymphohistiocytosis (FHL)patients present with an abnormal bleeding diathesis without anymor-phological changes to platelet granules. Unlike storage pool disordersthat rarely affect both α- and dense granules, some FHL subtypes affectall granule types, suggesting a common ‘core’ mechanism to regulatesecretion. In addition, systemic complications beyond platelets suggesta common mechanism of secretion in other haematopoietic cells.

Indeed, platelets share their secretory machinery, centred on theSoluble NSF Attachment Protein Receptor (SNARE) family of proteins,with other mammalian cells. In the simplest model, the universalSNARE-mediated fusion involves transport of the vesicle to the targetmembrane and ‘priming’ it for release, followed by calcium-mediatedconformational change in the SNARE complex that leads to the comple-tion of membrane fusion, and eventually to the release of granulecontents [58]. Four core SNARE proteins, characterised by 60 aminoacid coiled-coil SNARE domains, as well as SNARE-associated proteinssuch as Sec1/Munc18, Munc13-4 and small GTPase families regulatethat process [59], but our understanding of the machinery remainsincomplete. SNAREs were first identified in platelets in 1997 [60], andsince then, our understanding of the mechanisms of secretion has in-creased tremendously (see Fig. 2). However, some questions regardingthe identity of the central ‘players’ still remain to be addressed.

In FHL subtypes 3, 4 and 5, deficiency ofMUNC13-4 [61], syntaxin 11(STX11) [62] and MUNC18-2 [63,64], respectively, leads to substantialdefects in platelet granule secretion. Prior to the discovery of thosegenes, the mechanisms of SNAREs in human platelets were basedupon treatment of streptolysin-O permeabilised plateletswith function-al blocking antibodies and the correlative evidence of phosphorylationof certain proteins occurring at a similar rate to secretion [65–68].Gene knockout mouse models of a range of SNAREs (namely vesicle-associated membrane proteins, VAMPs) have also helped to identifythe potentially important players [69]. However, only studies on theFHL patients provided unequivocal evidence supporting the role ofthose three proteins in human platelets. Even the loss of the coreSNARE STX11 in FHL4 patients [62] or the knockout of VAMP8 coupledwith tetanus neurotoxin (TnT-LC) treatment in mouse platelets [69],does not lead to full ablation of secretion, suggesting ranked redundan-cy and compensation mechanisms [69]. As discussed in more detail inour recent review (Golebiewska and Poole [59]), it is likely thereforethat additional SNAREs are involved in the regulation of secretion.

5. Platelets — not only primary haemostasis

The roles of platelets are not limited to initial aggregation and plugformation. The cascade of events leading to vessel repair is summarisedin Fig. 3. Platelets are known to be involved at all the stages of thiscascade, including coagulation, immune cell recruitment and inflamma-tion, andwoundhealing, angiogenesis and remodelling. The importanceof platelets in each of the steps varies in different vasculatures. Inaddition, our understanding of the mechanisms of thrombus formationand haemostasis comesmainly from animal models where haemostasisis initiated by physical injury to the healthy vessel. In humans, themechanism by which ECM becomes exposed to blood flow, especiallyin the arterial circulation, is more often than not rupture of an athero-sclerotic plaque,which alsomeans the vesselmay be inflamed, stenosedand generally unhealthy, adding to the complexity of those interactions.Therefore themechanismsdiscussed here are likely to be anoversimpli-fication of the pathological arterial thrombosis. Different mechanismsare likely to contribute to venous and microvascular versus arterialthrombosis, with alterations in sheer differentially influencing throm-bus formation.

Fig. 3. Simplified illustration of events leading to vessel injury repair. Following the initial platelet plug formation, coagulation cascade activation results in production of fibrin thatreinforces the thrombus. Then leukocyte recruitment from the blood leads to an inflammatory response and antimicrobial response. Finally, wound healing and vessel wall remodellinglead to restoration of the continuity of the endothelium. Secretion of the 300+ bioactive substances from platelet intracellular stores is likely to be a major driver of these events.

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5.1. Platelets in inflammation

After the stable fibrin and platelet rich clot forms to stop the imme-diate bleeding, the further steps of the repair process begin. Close bidi-rectional cooperation between the haemostatic and immune systemsis required to ensure restoration of normal tissue function followingthe injury.

