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Cardiovascular & Haematological Disorders-Drug Targets, 2008, 8, 99-117 99 Beyond Hemostasis: The Role of Platelets in Inflammation, Malignancy and Infection Archibald McNicol1,2 and Sara J. Israels3,4,* 1 Departments of Oral Biology, 2Pharmacology & Therapeutics, 3Pediatrics & Child Health and 4the Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba Abstract: Platelets play a complex role in hemostasis and thrombosis. The expression of multiple membrane receptors, both constitutive and activation-dependent, mediates platelet adhesion and aggregation at sites of vascular lesion. Platelet activation leads to exocytosis of granular constituents, release of newly synthesized mediators, and discharge of membrane-bound transcellular signaling molecules. Many of the same mechanisms that play a role in hemostasis and thrombosis facilitate platelet participation in other physiological or pathological processes including inflammation, malignancy and the immune response. Platelet receptors such as GPIb/IX/V, P-selectin, P-selectin glycoprotein ligand 1, CD40 and the IIbß3 integrin, crucial to hemostasis, have been implicated in the progression of such inflammatory conditions as atherosclerosis, rheumatoid arthritis and inflammatory bowel disease, in the progression and metastatic spread of malignancies, and in the immune response to bacterial challenge. The release of platelet granular contents, including adhesive proteins, growth factors and chemokines/cytokines, that serve to facilitate hemostasis and wound repair, also function in acute and chronic inflammatory disease and in tumor cell activation and growth. Platelets contribute to host defence as they recognise bacteria, recruit traditional immune cells to the site of infection and secrete bactericidal mediators. The primary focus of this review is the “non-haemostatic” functions of platelets in physiological and pathological states. Key Words: Platelets, inflammation, metastasis, angiogenesis, immunity. INTRODUCTION Platelets are the smallest cells in circulating blood, have a biconvex disc structure with an equatorial diameter of 2-3 m, and are anucleate. They are derived from progenitor megakaryocytes in the bone marrow. Following their normal life-span of 8-10 days they are removed from the circulation during passage through the spleen. The role of platelets in hemostasis was recognised very early [1]; their role in thrombosis sometime later [2]. The consequences of platelet dysfunction, and the potential of the platelet as a therapeutic target, have been realized and exploited [3]. In invertebrates the functions of host defence and hemostasis are shared by a single cell type. The best-characterized examples of this are the nucleated amebocytes of the horseshoe crab--these aggregate and release their granule contents in response to such stimuli as exposure to foreign surfaces or bacterial endotoxin [4]. Non-mammalian vertebrate thrombocytes and mammalian platelets have retained non-hemostatic roles in host defence and inflammation. During the last decade, advances in functional analyses, knock-out models, microscopy and characterization of the platelet proteome have dramatically expanded our understanding of the role of platelets in a variety of patho-physiological states, including inflammatory disorders, tumor progression and infection. PLATELETS IN HEMOSTASIS AND THROMBOSIS The dynamics of blood flow dictate that circulating platelets are found primarily along the vessel wall, well posi*Address correspondence to this author at the Manitoba Institute of Cell Biology, Rm. ON2021A, 675 McDermot Ave. Winnipeg, Manitoba, Canada, R3E 0V9; Tel: (204) 787-4141; Fax: (204) 786-0195; E-mail: [email protected] 1871-529X/08 $55.00+.00 tioned for rapid response at sites of endothelial lesions. The platelet response to vessel wall injury with exposure of subendothelial collagen is characterized by the phases of adhesion, amplification and stabilization. This response is mediated by a variety of cell surface receptors, some of which are constitutively present in an active conformation, some of which require structural rearrangement to gain functional integrity, and others that translocate to the platelet surface following platelet activation [5]. The platelet glycoprotein (GP) Ib/IX/V complex acts as an initial adhesive receptor at the endothelial lesion by binding to collagen-associated von Willebrand Factor (vWF). Under low shear conditions, collagen binds to platelets via the 2ß1 integrin (GPIa/IIa); GPVI and CD36 also bind to collagen. Interestingly, both GPIb/IX/V and CD36 are platelet receptors for thrombospondin-1, an alternative to vWF in mediating platelet adhesion under defined conditions. Several other platelet surface integrins function as adhesive receptors, notably IIbß3 (immobilized fibrinogen), 5ß1 (fibronectin), Vß3 (vitronectin) and 6ß1 (laminin) [5]. Following adhesion, a number of soluble mediators engage specific receptors that result in platelet activation and amplification of the initial response as additional platelets are attracted to the site. Thrombin, a product of the coagulation cascade, is a powerful platelet agonist, mediated by the protease-activated receptors (PAR) 1 and 4, and by GPIb/ IX/V. Activated platelets release ADP and thromboxane (Tx) A2, both of which promote positive feedback stimuli via P2Y 1 and P2Y12, and via TP and TPß receptors, respectively. In addition, platelets express receptors for epinephrine, platelet activating factor (PAF), serotonin, vasopressin and immuno© 2008 Bentham Science Publishers Ltd. 100 Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 globulin G (IgG), each contributing to, and enhancing, platelet activation [5]. Additional platelets are recruited to the site of injury through two specific processes: First the release, by exocytosis from platelet dense granules, of serotonin and of a nonmetabolic pool of ADP- the latter, in particular, is an important secondary mediator of platelet activation [6]. Second the synthesis and release of TxA2 a pro-aggregatory mediator critical to the full haemostatic function of the platelet [5]. TxA2 is generated by the sequential actions of phospholipase A2, which liberates arachidonic acid from membrane phospholipids, cyclooxygenase-1, which converts arachidonic acid to prostaglandin endoperoxides (PGG2/PGH2), then thromboxane synthetase for the final synthesis of TxA2. Attenuation of either of these amplification pathways inhibits platelet function and significantly prolongs bleeding in vivo. Therapeutic ADP receptor antagonists, such as clopidogrel, and the cyclooxygenase inhibitor aspirin are effective anti-thrombotic agents. Additional platelets recruited by ADP- or TxA2-mediated mechanisms combine with the fragile layer of adherent platelets at the site of injury to form a consolidated hemostatic plug, or aggregate. This is facilitated by an activationmediated conformational change in the IIbß3 integrin, leading to the expression of its adhesive protein binding domain. Fibrinogen is the major physiological ligand for this domaina bivalent molecule, fibrinogen binds to IIbß3 molecules on adjacent platelets, establishing firm cross-linking to form a stable plug [5]. In addition to the release of dense granule constituents, two other exocytotic processes occur during platelet activation: the release of alpha granules and of lysosomal granules. Alpha granules contain a large number of proteins of diverse function, including adhesive proteins, chemokines, cytokines, coagulation factors and protease inhibitors (Table 1; note that this table provides the definitions for all alpha granule protein abbreviations that appear in the text) [7,8]. Lysosomal granules contain a variety of acid proteases, acid glycosidases, acid phosphatases and aryl sulfatases [5]. During the process of exocytosis, granular membranes fuse with the plasma membrane, resulting in the translocation of granular membrane proteins onto the platelet surface. In this way, increased numbers of constitutively-expressed proteins, and novel proteins, appear on the platelet surface. Proteins up-regulated on the platelet surface in this manner include the IIbß3 integrin, P-selectin (CD62P), CD63, and the CD40 ligand (CD40L; CD154) [5]. The phospholipid component of platelet plasma membranes is significantly altered following activation. The increase in expression of phosphatidylserine on the external leaflet of the plasma membrane provides a larger surface area for the assembly of coagulation factor complexes and generation of thrombin. Two populations of small membrane vesicles, microparticles and exosomes, are released from activated platelets [9]. Microparticles have a similar phosphatidylserine-enriched composition to