- Top of page
Platelet interactions with dendritic cells, T cells and B cells have been best studied in vasculitis and atherosclerosis, but similar mechanisms may contribute to acute and chronic vascular lesions in transplants. In acute inflammation, platelets adhere to vessels and release mediators that increase endothelial cell activation and leukocyte recruitment. Adherent platelets can also augment antibody and cellular immune responses. Activated platelets recruit T cells and initiate a feedback loop. In this loop, platelets secrete chemokines to recruit T cells, and then activated T cells stimulate platelets through CD40-CD154 interactions to secrete more chemokines thereby recruiting more T cells. The interaction of platelets and T cells is enhanced by P-selectin/PSGL-1 stimulation. Both helper and cytotoxic T cells are stimulated by platelets. Antibody production that is stimulated through increased helper T-cell function can activate complement. This sets up another activation loop because platelets express receptors for antibodies and complement. In addition to inflammation, platelets stimulate repair by releasing growth factors and chemokines to recruit circulating vascular progenitor cells. These repair mechanisms could promote the replacement of donor parenchmal cells with recipient cells and contribute to vascuplopathy. This review discusses the interplay of platelets and the immune system in relation to transplantation.
- Top of page
Platelets are typically characterized as simple cells that maintain vascular hemostasis, but platelets contain many pro-inflammatory mediators and surface ligands that are either released or expressed upon activation. Most relevant to transplantation are a number of chemokines, adhesion molecules, vasoactive mediators, costimulatory molecules and growth factors (Table 1). These mediators can be rapidly deployed from storage granules (Figure 1). However, platelets are not mere delivery packets. Even though the platelet is anucleate, it is now recognized that platelets contain, and when stimulated, splice pre-mRNA into translatable RNA (1,2). This mechanism allows platelets to increase expression of IL-1β and tissue factor.
Table 1. Partial list of granule contents and membrane expressed receptors on platelets that are relevant to organ transplants
|Location||Category||Molecule||Functions relevant to transplants|
|Alpha granule||Adhesion||P-selectin (CD62P)||Adhesion platelets, endothelial cells & leukocytes|
|vWf||Facilitates P-selectin-mediated rolling|
|Chemokine||MIP-1α (CCL3)||Monocyte & T-cell recruitment|
|RANTES (CCL5)||Monocyte, eosinophil & T-cell recruitment|
|MCP-3 (CCL7)||Monocyte, eosinophil & T-cell recruitment|
|PF4 (CXCL4)||Neutrophil & T-cell recruitment|
|PBP, β-TG NAP-2 (CXCL7)||Neutrophil recruitment|
|IL-8 (CXCL8)||Neutrophil recruitment|
|SDF-1 (CXCL12)||Lymphocyte & progenitor cell recruitment|
|Cytokine||IL-1β||Activates endothelial cells & lymphocytes|
|TNFα||Activates endothelial cells & increases vascular leakage|
|TGFβ||Cell proliferation, differentiation, apoptosis, fibrosis|
|Growth factor||PDGF||Angiogenesis & fibrosis|
|Dense granule||Nucleotides||ATP/ADP||Stimulate platelets & endothelial cells|
|Transmitter||Serotonin||Stimulates platelets & T cells|
|Membrane||Ligand||PSGL-1||Ligand for P-selectin|
|Receptor||PAR||Receptor for activation by thrombin|
|ADP receptor||Stimulates platelets|
|GPIa||Binds to collagen|
|GPIb||Binds to collagen|
|GPIIb/IIIa||Binds to vWf & fibrinogen|
|GPVI||Binds collagen strongly|
Figure 1. Surface receptors, granules and the canalicular system of platelets. Different glycoprotein (GP) receptors that are expressed on the membrane of platelets can cause shape changes though connections to the cytoskeleton. GPIa and GPVI mediate direct binding to subendothelial collagen and GPIb mediates indirect binding to collagen via vWf. GPIIb/IIIa binds to fibrinogen, vWf, fibronectin and vitronectin. Cyclooxygenase (COX) is localized in the dense tubular system compartment where it converts arachidonic acid substrate into thromboxane. Each platelet contains about 40 storage granules. The most numerous are α-granules that contain adhesion molecules, chemokines, cytokines and growth factors, some of which are listed in Table 1. Dense granules contain serotonin and ATP/ADP. The contents of the granules are derived from the megakaryocyte when platelets are formed and also taken up from the plasma via the canalicular system. In addition to uptake of molecules from the plasma, the canalicular system facilitates the rapid exocytosis of granule contents.