In the arterial circulation, the high shear environment leads to washout of any chemoattractant molecules from the site of the injury. There-fore platelets stably adherent to the ECM are required for ‘capturing’circulating immune cells and recruiting them to the thrombus. Plateletα-granules mediate inflammatory responses both by expressing adhe-sion receptors that facilitate interactions with endothelial cells andleukocytes, and by secreting a wide range of chemokines. P-selectintranslocates to the cell surface from α-granules on adherent plateletsand can recruit circulatorymonocytes, neutrophils and lymphocytes, in-ducing inflammatory responses in those cells [70]. Platelets also containabundant chemo- and cytokines, in particular CXCL4 (PF-4) and 7 atconcentrations 1000-fold greater than the plasma concentration [71].PF-4 has been shown to drive monocyte activation and differentiation[72], as well as neutrophil adhesion [73] and monocyte recruitment tothe endothelium [74].

Inflammatory cell infiltration of the arterial subendothelium, pro-moted by platelet cytokines, also contributes to atherosclerotic plaqueformation [75]. In addition, serotonin released from dense granulesupon activation by the inflamed endothelium further contributes torecruitment of immune cells to the vascular wall [76]. Transendothelialmigration of neutrophils both in human and mouse models of inflam-mation, is at least in part mediated by a platelet α-granule-derivedP-selectin and P-selectin glycoprotein ligand (PSGL)-1 interaction [77].In addition, the mechanism by which platelets are able to guide leuko-cyte migration through thrombi has also recently been identified [78].Release of NAP-2, the CXCR1/2 ligand, from platelet α-granules, leadsto a chemotactic gradient inside the growing thrombus. This in turnenables leukocytes to migrate through the physical barrier which is

the growing thrombus and towards the site of the injury and endothe-lium [78].

In addition to their essential roles in physiological responses to inju-ry, platelet–immune cell interactions are also likely to be very importantin the pathophysiology of atherosclerotic plaque formation. Modulationof platelet secretion could therefore help reduce plaque growth. Forexample, inhibition of platelet derived CXCR4–CCL5 heterodimerformation was shown to attenuate monocyte recruitment to inflamedvessel wall in ApoE−/− mice [79] and also reduce aneurysm formationin those animals.

5.2. Antimicrobial responses

Trauma to tissue or introduction of foreign objects into the vessel isthe most direct way by which bacterial and viral pathogens can invadethe body. Therefore it stands to reason that antimicrobial andhaemostatic responses are intimately linked. Platelets are the most nu-merous cells first to arrive at the site of injury, and the role of platelets incombating pathogens is increasingly apparent. It has been known formany years that platelets localize and adhere to sites of bacterial lesionsin the circulation, for example in endocarditis [80]. However, only in2002 was it shown that many of the cytokines secreted by plateletshave direct microbicidal properties [81], and that platelets can directlyinternalize pathogens [82]. At the same time, activation by pathogen-associated molecular patterns (via Toll-like receptors, TLRs) leads tothe release of more cytokines, such as PF-4 or CCL5 (RANTES) whichleads to recruitment of circulating inflammatory cells [83] and ensuresa rapid response to infection. Interestingly, TLR-mediated plateletresponses to different bacterial species vary — for example, secretorypatterns of PDGF (platelet-derived growth factor) and RANTES,but not P-selectin or PF-4 differs between platelets in response toEscherichia coli or Salmonella species [84]. The increasing importanceof platelets in the antimicrobial response has been reviewed exhaustive-ly by Yeaman [85]. The precise mechanisms of the platelet antimicrobialresponse and differential secretion remain to be elucidated.

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5.3. Wound healing

The final steps of the haemostatic response to external injury, orrupture of atherosclerotic plaque in the arterial circulation, lead torestoration of the integrity of the vascularwall. This process involves or-chestrated proliferation and migration of smooth muscle cells (SMCs),fibroblasts and endothelial cells. Platelets' wealth of growth factorsand chemokines is known to contribute to regulation of those processes.Platelet derived growth factor (PDGF) in particular is instrumental inregulating SMC proliferation and migration in both the arterial andvenous circulation [86]. Neointimal hyperplasia and restenosis, such asoften occurs following balloon angioplasty, were already shown to bemediated by PDGF 25 years ago [87]. Subsequently approachestargeting the PDGF signalling pathway developed but despite promisein animal models, PDGF inhibition has not yet shown to be effective inhumans [88].