that of the plasma McNicol and Israels membrane of activated platelets and serve to significantly increase the surface area available for thrombin generation. Thrombin activates additional platelets and cleaves fibrinogen to fibrin; polymerized fibrin stabilizes the platelet plug. Exosomes are smaller membrane vesicles released from activated platelets following fusion of multivesicular bodies and alpha granules with the plasma membrane [9]. Although exosomes do not have procoagulant function, they do express CD63 and prion protein, and have been implicated in the inter-cellular transfer of information. PLATELETS AND INFLAMMATION Atherosclerosis The first suggestion that platelets have a role in inflammation came, not surprisingly, from a pair of observations related to atherosclerosis [10,11]: that atherosclerosis was an inflammatory lesion, and that platelets were not merely passive constituents trapped in the atherosclerotic plaque. The etiology of atherosclerosis is highly complex [12,13] and a detailed description is beyond the scope of this review. Briefly, increased levels of circulating low density lipoprotein (LDL) diffuse through endothelial cell junctions into the subendothelial matrix. The trapped LDL undergoes a variety of structural modifications, including lipid oxidation, resulting in the formation, and trapping, of minimally oxidized LDL. The accumulation of minimally oxidized LDL triggers the activation of the overlying endothelial cells. This activation is characterized by a loss of vascular integrity, cytokine production, and the expression and shedding of leukocyte adhesive molecules [14], leading to the recruitment and binding of monocytes and lymphocytes. These leukocytes “roll” along the endothelium and pass through endothelial cell junctions in a classic inflammatory response. The intensity of the response is regulated by multiple factors, including genetic factors, elevated levels of free radicals generated by smoking, hypertension or diabetes mellitus, elevated levels of homocysteine, sex hormones and infections (e.g. herpes virus, Chlamydophila pneumoniae). Sub-endothelial monocytes proliferate and differentiate into macrophages that engulf highly oxidized LDL to form foam cells - accumulation of these cells appear as fatty streaks; they release extracellular lipids to form a necrotic core. Under the influence of cytokines and growth factors a mature atherosclerotic fibrous plaque is formed as the necrotic core enlarges, becomes calcified and is infiltrated by smooth muscle cells [12,13]. Although it has been long appreciated that platelets are incorporated into the thrombus that forms at the site of mature plaque, their role in the early stages of atherosclerosis has been recognised more recently [15; Fig. (1)]. The characterization of the platelet releasate has identified a large number of proteins with significant pro-inflammatory properties, including PF4, interleukin (IL)-1ß and RANTES [8]. Studies using a variety of murine knockout models established that quiescent platelets adhere to, and roll on, stimulated endothelial cells and demonstrated that there was a delay in the progression of the disease when certain adhesive proteins were absent [16]. Beyond Hemostasis Table 1. Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 101 Platelet Alpha Granule Constituents Adhesive Proteins Fibrinogen chain Fibrinogen chain Fibrinogen chain Fibronectin Thrombospondin 1 Vitronectin Von Willebrand Factor (vWF) Chemokines Other Connective Tissue-Activating Peptide (CTAPIII; CXCL7) Epithelial Neutrophil Activating Peptide (ENA-78; CXCL5) GRO- (CXCL1) I-309 (CCL1) Interleukin-8 (IL-8; CXCL8) Macrophage Inflammatory Protein 1 (MIP-1; CCL3) Monocyte Chemoattractant Protein-1 (MCP-1; CCL2) Monocyte Chemoattractant Protein-3 (MCP-3; CCL7) Neutrophil-Activating Peptide-2 (NAP-2; CXCL7) Platelet Basic Protein (CXCL7) Platelet Factor (PF4; CXCL4) Platelet Factor 4 variant 1 (PF4alt; CXCL4L1) Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES; CCL5) Stromal Cell-Derived Factor (SDF-1; CXCL12) Thymus and Activation-Regulated Chemokine (TARC; CCL17) -Thromboglobulin (-TG; CXCL7) Cytokines Growth Factors Interleukin-1 (IL-1) High Mobility Group Box Chromosomal Protein-1 (HMGB1) Basic fibroblast growth factor (bFGF) Epidermal growth factor (EGF) Hepatocyte growth factor (HGF) Insulin-like growth factor-1 (IGF-1) Insulin-like growth factor-2 (IGF-2) Platelet-derived endothelial cell growth factor (PD-ECGF) Platelet-derived growth factor (PDGF) Transforming growth factor (TGF-) Vascular endothelial growth factor A (VEGF-A) Vascular endothelial growth factor C (VEGF-C) Albumin Amyloid -A4 protein Angiopoietin-1 Angiostatin Clusterin Endostatin Factor V Factor XI Factor XIII High-molecular weight kininogen (HMWK) Matrix metalloproteinase-2 (MMP-2) Metalloproteinase inhibitor-1 Multimerin Osteonectin Plasminogen Plasminogen activator inhibitor (PAI-1) Protein C inhibitor Protein S Secretory granule proteoglycan core protein (SGPCP) Thrombocidin-1 (TC-1) Thrombocidin-2 (TC-2) Thymosin -4 Tissue inhibitor of metalloproteinase (TIMP-4) von Willebrand antigen-II 2 macroglobulin 2-antiplasmin -actinin 1 -actinin 2 -actinin 4 Protein and peptide contents of alpha granules that are released upon platelet activation, in response to a variety of stimuli. From Harrison and Cramer [7] and Coppinger et al. [8]. The process underlying the adhesion of quiescent platelets to activated endothelial cells has been the subject of considerable study. In vitro studies using activated human umbilical cord endothelial cells (HUVECs) demonstrated that antibodies to either vWF or IIbß3 attenuate platelet adhesion, supporting the role of these proteins in the process [17]. Furthermore, blocking antibodies to the primary platelet vWF receptor GPIb/IX/V inhibited atherosclerotic plaque formation in an apoE-/- murine model, establishing vWF as a mediator of platelet adhesion to dysfunctional (i.e. where endothelial cell-dependent vasodilation is impaired [14]), and to activated endothelial cells [16,18]. P-Selectin P-selectin has been implicated as a factor in the plateletrelated development of atherosclerosis. P-selectin is expressed only on the surface of activated platelets and, similarly, is expressed following activation of endothelial cells. Intravital microscopy studies in mice have demonstrated that quiescent platelets initially bind loosely to, and then roll on, endothe- lial cells; these processes are mediated by the expression of P-selectin on endothelial cells, but not on the platelets [16,17,19]. P-selectin glycoprotein ligand 1 (PSGL-1; CD15) and GPIb/IX/V have both been implicated as P-selectin counter-receptors on the platelet surface. Both PSGL-1 and GPIb/IX/V are constitutively expressed on the surface of quiescent platelets and probably function in tandem to mediate the initial interaction of platelets with endothelial cell Pselectin [16,17,19]. Regardless of the counter-receptor(s) involved, the P-selectin-mediated adhesion is fragile and reversible (Fig. (1b)). During the adhesion process, activation of platelets initiates a number of events with important pro-inflammatory implications. Activated platelets express several integrins in their “open” conformation, of which IIbß3 is the most important [16,17,19]. The IIbß3 binds adhesive proteins such as fibrinogen, vWF and fibronectin which tether the platelets to ICAM-1 and Vß2 on activated endothelial cells, thereby consolidating platelet adhesion (Fig. (1c)). Studies in an apoE/IIb double-deficient murine model showed signifi- 102 Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 McNicol and Israels Fig. (1). Role of platelets in the early development of atherosclerosis. d) e) PGSL-1 CD40 PGSL-1 Microparticles CD40 a-granule exocytosis CD40L I PGSL-I IIE,03 II II (T1b/IN/V I1i aIIbb3 11 Fbgn. I Aen. vWF Vbs.; IC.AM-] CD40L P-selectin P-sclectin GP1b/IX/V a IIE PGSL-1 0 0 P-sclectin Beyond Hemostasis Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 103 0 (Fig. 1. Contd….) Microparticles PGSL-1 RANTES CCRI/ CCR5 P-selectin Fig. (1 a). The progression of platelet involvement during the early stages of atherosclerosis. Quiescent, discoid platelets bind to the dysfunctional endothelium (broken line) and become activated, losing their discoid appearance and extending filopodia. Monocytes bind to, then roll on, the activated platelets and finally migrate through the endothelial layer. Platelet-derived transcellular mediators stimulate changes in the endothelial cells facilitating the direct binding and rolling of monocytes on the luminal surface. This progression is seen in more detail in figures 1b-1f. Fig. (1b). Platelet receptors (GPIb/IX/V, IIb3, PSGL-1), endothelial cell receptors/counter receptors (ICAM-1, V3, P-selectin), and