Download figure to PowerPoint
Through mediators and ligands, platelets cause activation of endothelial cells as well as activation and recruitment of leukocytes (3). Platelets have also been demonstrated to mediate migration of endothelial progenitor cells to the site of vascular lesions (4,5). The potential for platelets to interact with other cells in the circulation and on the vascular surface is enhanced by the extremely large concentration of platelets in the blood (about 1.5–4.5 × 105/μL) and their highly reactive state. Platelets are known to contribute to the progression of atherosclerosis (6), and they may contribute to acute and chronic vascular changes in transplants.
Early pathological descriptions of transplants recognized platelets as components of hyperacute and acute transplant rejection (7). Subsequent to these reports a number of studies followed in which platelets were labeled with indium to track their accumulation in renal transplants (8). Results of these studies demonstrated platelet accumulation to be an early indicator of transplant rejection. However, no clear mechanistic links were identified. More recent studies have also described intravascular platelet aggregates in clinical and experimental models of antibody-mediated rejection (9–12). Beyond their possible effects in the transplant, Kirk and colleagues have made a critical observation that CD154 shed from platelets remote from the transplant can serve as costimulatory molecules to induce rejection of murine cardiac allografts (13).
In light of the recent knowledge accumulated about the diverse roles of platelets in vascular maintenance and inflammation, a renewed examination of platelets in transplants is warranted. This review will provide an overview of platelet function in vascular inflammation, and then discuss the role of platelets in aspects of innate and adaptive immunity that relate to transplant rejection.
Platelets and endothelial cells
Endothelial cells are the primary interface between the transplanted organ and the leukocytes of the recipient. Platelets have evolved to orchestrate rapid responses to changes in the vascular endothelial cell barrier. Obviously, severe rejection disrupts the endothelial cell layer and exposes extracellular matrix components such as collagen that interact with platelet receptors such as GPIa (Figure 1). Exposed collagen, platelet phospholipids and tissue factor initiate the coagulation cascade that generates platelet agonists, most importantly thrombin. Thrombin activates endothelial cells, platelets, monocytes, dendritic cells and T cells through a family of thrombin protease-activated receptors (PARs). The multiple effects that coagulation factors and PARs have on ischemia-reperfusion and antibody-mediated rejection have been the subjects of a recent minireview (14). This response is expanded rapidly because activated platelets and endothelial cells release factors (e.g. ADP, thromboxane), which activate other platelets. Activation of platelets through ADP is regulated by CD39, an ectonucleotidase expressed on the membranes of normal vascular endothelium. CD39 hydrolyses ADP released from dense granules of activated platelets. In chronic rejection, CD39 activity is decreased on the vasculature of grafts suggesting a potential role for ectonucleotidases in transplant rejection that has been validated using genetically modified mice (15,16). Cardiac grafts from transgenic mice that overexpress CD39 have been found to be protected against antibody-mediated rejection (15). Methods to deliver soluble ectonucleotidases have been developed and present a novel potential therapeutic strategy to limit vascular inflammation and prolong graft survival (16).
Severe rejection is not required to initiate interaction of platelets with endothelium. Merely activating endothelial cells to express increased surface P-selectin as well as integrins such as ICAM and VCAM will engage platelets. P-selectin on endothelial cells transiently localizes platelets to the site of vascular inflammation through interactions with P-selectin glycoprotein ligand-1 (PSGL-1) on platelets. Endothelial P-selectin also concentrates exocytosed von Willebrand Factor (vWF) to the site and promotes platelet localization via interactions with GPIb (17). Following these first interactions, GPIIb/IIIa undergoes a receptor conformational change that permits binding to fibrinogen. GPIIb/IIIa binding to fibrinogen aids in firm platelet adhesion by crosslinking platelets and facilitates GPIIb/IIIa interaction with ICAM-1 (18). The cycle of interactions between platelets and endothelial cells leads to exocytosis of granules containing pro-inflammatory mediators from platelets, further endothelial cell activation, and leukocyte recruitment (Figure 2).