Another important mediator in healing and remodelling is platelet-derived SDF-1α which mediates CD34+ bone marrow derived progen-itor cell recruitment to the injury site and their differentiation intoendothelial progenitor cells [89–91]. Inhibition of SDF-1α binding toits receptor CXCR4 was shown to retard diabetic wound healing in ex-perimentalmodels by impairing cellularmigrationwhile concomitantlyprolonging the inflammatory response [92]. At the same time, platelet-mediated differentiation of CD34+ progenitor cells into mature foamcells is of particular importance in atherosclerotic plaque formation[93]. SDF-1α is also a potent pro-angiogenic mediator, enhancingCXCR4-expressing endothelial cell proliferation, differentiation,sprouting and tube formation in vitro and in vivo [94–96]. Other pro-and anti-angiogenic factors such as VEGF (vascular endothelial growthfactor) and endostatin are also released from platelets [97], supportingtheir crucial role in regulating the revascularisation of the damagedtissue.

In addition to angiogenic factors, platelets also store and secrete anumber of tumour necrosis factor (TNF)-α-related apoptosis regulatorssuch as CD95, Apo2-L and Apo3-L which can induce inflammatoryresponses and apoptosis in other circulating cells, as well as anti-apoptotic molecules [98]. The balance between pro-apoptotic andanti-apoptotic molecules, adequately promoting survival or eliminatingthe cells from the wound site, is also crucial for regulation of healing.

In summary, the increasing understanding of the role of platelet se-cretion in wound healing and tissue regeneration led to the develop-ment of multiple applications, especially in trauma management.Autologous platelet-rich plasma gels and growth factor concentratesare used in a number of clinical scenarios, from healing of acute skinwounds and diabetic ulcers to regeneration of tendon, ligament andnerve tissue [99]. Modulation of secretion for clinical benefit on theother hand is the area that remains to be further investigated.

6. Platelet secretion and disease

Platelets' wealth of potent bioactive molecules is essential forthe regulation of the complex physiological processes outlined so far.However, their ability to influence other cells means that they are alsocentral to the pathophysiology of disease. Platelet secretion can causedisease beyond the obvious thrombotic scenarios, such as atherosclero-sis, or heart attacks and strokes resulting from occlusive thrombusformation in circulation. Multiple roles in conditions from cancerprogression and metastasis, to chronic inflammatory conditions, sepsisand acute lung injury have also been proposed.

6.1. Malignancy

Early evidence for platelet function in cancer progression wasoriginally derived from studies in a mouse model of severe thrombocy-topenia [100]. In 1968, Gasic et al. reported that platelet depletioncaused by neuraminidase pre-treatment led to a significant decrease

in number of lung metastases following tumour inoculation [100]. Itwas later found, that infusion of resting, but not degranulated platelets,could rescue the tumour metastatic potential, in addition to preventingthrombocytopenia-induced tumour bleeding [101]. Thus, platelet secre-tion appears to be the essential permissive and protective factor fortumour metastasis.

Platelet α-granules contain a number of growth factors that are im-portant in physiological wound healing and angiogenesis includingVEGF, PDGF, epidermal growth factor (EGF), and transforming growthfactor beta (TGFβ) [102]. The same molecules can be ‘hijacked’ by can-cer cells to potentiate tumour survival andmetastasis, which is essentialfor tumour survival beyond 1–2 mm in size. PDGF as a potent mitogenfor mesenchymal cells including fibroblasts, smooth muscle cells, andglial cells, was a prime suspect in mediating cancer metastasis [103]. In-deed, overexpression of the PDGF receptor on tumour cells correlateswith increased metastatic potential in breast cancer [104]. PDGF/PDGF-receptor interaction was also found to promote lymphangiogenesis andlymphatic extravasation in thyroid cancer [105]. Circulating VEGF, most-ly derived from platelets, not only facilitates angiogenesis and increasesvascular permeability, which promotes tumour cell extravasation [106],but was also recently implicated in the function of cancer stem cellsand tumour initiation [107]. Platelet-derived TGFβ, in synergy withother platelet-bound factors, was recently shown to be an essentialpermissive factor for metastasis, specifically in epithelial–mesenchy-mal-like transition [108]. On the other hand, platelets also containanti-angiogenic factors (such as PF-4, TSP-1 and endostatin) that canlimit cancer survival and growth [109]. This again provides evidencefor themultifaceted and complex roles of platelet secretion in patholog-ical processes. Regulating platelet secretion could be a viable target foranti-metastatic therapies, and indeed, PF-4 is being developed as anovel anti-angiogenic cancer therapy [110].