adhesive proteins (vWF) mediate the binding of quiescent platelets to dysfunctional endothelial cells. Fig. (1c). The platelet is activated, the IIb3 integrin is in an open, fibrinogen binding conformation, and the platelets express P-selectin and CD40L. Fig. (1d). Monocytes, via PSGL-1 and CD40, bind to the adherent, activated platelet Fig. (1e). Monocytes roll along the adherent platelets to an endothelial cell junction. Concomitantly, platelets release microparticles and secrete -granule contents. Microparticles serve to deliver multiple transcellular mediators to the endothelial cell including RANTES. Fig. (1f). RANTES binds to its serpentine receptor (CCR1/CCR5) leading to an upregulation of the luminal expression of monocyte-binding adhesive receptors. cantly reduced atherosclerotic plaque development [17,20]. As outlined above, activated platelets also express P-selectin on their surface, which serves multiple pro-inflammatory roles [16,17,19,21,22]. First, P-selectin mediates the adhesion and rolling of PSGL-1-expressing immune cells (monocytes, neutrophils, lymphocytes) on the adherent platelets (Fig. (1d)). Second, P-selectin-mediated signaling leads to the clustering of IIbß3 on the platelet surface, enhancing the anchoring capacity of the integrin and facilitating firm platelet adhesion to the endothelium (Fig. (1c,d)). Third, the engagement of P-selectin appears critical in the exocytosis of platelet alpha granule-derived chemokines (Fig. (1e); Table 1) some of which have been shown to participate in the development of the atherosclerotic lesion. Fourth, P-selectin facilitates the delivery of platelet-derived chemokines, notably RANTES (see below), to the endothelium (Fig. (1e)). CD40/CD40 Ligand Platelet adhesion/activation is associated with the upregulation of CD40L, a member of the TNF family, which is also found on activated T lymphocytes, monocytes, endothelial cells and vascular smooth muscle cells [16,19,23,24,25]. The CD40L receptor is CD40, an integral membrane protein expressed on a variety of cells including B cells, monocytes, endothelial cells, fibroblasts, vascular smooth muscle cells and platelets. A proportion of CD40L is shed from activated platelets in a soluble form (sCD40L); it has been estimated that 95% of circulating sCD40L is platelet-derived [24]. The 104 Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 McNicol and Israels plasma level of sCD40L is regarded as predictive of recurrent cardiovascular disorders, such as myocardial infarction and stroke, but not of the development of atherosclerosis [25]. the RANTES onto the activated endothelium (Fig. (1f)). The binding of RANTES to the endothelium enhances monocyte recruitment and activates monocytic integrins, consolidating their attachment and rolling at the atherosclerotic lesion [29]. In atherosclerosis, membrane-bound CD40L, but not sCD40L, interacts with endothelial cells and induces the expression of leukocyte adhesive molecules (e.g. VCAM, ICAM, E - selectin), pro-inflammatory cytokines (e.g. IL-6, IL-8, MCP-1) and matrix metalloproteinases [24,25]. In addition monocytes and macrophages are activated to release a range of pro-inflammatory mediators. However, the association between platelet-derived CD40L and the early stages of atherosclerosis is less well defined than, for example, the role played by T lymphocyte-derived CD40L. In contrast, both platelet membrane-bound CD40L and platelet-derived sCD40L play important roles in the later stages of atherothrombosis where they induce tissue factor expression on endothelial cells and monocytes, and bind directly to IIbß3 to stabilize thrombi [23,24,25]. The engagement of platelet CD40 by either sCD40L or CD40L-expressing T-cells leads to the activation of platelets and the exocytosis of RANTES which, as outlined below, appears to be an important proinflammatory mediator [26]. PF4 was the first member of the chemokine family identified in platelets. It is released from activated platelets in a P-selectin-dependent manner and, depending on the presence of individual co-stimuli, plays multiple pro-inflammatory roles [19]. In the presence of tumor necrosis factor- (TNF), platelet-derived PF4 stimulates both neutrophil exocytosis and firm adhesion to the endothelium. It also facilitates RANTES-induced adhesion of monocytes to the endothelium. The presence of PF4 and RANTES together is associated with the progression of atherosclerosis, and conversely antagonism of RANTES receptors significantly decreases the size of atherosclerotic lesions. The effects of PF4 are not limited to direct interactions with leukocytes. PF4 has been shown to inhibit the degradation of the LDL receptor, thereby limiting lipoprotein removal with associated pro-atherogenic consequences [17]. PF4 also enhances the uptake of oxidized LDL by macrophages-it is found associated with macrophages in early, and with foam cells in late, atherosclerotic lesions [17]. Studies using either CD40L-deficient mice or anti-CD40L blocking antibodies showed both decreased overall atherosclerotic plaque development and a more stable, collagenrich, macrophage- and T lymphocyte-poor plaque structure [23]. Thus the CD40/CD40L system is implicated in atherosclerosis, and in a number of other inflammatory disorders (e.g. arthritis, psoriasis, multiple sclerosis, Crohn’s disease). Chemokines/Cytokines Interestingly, platelets also express receptors for a variety of chemokines, notably for MCP-1 (CCR2), IL-8 (CXCR2), fractalkine (CX3CR1), RANTES (CCR1/CCR5), SDF-1 (CXCR4), and TARC and MDC (both CCR4) [19]. Platelet activation has been reported to occur in response to SDF-1 and fractalkine [27]; murine platelets lacking the fractalkine receptor CX3CR1 display a decreased atherosclerotic phenotype [28]. In contrast, the engagement of CXCR4 or CCR4 by their respective chemokine, although not leading to platelet activation per se, significantly enhances the effect of low levels of platelet agonists, such as thrombin or ADP [19]. Thus, these chemokines may play a role in the post-adhesion platelet activation early in the development of the atherosclerotic lesion. These platelet receptors may also serve to accumulate chemokines at the lesion, as has been postulated for CCR2. CCR2 is a low affinity MCP-1 receptor on platelets; the engagement of CCR2 by MCP-1, however, has no effect on platelet function. Platelets transport MCP-1 to the lesion where it dissociates from CCR2 and accumulates at that site [22]. RANTES is a major contributing factor to the vascular inflammation associated with atherosclerosis. It is stored in platelet alpha granules and released following activation as a component of microparticles [19]. The microparticles provide an efficient, localised transfer mechanism, depositing Platelets store and, upon activation, release a number of other chemokines including MIP-1 , MCP-3, TARC, GRO and ENA-78 (Table 1; Fig. (1e)). In addition, ß-TG is a precursor of CTAPIII, PBP and NAP-2. These molecules are likely to have an impact on the recruitment and activation of leukocytes during the progression of atherosclerosis [19,22]. Several cytokines are secreted by activated platelets [17,19], of which IL-1 has been the most widely studied. Platelets store IL-1ß in its precursor pro-IL-1ß form [17,19]. Platelet activation leads to the rapid release of active IL-1ß in both a soluble form and in association with microparticles [19] (Fig. (1e)). Although both forms enhance endothelial cell adhesiveness for neutrophils, microparticle-associated IL-1ß is significantly more effective, possibly because of the presence of additional pro-inflammatory factors, such as Pselectin, on microparticles [30]. The conversion of pro-IL-1ß to IL-1ß, and subsequent release of IL-1ß from platelets, occurs in a ß3 integrin-mediated process and, as demonstrated in vitro, continues for several hours [30]. This suggests that platelets play a significant role in the chronic inflammatory response. Growth Factors Several other atherogenic mediators are released by activated platelets that may have a role in the chronic stages of atherosclerosis, although in most cases the evidence is currently circumstantial. PDGF and TGF-ß, both secreted from -granules, lead to smooth muscle cell proliferation and biosynthesis respectively [31]. Consequently, both are likely to be involved during the stages of plaque development where smooth muscle cells proliferate and infiltrate the plaque [17]. The contribution of platelets to inflammation associated with other disorders is less well delineated, however platelets have been implicated in the etiology of conditions such as Beyond Hemostasis Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 105 inflammatory bowel disease (IBD), rheumatoid arthritis, systemic lupus erythematosus, psoriasis and migraine [32]. Inflammatory Bowel Disease Several lines of evidence support a role for platelets in ulcerative colitis and Crohn’s disease, the two most common forms of IBD [24]. The platelets of individuals with both conditions express significantly elevated levels of activation markers, such as P-selectin and CD40L, in combination with elevated levels of plasma ß-TG, PF4, and sCD40L, consistent with platelet activation [24]. Interestingly, the P-selectin expression on platelets has been reported to be found primarily in mesenteric rather than in peripheral venous blood. Histopathological examination of rectal mucosal capillaries from patients with IBD showed the presence of microthrombi, although their presence is unrelated to the severity of inflammation. Platelets readily adhere to human intestinal microvascular endothelial cells (HIMECs) incubated in vitro with IL-1ß. The accompanying expression of platelet CD40L serves to facilitate platelet binding to the endothelial cells. CD40L stimulates endothelial cells to upregulate surface levels of VCAM-1 and ICAM-1, two primary leukocyte receptors, and to release the neutrophil chemoattractant IL-8 [33]. Platelet-derived sCD40L also stimulates intestinal cells to increase adhesive receptor expression and chemokine release. The platelets of individuals with IBD also release RANTES, which in in vitro studies, has been shown to be highly bound to HIMECs [24]. Based on these studies, Danese and colleagues have suggested a sequence of events whereby platelets adhere to intestinal microvascular endothelial cells activated by IBD. The platelets release sCD40L, RANTES and potentially other chemokines, which are localized to the endothelium. This results in the increased expression of adhesive receptors, leukocyte adhesion and migration into the interstitium where inflammation occurs [24]. Rheumatoid Arthritis It is well-documented that platelets accumulate in the synovial fluid of individuals with rheumatoid arthritis, although the significance is not well understood. The platelets from individuals with rheumatoid arthritis express increased levels of IIbß3 and form increased numbers of complexes with monocytes, neutrophils and lymphocytes. However, their platelets do not express more P-selectin, nor does their plasma have elevated levels of TGF-ß [34]. Studies using an animal model of arthritis have shown that platelets adhere to, and roll on, arthritic endothelium [35]. Thus it is unclear whether platelets contribute to the disease progression or respond to the pathological setting of rheumatoid arthritis. For example, platelets are responsive to both platelet activating factor and IgG, both of which are elevated in rheumatoid arthritis. Psoriasis It has been recognized for 30 years that psoriasis is a risk factor for thrombosis. The circulating blood of patients with psoriasis has significantly more platelet microaggregates and elevated ß-TG levels, when compared to blood of control individuals [36]. The platelets of individuals with psoriasis are hypersensitive to soluble agonists such as ADP, collagen and arachidonic acid due, at least partly, to enhanced cyclooxygenase activity [37]. It was suggested that platelet hyperactivity led to the release of mitogenic and inflammatory mediators that contributed to the psoriasis [36]. More recent studies in a murine model demonstrated that activated platelets could roll on skin venules in a P-selectin- and IIbß3 mediated manner [38]. Furthermore platelet P-selectin expression is increased in individuals with psoriasis [38]. Although many platelet-associated inflammatory mediators are implicated in the progression of psoriasis (e.g. CD40, CD40L, fractalkine) the role, if any, that platelets play in the development of the disease remains conjecture. Migraines For several decades platelets have been implicated as one of the principle etiological factors underlying migraine headache [3 9,40]. This was based largely on the hypothesis that migraine was considered to be a disorder of vascular tone, that serotonin was the major contributing factor, and that the platelets of these patients abnormally handled serotonin [40]. There is now evidence that migraine can be considered as an inflammatory disorder specifically within cephalic tissue. It is proposed that the release of inflammatory mediators activates peripheral nocioceptors associated with sensory fibres innervating the meninges [41]. A variety of contributing mediators have been proposed, including protons, free radicals, complement, prostaglandins, kinins and cytokines [41], including several platelet-derived cytokines (e.g. IL-1, ß-TG). Although there is no conclusive evidence linking the exocytosis of cytokines from platelets with the onset or progression of migraines, it is interesting to note that platelet hyperactivity has long been recognized as a hallmark of this condition; indeed there is a small, but significant, increase in the incidence of stroke associated with migraines. Thus it is plausible that enhanced platelet release may provide inflammatory mediators that synergize with other mediators of migraine. Furthermore, in common with other inflammatory conditions, there is now evidence that patients experiencing migraine without aura, in contrast to either those with aura or controls, have significantly increased formation of plateletleukocyte complexes [42]. The identity of the receptors involved has not been determined. Inflammatory Pulmonary Disease Finally, a role for platelets in various pulmonary conditions has been postulated. The platelets of individuals with asthma have higher than control levels of expressed Pselectin [43], and platelet-derived P-selectin functions to recruit leukocytes to their lungs [44]. Studies in human ex vivo asthma models demonstrated that antibodies to P-selectin attenuate platelet-leukocyte complex formation and subsequent pulmonary recruitment [45]. O’Sullivan and Michelson have summarized reports of an intrinsic platelet dysfunction underpinning cystic fibrosis 106 Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 McNicol and Israels [46]. The platelets from these individuals appear hyperactive with increased production of both TxA2 and sCD40L. The relationship between platelets and the progression of cystic fibrosis remains to be fully addressed [46]. number of inflammatory conditions, the specific beneficial anti-microparticle effects of these drugs have not been addressed. A role for platelet-derived P-selectin in the development of inflammatory lung disease has been recently postulated [47]. In a murine model of acute lung injury, P-selectinmediated platelet-neutrophil interactions were critical to disease progression, and pulmonary inflammation was attenuated by blocking P-selectin. Furthermore antagonism of the platelet TxA2 receptor significantly inhibited platelet-neutrophil interactions and subsequent neutrophil adhesion to, and activation of, endothelial cells. Thus, it appears the platelet cyclooxygenase pathway modulates the progression of inflammatory lung disease. Platelets play a significant role in the pathogenesis of malignancy. There is now evidence for the contribution of platelets to tumor cell proliferation, metastasis, and tumor angiogenesis, in animal models, and in a variety of tumors types including carcinomas of breast, colon, lung, ovary and melanoma. The association between malignancy and thrombosis was first recognized in 1865 by the French clinician Armand Trousseau who made the observation that migratory thrombophlebitis could be an indicator of occult malignancy; he subsequently diagnosed his own fatal pancreatic carcinoma by this sign [50,51]. Trousseau’s syndrome, the clinical manifestation of the hypercoagulable state, seen particularly with advanced carcinomas, is the result of a complex interaction among tumor cells, inflammatory cells, endothelium and platelets. A decade later, the German surgeon Theodor Billroth described tumor thrombi within the blood vessels of patients with cancer, and surmised that tumor emboli played a role in the development of metastatic disease [52]. Therapeutic Implications for Inflammation Anti-platelet drugs (i.e. those agents which affect platelet function) play a well-defined role in the primary and secondary prevention of arterial thrombotic disorders. Inhibition of cyclooxygenase-1 by aspirin with resultant decreased TxA2 generation, and antagonism of the ADP receptor by clopidogrel or ticlopidine attenuate the amplification pathways of platelet activation. In addition, the blockade of the active site of IIbß3 by humanized antibodies such as abciximab significantly reduces thrombotic complications associated with percutaneous coronary intervention. The development of safe, effective, orally active IIbß3 antagonists