In addition, Schumacher et al. recently showed that Unc13dJinx mice,that lack expression of Munc13-4, were protected from lungmetastasesand suggested that lack of ATP secretion from platelet dense granuleswas responsible for that protection [111]. Although we and others [12,14,112] had previously shown a defect in α-granule secretion in thesemice, consistent with an amplificatory role for secreted ADP(as described earlier in this article andHarper et al. [112]), they convinc-ingly argue that ATP acting via P2Y2 receptors on endothelial cellscontributes another mechanism that increases endothelial cell perme-ability and facilitates tumour cell extravasation, additional to the well-established pro-angiogenic functions of α-granule releasate.

6.2. Other conditions

Due to their ability to initiate and sustain inflammatory responsesplatelets are often one of the main culprits and reasons for mortalityin a series of other conditions. Therefore in some cases, platelet inhibi-tion or depletion correlates with better outcomes. The exception tothat is sepsis, where a role for platelets in exuberating the disease wasproposed. On the one hand, platelet accumulation in vital organs cancause inflammatory responses by platelet-mediated immune cell re-cruitment [113] and can lead to acute organ injury. On the other hand,disseminated intravascular coagulation leads to thrombocytopeniawhich in turn increases vascular permeability, normally stabilised byplatelet secreted VEGF [114]. This in turn can predispose to edema,shock and organ failure that more often than not lead to death in severesepsis [115].

Theplatelet P-selectin/PSGL interactionmediating platelet–neutrophilinteraction in infection, is also a proposed mechanism for monocyterecruitment accompanying transplanted graft rejection [116] andblocking P-selectin was recently shown to protect mice fromantibody-mediated rejection [117]. Similarly, P-selectin mediatedneutrophil recruitment [118] and platelet-derived CXCL4–CCL5 hetero-dimer formation [119] are thought to be major pathogenic contributorsto the acute lung injury.

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Via their pro-inflammatory potential, platelets are also linked to in-flammatory bowel disease (elevated levels of RANTES are found inCrohn's disease patients) [120], migraines (IL-1 and β-thromboglobulinare implicated in their aetiology) [121] and asthma (increased P-selectin mediated leukocyte recruitment thought to contribute topulmonary inflammation) [122]. Therefore it is reasonable to hypothesisethat many further conditions associated with systemic inflammationcould be linked to platelet function or dysfunction.

7. So… how is it all regulated?

As outlined in this review, the roles of platelets extend beyond initialplatelet adhesion and aggregation at the site of injury. Platelets are not‘merely’ contributing to processes such as coagulation, inflammation,and antimicrobial responses or wound healing, they are arguably cen-tral and essential in themajority of those processes. As outlined, plateletsecretion of 300+ molecules from intracellular stores is likely to be amajor driver of those interactions (summarised in Fig. 4). Yet, clearlyour current understanding of platelet secretion at the molecular levelis insufficient to explain how the careful balance between all the potentmitogenic, pro-angiogenic, anti-angiogenic, pro-inflammatory, anti-inflammatory and adhesive factors released from granules undercertain activation conditions is achieved. If all the contents of theintracellular stores were released in an uncoordinated manner, thethrombus growth and all the following events would be similarly unco-ordinated. It seems implausible that just a handful of SNARE proteinscan achieve that. Therefore several other mechanisms and avenueshave been investigated recently.

One of the hypotheses put forward is that platelet granules are notuniform, and that certain factors, especially α-granule cargoes, may bedifferentially packaged and thus released ‘thematically’. Ma et al.observed that platelet stimulation with specific protease-activatedreceptor (PAR) 1 or PAR4 agonist resulted in the preferential releaseof VEGF or endostatin, (anti- and pro-angiogenic factors, respectively)[123]. Differential packaging of granules was a very attractive explana-tion for that observation, and indeed, spinning-disk confocal micro-scope images of resting platelets, clearly showing distinct localisationof two of the main α-granule cargoes, fibrinogen and vWF, have beenpublished [124]. Immunogold labelling by the Italiano group alsoshowed α-granule populations containing either endostatin or VEGFbut not both [125]. More recently however, a super-resolution analysisof 15 platelet α-granule cargoes using 3D structured illumination

Fig. 4. Summary of some of the platelet granule cargoes and their functional classification.α-Grcoagulation-related factors), hence amechanism(s) ensuring tight spatial and temporal regulatiof the physiological (green) and pathological (red) processes that are now known to be affecteMany of those physiological and pathological processes can be affected by a combination of fcargoes have multiple roles, while the roles of others still have not been fully elucidated.Adapted from Golebiewska and Poole [59].