as antithrombotics has proved to be more challenging [48]. The multiple effects of platelets in inflammatory diseases suggest that anti-platelet therapy may produce clinical benefit in these disorders. Aspirin is well accepted as an antiinflammatory agent, however its anti-platelet actions are observed at doses (81-325 mg/day), significantly lower than those required for traditional anti-inflammatory effects (1500 mg/day). Some of the beneficial effects of aspirin in cardiovascular disease, including atherosclerosis, are clearly not a result of an anti-platelet action. For example, aspirin inhibits reactive oxygen formation, increases nitric oxide synthase and reduces C-reactive protein levels, each of which is cardioprotective but platelet-independent. Thus the complexity of the situation in atherosclerosis makes it difficult to assign specific benefit to the anti-platelet actions of aspirin in early stages of atherosclerosis. In contrast, platelet inhibition is of fundamental importance to aspirin’s actions in athero-throm-bosis. Few studies have addressed traditional anti-platelet drugs in non-atherosclerotic inflammatory conditions. At present, there is little evidence to support a specific anti-platelet action of aspirin in inflammatory disorders. Interestingly, a number of drugs in addition to traditional anti-platelet agents (e.g. aspirin, ticlopidine, clopidogrel, abciximab) have been shown to decrease the release of microparticles specifically from platelets, including: pravastatin, eprosartan, nifedipine, digoxin, iloprost and vitamin K [49]. Although microparticles have been implicated in a PLATELETS AND MALIGNANCY The interaction of platelets and tumor cells is bidirectional, with tumor cell-expressed proteins activating platelets, and activated platelets promoting tumor survival and progression by a number of different mechanisms discussed below. The clinical importance of understanding the details of these interactions is to determine whether it is possible to use targeted therapy that prevent the contribution of platelets or their releasates without incurring significant bleeding. Some of these potential targets are common to inflammation and malignancy; inhibiting those targets could abrogate the contribution of platelets in both settings. The role of platelets in the development of experimental pulmonary metastases was originally recognized by Gasic and colleagues in the 1960s. Thrombocytopenic mice were protected from development of pulmonary metastases following tail vein injection of tumor cells [53]; this benefit was reversed by platelet transfusion [54]. Subsequent studies have confirmed a role for platelets in the development of metastases, the formation of tumor emboli and the arrest of tumor cells at sites on the vessel wall [55]. Tumor Cell-Platelet Aggregate Formation The formation of platelet-tumor aggregates confers a survival advantage to malignant cells by protecting them from immune surveillance [56,57], allowing their persistence in the vascular compartment and enhancing metastatic potential [53,54,58,59]. Platelets facilitate the adhesion of tumor cells to vessel wall and release growth factors that may enhance tumor cell growth and angiogenesis at sites of extravasation (Fig. (2)). Activation of platelets is the result of multiple mechanisms available to tumor cells. Thrombin, cancer procoagulant or cathepsin B, and matrix metalloproteinase-2 released Beyond Hemostasis Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 107 Fig. (2). The role of platelets in tumor progression and metastases. Fig. (2a). The progression of platelet binding to tumor cells (TC). Tumor cells activate platelets using a variety of mechanisms (e.g. tissue factor (TF) and thrombin) leading to changes in membrane composition, including the expression of IIb3 in its open conformation and Pselectin. The binding of platelets to tumor cells to form heterotypic aggregates is mediated by integrin/adhesive protein combinations (e.g. platelet IIb3, tumor cell v3, fibrinogen) and by complexes of tumor cell mucins and P-selectin. Tumor cells tether, roll and adhere in a manner similar to leukocytes shown in Fig. 1. Fig. (2b). Activated platelets promote survival of tumor emboli in the circulation by protecting tumor cells from clearance by natural killer cells (NK), allowing their adhesion and extravasation at metastatic sites. Release of platelet-derived factors, stimulate tumor growth (e.g. LPA) and modulate neoangiogenesis (e.g. VEGF and endostatin). from cancer cells can all activate platelets. Tumor-generated thrombin also has an autocrine effect on tumor cells, and stimulates heterotypic aggregate formation. A second mechanism depends on the constitutive expression of tissue factor on the surface of some tumor cells leading to activation of the coagulation cascade and thrombin generation on the procoagulant platelet surface. The potential to activate platelets varies among tumor cell lines, but the capacity of tumor cell lines to induce platelet aggregates in vitro correlates with increased in vivo thrombosis and metastases [55,60]. Platelet-tumor cell aggregates result from the cross-linking of platelet integrins, primarily IIb3, with integrins expressed on tumor cells, such as v3 [61], by adhesive ligands such as fibrinogen, fibronectin and vWF. In some tumor cell lines, including breast, prostate, colon and melanoma, ectopic expression of megakaryocytic genes results in the expression of functional IIb3 [62-65]. Expression of IIb3 on tumor cells also may result from its transfer by microvesicles, released from activated platelets, to the surface of malignant cells [66,67]. GPIb/IX/V is involved, and blocking of its interaction with vWF reduces platelet-tumor cell interactions [68-70]. Participation by other receptors (31, 51) and adhesive proteins (laminin, vitronectin, thrombospondin, and collagen) also has been identified [71]. Tumor cell-platelet adhesion mimics that of inflammatory cells, with initial tethering and rolling mediated by P-selectin 108 Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 followed by firm adhesion to 3 integrins by fibrinogen and vWF [72](Fig. (2a)). P-selectin, which plays a critical role in the interaction of platelets with inflammatory cells, is also emerging as a pivotal receptor in platelet-tumor cell interaction and the formation of platelet-tumor cell aggregates. The binding of Pselectin to carcinoma mucins mediates this interaction. Although some tumor cells, such as HL-60, express PSGL-1 [21], in most cases, the selectins recognize aberrantly glycosylated mucins [73-75] or the GPI-linked surface mucin CD24 [76], which carry sialylated, fucosylated and sulfated carbohydrates [77,78]. Specific cleavage of these mucins blocked platelet binding to the tumor cells [77]. Heparan sulfate-like proteoglycans expressed on some tumors can also function as P-selectin ligands [77,79], and this finding has prompted the investigation of heparin therapy targeted at the selectin-ligand association (see below). Platelets and Tumor Metastases The role of platelets in promoting experimental metastases in mouse models has been demonstrated using a variety of techniques to produce thrombocytopenia. More recently, NF-E2 mice with marked thrombocytopenia have been shown to be relatively protected from pulmonary metastases following tail vein injection of melanoma cells [80]. Fibrinogen deficiency also is protective [80,81]. Platelets play an active role in these models as inhibition of platelet activation or aggregation abrogates metastatic potential. Inhibition of IIb3-mediated fibrinogen binding by monoclonal antibodies, pharmacological inhibitors or by the genetic loss of integrin 3 decreases metastases [82,83,84]. Inhibition of platelet activation by prostacyclin blocks tumor-induced platelet activation and experimental metastases [85]. Genetic alterations resulting in thrombin receptor PAR-4 deficiency [80] or G protein Gq deficiency [86], give rise to mice with significant defects in agonist-induced platelet aggregation and thrombus formation despite normal platelet counts, and protection from hematogenous metastases in comparison to wild-type mice. Similar protection is observed in P-selectindeficient mice when injected with mucin-producing carcinomas [87]. The contribution of activated platelets to metastatic potential is likely multifactorial (Fig. (2b)). The mechanisms by which platelets contribute to metastases include: (1) The arrest of tumor cells at sites of vascular damage by tethering and rolling on adherent platelet and or endothelial cell expressed P-selectin, followed by integrin-mediated stable adhesion [72,88]. The interaction of platelets and tumor cells at the vessel wall bears many similarities to the interaction of