microscopy (3D-SIM) failed to confirm any functional co-localisation[126]. van Nispen tot Pannerden et al. identified different classes of α-granules, observing snapshots of resting platelets under EM [127].They reported a distinct population of approximately 50 nm-wide‘tubes’ that they designated ‘tubularα-granules’. They contained fibrin-ogen but not vWF, as opposed to the usual, ‘spherical’α-granuleswhichcontained both. Differential packaging of α-granule cargo remains anattractive explanation for the observed separation of pro- and anti-angiogenic releasates, but the difficulties associated with platelet re-searchmean that we still lack definitive confirmation of this hypothesis.No similar studies on dense granule secretion exist.

In 2012, the Whiteheart group performed a systematic quantifica-tion of granule secretion using micro-ELISA arrays for 28 distinctα-granule cargo molecules in response to 4 different agonists [10],quantifying the kinetics of secretion of each cargo. This was the firsttime that kinetics of secretionweremeasured— no conclusive evidencefor the ‘fast’ dense granule, ‘slower’ α-granule and ‘slowest’ lysosomesecretion model previously existed. They found that not only secretionof different cargoes varied depending on the agonist used, but thatthere was also an overlap in secretion kinetics of cargoes of opposingfunctions. In conclusion, they proposed that the kinetics of releasecould not independently explain differential secretion and granuleshape, proximity to the membrane or cargo solubility is likely to influ-ence the kinetics [10]. It is also likely that different SNARE complexescould drive the fusion with different kinetics— another reason to ques-tion the ‘one sizefits all’ SNARE complex in platelets that is currently ac-cepted (SNAP23–VAMP8–STX11). SNARE redundancy and functionalspecialisation have been described in other cells. For example twoendosomal SNARE complexes exist, with one mediating the early tolate endosome transport, while the other — late endosome to surfacestep [128]. Similarly, human neutrophils have several different SNAREsthat regulate secretion of specific granules [129]. Therefore it is possiblethat differential secretion of cargoes in platelets is also mediated by notone, but several different SNARE complexes. In addition, it is thoughtthat platelet granules may fuse with each other or with the open cana-licular system (OCS) rather than directly with the plasma membrane,which could accounts for different secretion rates depending uponthe stimulation conditions [130]. Signalling cascades between agoniststimulation and SNARE-mediated secretion events also remain largelyunknown. The accepted model of platelet signalling principallyconverges on the increase in cytosolic calcium to drive the cellular ma-chinery upon activation, but the signalling downstream of receptors is

anules (left hand side) contain cargoes with often opposing actions (e.g. angiogenesis andon of secretion is likely to be in place to allow platelets to exert theirmany functions. Somed by platelet secretome are also listed (amber indicates both depending on the scenario).actors. It should be noted that although functions are assigned to each cargo here, many

160 E.M. Golebiewska, A.W. Poole / Blood Reviews 29 (2015) 153–162

likely to diverge at some point to appropriately drive different secretoryevents. Again, the nature of plateletsmeans that studying cell signallingin real time is problematic. Finally, of course, dissecting platelet functionin vitro or in animal models of thrombosis does not necessarily reflecttheir function in human health and disease, and this remains a majorchallenge for future research. In summary, our understanding of plate-let secretion is increasing, but remains rudimentary at the momentwith no clear answers to the question of how the remarkable complex-ity is achieved.

8. Conclusions

Platelet secretion is undoubtedly of pivotal importance for regulat-ing not only canonical platelet functions, but also mediating theirimpact on other cells, and understanding platelet secretion may haveconsequences reaching far beyond the ‘platelet field’. Yet platelets re-main lesswell studied in terms ofmechanisms governing their function,relative to other secretory cells such asneuroendocrine or immune cells.In this review we have shown examples of the ways in which plateletsecretion is essential for sustaining haemostasis and ensuring anadequate response to injury, but also how the same mechanismsmay mediate pathological responses. This means that any approachestargeting platelets or platelet secretion have to be carefully tailored foreach specific application. Dissectingmechanisms of differential secretionof different cargoes may be helpful in designing therapies that can avoidcurrently common bleeding side effects while limiting pathologicalinflammation, tissue hypertrophy or cancer cell metastasis.

Conflict of interest statement

None.

Acknowledgements

This work was supported by the British Heart Foundation(Programme Grant RG/10/006/28299, FS/09/009/26444). EMGand AWP contributed equally to the production of this paper.

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