platelet/leukocyte responses to inflammation. (2) The provision of a physical barrier protects tumor cells from elimination by the immune surveillance of natural killer cells. Common to several of the knock-out murine models described above, was the observation that impaired platelet function did not alter the initial localization of tumor cells in the lungs, but did affect the survival and growth of the tumor emboli [86,87]. In the Gq -/- mice this survival advantage McNicol and Israels was lost following depletion of NK cells using anti-asialo GM1 antibodies [86], suggesting that activated platelets may prevent the direct cell-cell contact required for NK cellmediated tumor lysis [56,57,89]. Platelets and Tumor Growth Activated platelets also contribute directly to tumor cell proliferation and survival at both primary and metastatic sites, by contributing to thrombin generation, release of soluble mediators, and the generation of microparticles and exososmes that transfer other growth mediators to adjacent tumor cells. Thrombin has been demonstrated to promote tumor growth and metastases in murine models of spontaneously metastasizing breast cancer. Pretreatment of mice with hirudin decreased tumor growth by ten-fold, and metastases more significantly [90]. Endogenous thrombin generation results from the expression of tissue factor on the surface of tumor cells activating coagulation factors on the procoagulant surface of activated platelets. Ectopic expression of the thrombin receptor PAR-1 on tumor cells and its activation by thrombin enhances tumor cell growth and metastases [71]. This cycle of tumor-induced thrombin generation by platelets and thrombin-induced tumor cell activation and growth can be interrupted in vitro by either direct thrombin inhibitors [71] or PAR-1 inhibitors [91]. Lysophosphatidic acid (LPA) is a bioactive water-soluble phospholipid that has recently been shown to have a significant role in cancer cell proliferation, migration and metastases. LPA interacts with G - protein-coupled LPA receptors that feed into multiple effector pathways by activating three G proteins, Gq, Gi and G12/13. In tumor cells these pathways enhance a variety of biological activities including cell proliferation, survival, migration, invasion, and the inhibition of differentiation [92,93]. There appear to be several sources for the LPA found in the tumor milieu, including generation of LPA by a tumor cell ecto-enzyme autotaxin, a lysophospholipase that cleaves lysophosphatidylcholine. LPA also is generated and released by platelets, the product of phospholipase A2 cleavage of phosphatidic acid. Activation of platelets by tumor cells stimulates LPA production and release, which then binds to tumor cell LPA receptors (Fig. (2b)). Using a breast cancer cell line that over-expressed the LPA receptor, tumor cells could be stimulated by the supernatant from activated platelets, and this effect was abolished by treatment of the supernatant with LPA degradation enzymes [94]. In this model, platelet LPA also stimulated tumor cell release of IL-6 and -8, enhancing osteoclast activity and bone resorption at sites of bony metastases [94]. Platelets release growth factors, adhesive proteins and chemokines that stimulate tumor activity directly, or recruit inflammatory cells that release soluble mediators such as TNF that inhibits tumor cell apoptosis [95], promoting tumor cell survival. A potent source of mediators that can influence tumor cell growth is platelet microvesicles, both surface-derived microparticles and exosomes, which have been shown to increase the growth and invasive potential of lung Beyond Hemostasis Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 109 and breast cancer cells. The effects of isolated platelet microvesicles on tumor cells in vitro included activation of proliferative signaling pathways, and enhanced proliferation and survival of cells in serum-free cultures [66,67]. Increased numbers of circulating peripheral blood microvesicles have been correlated with poorer prognosis in patients with gastric carcinoma [96]. Tumor Angiogenesis Platelet granules contain both pro-angiogenic and antiangiogenic factors (Table 2). These factors are released following platelet activation and secretion (Fig. (2b)); the balance appears to favour platelets providing a pro-angiogenic stimulus in the context of malignancy [97,98]. Recent evidence suggests that pro- and anti-angiogenic mediators may be packaged in distinct subsets of alpha granules [99], and released by specific stimulation of one proteinase-activated receptor, (PAR-1 induces release of VEGF) versus another (PAR-4 induces release of endostatin) [100]. Table 2. Angiogenic Factors in Platelet Alpha Granules Pro-Angiogenic Mediators Anti-Angiogenic Mediators VEGF-A and VEGF-C PF4 (CXCL4) bFGF Thrombospondin- 1 HGF Angiostatin IGF-1 and -2 Endostatin EGF TGF- PDGF PAI-1 PD-ECGF TIMP-4 Angiopoietin- 1 MMP-2 Therapeutic Implications for Malignancies The complex interaction of platelets with tumor cells makes the platelet and platelet-generated effector molecules apt targets. Despite in vitro studies showing inhibition of tumor-cell -induced platelet aggregation, aspirin, ticlopidine and prostacyclin failed to affect experimental metastases in mouse models [54,68]. More success was seen in experimental models with agents such as the chimeric antibody abciximab and the peptide epifibatide that block integrin binding to adhesive proteins. Integrin blockade has been shown to inhibit platelet-tumor cell adhesion, experimental metastases and platelet-induced sprouting of endothelial cells [68,82, 106]. Inhibitors of thrombin generation, including coumarins, heparins, and direct thrombin inhibitors such as hirudin, also decreased experimental metastases [55,71,107]. At present, the most promising therapeutic agent is heparin. Clinical trials have demonstrated a survival advantage for unfractionated and low molecular weight heparins over placebo in subgroups of cancer patients without thrombosis [108,109], and over warfarin in cancer patients with venous thrombosis [110]. The advantage of heparin may be that its effects on tumor growth and metastases are multiple, including inhibition of thrombin generation, fibrin formation, tumor cell stimulation through PAR-1 receptors, and platelet activation (Table 3). Heparin also blocks binding of tumor cell glyscosaminoglycans to selectins on platelets and endothelial cells, inhibiting formation of heterotypic aggregates and their adhesion to the blood vessel wall [77,111]. Table 3. Anti-Malignant Properties of Heparin Inhibition of thrombin and fibrin formation Inhibition of tumor cell adhesion to platelets and endothelial cells Interference with selectin binding to tumor cell glycosaminoglycans Inhibition of angiogenesis The most significant link between platelets and tumor angiogenesis may be VEGF. Platelets provide a large reservoir of VEGF that is released following tumor cell-induced activation. The concentration of platelet-derived VEGF is a better predictor of tumor progression than serum concentrations of VEGF [101]. In vitro studies have shown that platelets stimulate endothelial cell proliferation and tube formation [102,103], and inhibitors of IIb3 such as abciximab block tumor cell-induced platelet VEGF secretion and sprouting of endothelial cells [104]. VEGF stimulates the endothelial cell expression of tissue factor and release of vWF, enhancing platelet adhesion to tumor vessel wall. The abnormal tumor vasculature and resultant turbulent flow also may contribute to platelet adhesion, degranulation and increased release of angiogenic factors within the tumor [105]. The release of platelet-derived microparticles under these conditions also may play a role in angiogenesis; platelet microparticles stimulate tumor cell expression of angiogenic factors including IL-8, VEGF and HGF and membrane metalloproteinases involved in degradation of extracellular matrix and remodeling of the basement membrane [66]. Stimulation of apoptosis Immune system modulation The ideal therapeutic agent(s) will be one that inhibits platelet adhesion and release without increasing the risk of bleeding beyond a tolerable range. We know little about the optimal timing or duration for these agents to be effective in preventing tumor progression and metastases, as most experimental models are not true reproductions of clinical tumor progression. PLATELETS AND IMMUNITY The potential association of infections, both bacterial and viral, with the development of cardiovascular disease has long been suspected. The best characterized is infective endocarditis [112], although a variety of infectious agents also have been implicated in the etiology of conditions such as atherosclerosis [113], myocardial infarction [114] and stroke [115]. Only recently have mechanistic relationships been 110 Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 partially elucidated, and platelets have been implicated as major contributors to the development and progression of infection-associated cardiovascular disease. It is becoming clear, however, that in addition to contributing to adverse outcomes, platelets play a host-defence role against bacterial pathogens in particular [19]. Platelets and Bacteria The interaction between platelets and bacteria has recently been extensively reviewed [116]. Bacterial species cause platelet aggregation utilizing a variety of the mechanisms; even within individual species there may be strainspecific mechanisms of platelet activation. For example Staphylococcus aureus, the most frequent causative pathogen responsible for infective endocarditis [117], activates platelets by differing mechanisms depending on the presence of specific proteins on the bacterial cell wall. S. aureus can stimulate platelets via mechanisms involving: IgG and its associate platelet receptor Fc RIIA, fibrinogen and its receptor IIbß3, vWF and its receptor GPIb/IX/V, and complement [116]. Indeed, the specific process invoked by individual strains of S. aureus likely dictates the speed of the platelet activation response [116]. A number of other pathogens implicated in infective endocarditis, including species of the Staphylococcus (S. epidermidis, S. capitis) and Streptococcus (S. sanguis, S. agalactiae, S. pyogenes, S. gordonii, S. pneumoniae, S. mitis) genii, as well as Neisseria gonnorrhoeae and Pseudomonas aeruginosa, have been shown to activate platelets using similar strategies to those utilized by S. aureus [116]. The relationship between bacterial infections and coronary artery disease is less clear and somewhat controversial. Individuals with active infections are at a higher risk and those who have received antibiotics in the previous year are at lower risk for acute cardiovascular events [116]. Ott and colleagues identified the presence of DNA from a number of different bacterial species in atherosclerotic plaques [113]. Consequently, it has been suggested that coronary artery diseases, such as atherosclerosis [113] and myocardial infarction [114], are not associated with any single pathogen but rather with the cumulative effects of multiple organismsthe concept of total pathogenic burden [113,114,116]. Helicobacter pylori, coincidentally first reported by Giulio Bizzozero [118] who also shares credit for the original identification of platelets [2], is now accepted as the causative organism of many gastric disorders, and may also increase the risk of myocardial infarction and atherosclerosis [119]. Similarly, Chlamydophila pneumoniae (previously known as Chlamydia pneumoniae) has been reported to increase the risk of myocardial infarction, although prophylactic antibiotic therapy failed to prevent acute events in a highrisk population group [120-122]. Transient bacteremias with normal orally-derived bacteria are common, not only after dental procedures (extraction, endodontic treatment, periodontal surgery, root scaling) [123,124] but following regular “non-invasive” procedures such as brushing [125] (notably with the use of electronic McNicol and Israels toothbrushes [126]), and in individuals with periodontal disease [127]. Such transient bacteremias, which involve both anaerobes, such as Porphyromonas gingivalis, and aerobes, such as S. sanguis, would contribute significantly and regularly to total pathogenic burden. P. gingivalis and S. sanguis have both been isolated from atherosclerotic plaque [128], although only P. gingivalis is a perio-pathogen [129]. Consequently there has been considerable interest in the relationship between oral disease, primarily periodontal disease, and cardiovascular disease. A systematic review completed in 2003 suggests an overall moderate association between periodontal disease and atherothrombosis, including coronary artery disease, stroke and peripheral vascular disease [130]. Furthermore, studies in pigs and in mice have shown that infection with P. gingivalis is as effective as a high cholesterol diet at inducing atherosclerosis [131,132]. Invasive, perio-pathogenic strains of P. gingivalis have been shown to upregulate the expression of adhesive receptors on [133], and the release of chemoattractants from, cultured endothelial cells [134]. This provides a potential mechanism for the genesis of atherosclerotic lesions, including the adhesion of platelets to the activated endothelial cells, as outlined above. Interestingly an invasive strain of P. gingivalis enhances tissue factor expression and activity, but attenuates tissue factor pathway inhibitor levels [135], suggesting multiple targets in addition to platelets. Individual strains of H. pylori, S. sanguis and P. gingivalis have been shown to activate platelets in vitro [116]. In each case, the presence of IgG and its platelet receptor Fc RIIA and probably, vWF and its platelet receptor GPIb/IX/V, are essential for activation to occur. Fitzgerald and colleagues have proposed a general scheme by which platelet activation can be stimulated by bacterial pathogens (Fig. (3a)). This involves the initial adhesion of the bacterium to the platelet - this may involve either the direct interaction of a bacterial wall or membrane protein (e.g. clumping factors A and B, fibronectin binding proteins A and B, serine-rich protein, serine-aspartate repeat protein), with a platelet receptor (e.g. GPIb/IX/V, IIbß3) or via an intervening plasma protein (e.g. vWF, fibrinogen). Subsequently, circulating antibodies to proteins on the bacterial surface activate the adherent platelets by engaging Fc RIIA [116]. Initial activation includes the synthesis and release of TxA2 (most bacteria-induced platelet aggregation is aspirin-sensitive), and the subsequent expression and occupation of fibrinogen binding sites on IIbß3 for full aggregation to take place. It should be noted that a role for factors excreted by bacteria in platelet activation cannot be excluded [116] (Fig. (3)). Platelets and Innate Immunity The response to infection comprises a rapid, antigenindependent innate response and a delayed, prolonged antigen-specific adaptive response. The innate response to bacterial infections is multi-factorial and has been reviewed for several specific invading organisms [136,137]. In general, bacteria secrete and/or express ligands (e.g. gram negative bacteria-derived LPS, gram positive-derived lipoteichoic ac- Beyond Hemostasis Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 111 Fig. (3). Association of platelets with bacteria Fig. (3a). Schematic representation of the association of platelets with bacteria (Bact), based on the model proposed by Fitzgerald and colleagues [113]. Bacterial cell wall or membrane proteins interact directly, or via adhesive proteins (e.g. vWF), with platelet membrane proteins, notably the GPIb/IX/V complex. IgG, bound to bacterial antigens engage FcRIIa on the platelet, stimulating signaling pathways culminating in TxA2 release and full platelet activation. Activated platelets engulf the bacteria in a heterogenous aggregate. Fig. (3b). Transmission electron micrograph of platelets and S. sanguis (B) stirred together in a platelet aggregometer, demonstrating platelet activation and extension of filopodia (arrows) that surround the bacteria. Scale bar = 0.5m. ids, bacterial cell wall peptidoglycans) for the toll-like receptor (TLR) family of membrane proteins [138,139]. To date 12 TLRs have been described in humans and have been localized primarily to phagocytic cells (e.g. monocytes, macrophages), epithelial cells and, as discussed below, platelets. Several of the TLRs, including TLR-1, TLR-2, TLR-4 and TLR-6 are expressed on the cell surface. TLR-2 forms heterodimeric complexes with TLR-1 or TLR-6 and binds lipoproteins and lipoteichoic acids, whereas TLR-4 recognises a variety of ligands, although LPS and viral proteins appear to be its primary agonists. In contrast, some TLRs such as TLR-8 and TLR-9 are located intracellularly and serve as receptors for single stranded RNA and DNA, respectively [138,139]. Engagement of TLR-expressing cells leads to cell-specific release of transcellular mediators, pri- marily chemokines and cytokines that mediate the immune response to invading organisms [138,139]. Interestingly platelets have been shown to express several TLRs, including TLR-1, TLR-2, TLR-4, TLR-6, TLR-8 and TLR-9 [140,141] with increased surface expression of TLR-2 and TLR-9 following activation [141]. The role of platelet TLRs remains unclear. Studies in mice demonstrated that engagement of TLR-4 by LPS stimulated an IIbß3-mediated binding of platelets to fibrinogen in the absence of Pselectin expression [142]. Furthermore LPS binding to TLR-4 caused thrombocytopenia in mice and, in the presence of anti-platelet antibodies, was associated with decreased TNF production [143]. Recently Semple and colleagues demonstrated that platelet-bound LPS, presumably via TLR-4, syn- 112 Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 ergizes with anti-platelet antibodies thereby enhancing Fcreceptor-mediated phagocytosis [144]. The effects of LPS on IIbß3-mediated fibrinogen binding may suggest a role for TLRs in platelet activation. The LPSinduced release of sCD40L from platelets was attenuated by blockade of TLR-4 but not of TLR-2 or TLR-9 [145], consistent with the elevated levels of sCD40L observed in the plasma of patients with meningococcal sepsis [146]. In contrast, Ward and colleagues were unable to show an effect of ligands for TLR-2 or TLR-4 on cytosolic calcium mobilisation, P-selectin expression or aggregation, either alone or in combination with traditional platelet agonists such as platelet activating factor, epinephrine or ADP[147]. Anti-Bacterial Efects of Platelets Intriguingly platelets may serve an anti-bacterial function. It has been proposed that, as platelets rapidly accumulate at sites of injury/infection, they may play similar “surveillance” roles to that of traditional immune cells such as macrophages, mast cells and dendritic cells [148]. The upregulation of CD40L on the platelets surface (in response to vascular damage, soluble agonists or bacteria) has effects on adaptive immunity, including accelerating dendritic cell maturation, stimulating IgG production by B cells and enhancing T cell activity [149]. Bacteria-induced platelet activation results in the secretion of granular contents, including various chemokines that, as outlined above for inflammation, serve to attract immune cells (monocytes, basophils, NK cells, macrophages) to the site of infection [148]. Finally, platelets adhering to and aggregating around bacteria may promote bacterial clearance [148]; indeed activated platelets may internalize bacteria thereby removing them from the circulation [19]. Platelet alpha granules also contain thrombocidins, members of a family of anti-bacterial proteins that are found primarily in neutrophils and play a central role in the innate immune system [19]. Two thrombocidins have been characterized, TC-1 and TC-2, which are variants of neutrophilactivating peptide-2 (NAP-2) and connective tissue-activating peptide-III (CTAP-3) respectively. TC-1 and TC-2, but not the parent molecules, are bactericidal for several organisms including E. coli and S. aureus, and are fungicidal for Cryptococcus neoformans [150]. Studies in rabbits have shown thrombocidins to be important for host defence against Streptococci-induced endocarditis [151]. Furthermore, the thymosin- 4, PBP, RANTES and PF-4 released from activated platelets have been reported to have bacteriocidal effects against E. coli and S. aureus when tested in vitro [152]. Platelets and Viruses Associations between platelets and several viral infections have been proposed. The hepatitis B virus (HBV) will bind to GPVI on the platelet membrane which, it has been suggested, may serve to facilitate viral transport and promote viral survival [153]. Studies, primarily in animal models of HBV infection, have demonstrated that platelets facilitate the McNicol and Israels accumulation of cytotoxic T lymphocytes in intrahepatic sites of necrosis and inflammation It is unclear whether this is the result of the interaction of platelets with HBV particles or platelet participation in the inflammatory response as outlined above. Platelets have been shown to engulf viruses, most notably human immunodeficiency virus type 1 (HIV-1); a significant proportion of circulating HIV-1 is associated with platelets. The adhesion of HIV-1 to platelets is mediated by two platelet membrane proteins, dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN), and C-type lectin-like receptor 2 (CLEC-2) [156]. DC-SIGN facilitates the endocytosis of HIV-1 by platelets [157], whereas CLEC-2 is linked to Src kinase/phospholipase C 2 pathways and is unlikely to be involved in HIV-1 uptake [156]. The importance of these platelet receptors, viral uptake and intracellular signaling to the progression of HIV-1 disease is unknown. Platelets and Prions The transmissible spongiform encephalopathies (TSE) are a family of neurodegenerative disorders that include bovine spongiform encephalopathy in cattle, scrapie in sheep, and Creutzfeldt-Jakob Disease and Gerstmann-StrausslerSchienker syndrome in humans. Infectious forms of TSE are due to a variant protease-resistant form (PrPres) of a normal cellular protein, prion protein (PrP) [158]. PrP is a glycophosphatidylinositol-linked membrane protein of unknown function. However, its conversion to PrPres renders the protein highly infectious, affecting health care and agricultural policies worldwide. PrP is present on the membrane of platelet alpha granules [159] and is released on exosomes following platelet activation [160]. The presence of PrPres in platelets has not been reported, however its sequestration, and subsequent exosomal release, could have significant implications for TSE disease transmission, notably by blood transfusions [161]. Therapeutic Implications for Infections The therapeutic implications of manipulating interactions between platelets and bacteria have not been widely addressed, although several antibiotics, notably penicillin, may have anti-platelet side-effects. The increased understanding of the role of both bacterial pathogens and platelets in the development of atherosclerosis has led to the identification of specific potential targets. The IgG receptor FcRIIA is a common mediator of platelet responses to multiple bacterial species. Antagonism of this receptor represents a logical strategy [116], particularly in keeping with the concept of total pathogenic burden. Furthermore, FcRIIA antagonists are unlikely to compromise the hemostatic function of platelets, avoiding the bleeding risks associated with traditional antiplatelet drugs. The anti-bacterial potential of platelets could be utilized by selectively releasing, or mimicking, thrombocidins. The role of platelet interactions with T lymphocytes in the progression of hepatic necrosis and inflammation caused by Beyond Hemostasis Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 113 HBV, has raised the possibility that anti-platelet drugs may reduce the severity of chronic liver disease in HBV infected individuals [154] Studies are currently underway to determine the beneficial effects of aspirin and clopidogrel in HBV-induced liver disease. [6] [7] [8] CONCLUSION Platelets have retained many of the functions of the primitive multi-functional hemocyte. The predominant function of platelets is considered to be hemostasis, where they play a complex role integrated with other soluble and cellular participants. Platelets adhere to the exposed sub-endothelial collagen of a damaged vessel wall, are activated, up-regulate additional adhesive receptors and secrete transcellular mediators, contributing to the formation of a stable platelet aggregate. Defective activation of platelets compromises hemostasis and can result in excessive clinical bleeding. In contrast inappropriate platelet activation is associated with arterial thrombotic conditions such as myocardial infarction and stroke. Platelets are a primary target for the prevention of recurrent cardiovascular thrombosis. It is now clear that platelets have a wider repertoire of physiological roles and therefore are implicated in more pathological conditions than thrombosis; they are active participants in the immune response and host-defence. In these situations, platelets function like traditional immune cells such as macrophages and mast cells, binding to bacteria, secreting chemokines, and clearing invading organisms from the circulation. Many of the underlying mechanisms used by platelets in their immune response are identical to, or extensions of, the hemostatic processes. Their contributions to pathological lesions in inflammatory disorders, and to tumor progression, are the result of these responses at the wrong place and time. [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] The challenge for therapeutic intervention in these diseases will be to identify drugs that preferentially block specific targets involved in the complex contribution of platelets to inflammation or tumor progression, while leaving their hemostatic function at least partially intact. [22] ACKNOWLEDGEMENTS [23] The authors would like to thank Dr. E. D. Israels for helpful suggestions during the preparation of this manuscript and E.M. 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