Figure 2. Platelet interactions with endothelial cells and mononuclear cells. Activated platelets in vein of rat cardiac allograft undergoing rejection (top panel). No platelets are attached to the intact endothelial cell layer on one side of the vein (upper side). In contrast, platelet aggregates (arrow heads) are attached to the inflamed side of the vein (lower side). The platelets are stained brown for vWF that anchors them to the wall of the vessel. Interspersed between the platelet aggregates are mononuclear cells (arrows). Platelet aggregates also fill some capillaries in the myocardium. The diagram illustrates some of the molecular interactions among platelets, endothelial cells, monocytes and T lymphocytes (bottom panel).
Download figure to PowerPoint
Once activated, platelets also initiate interactions with quiescent endothelial cells. Huo and co-workers have demonstrated that activated platelets injected into an ApoE null mouse will exacerbate and accelerate the onset of atherosclerotic disease by a process that requires platelet expression of P-selectin (19). Subsequent work by Wagner and colleagues has further demonstrated that activated platelets induce exocytosis of P-selectin and vWF by endothelial cells (20). These reports demonstrate that platelets can directly stimulate an intact endothelium by a P-selectin-dependent mechanism. Obviously, the pro-inflammatory effects of platelets on endothelial cells could be of critical importance in transplantation, but this remains to be tested.
Vasoactive effects of platelets caused by lipid-derived inflammatory mediators
Inflammatory cells and platelets produce and respond to potent lipid mediators such as thromboxane and leukotrienes that have vasoactive effects. Experimental models indicate that some of these inflammatory mediators can have effects in transplantation ranging from cytoprotection to accelerating rejection. Platelets express cyclooxygenase (COX) and lipoxygenase (LOX) enzymes that use arachadonic acid substrates to produce pro-inflammatory lipid mediators (21). Many studies have indicated that platelets only contain COX-1, however, IL-1β and TNF-α increase COX-2 expression in megakaryocytes, and consequently in platelets (22).
Upon initial platelet tethering to an inflamed vascular endothelium, platelets activate COX and LOX and produce thromboxane and leukotrienes. These can then function in either an autocrine or paracrine fashion to exert effects on other receptor expressing cells including endothelial cells and leukocytes. During acute rejection, production of prostanoids, such as thromboxane, is increased within an allograft (23). These molecules may affect graft function by modulating vascular tone, capillary permeability and platelet aggregation. Thromboxane may also directly modulate T lymphocyte and antigen-presenting cell function within the transplant. Studies have suggested that activation of thromboxane receptors promotes T-cell proliferation and contributes to immune-mediated tissue injury in mouse cardiac transplants. Mice that lack thromboxane receptors have a modest prolongation of cardiac allograft survival (24). However, the same group of investigators found mice that lack 5-lipoxygenase have an increased rate of renal allograft rejection (25). It is unclear if these results are a reflection of a difference in organ type (cardiac vs. renal transplant) or a reflection of a difference in the roles of thromboxane and leukotrienes in the inflammatory response. It is interesting to note that role of thromboxane in accelerating atherogenesis is not associated with thromboxane receptor signaling in bone marrow cells, and therefore depends on the receptors in the tissue bed (26). This may also account for potential differences in responses by renal and cardiac transplants to arachidonic acid-derived mediators.
Recently, Flavahan has challenged the simplistic idea that pro-inflammatory COX-1-dependent generation of thromboxane in platelets is balanced by COX-2-generated prostacyclin in endothelial cells (27). COX-2 derived prostacyclin has been noted to affect vascular remodeling and COX-2 inhibitors have been associated with increased risk of heart attack and stroke (28,29). This makes it interesting to speculate whether COX inhibitors likewise may be helpful or harmful in the setting of transplantation. Studies such as these indicate a need for further investigation into the role of platelet-derived lipid mediators in transplant rejection.
Platelets and innate immune responses
The secretory phase of platelet activation results in the release of numerous pro-inflammatory molecules that are key in the recruitment and activation of the innate immune system (Table 1). Interestingly, platelets are the source of the first described CXC class of chemokines, Platelet Factor 4 (PF4 or CXCL4), which is the protein secreted in the greatest amount by platelets (30). Secreted chemokines, such as PF4 and β-thromboglobulin (β-TG), recruit neutrophils. β-TG is a cleavage product of Platelet Basic Protein and it can be further cleaved to yield neutrophil-activating peptide 2 (NAP-2 or CXCL7). Secreted mediators such as RANTES (CCL5) and MCP-3 (CCL7) arrest monocytes at the site of platelet deposition (31). Adherent platelets also exteriorize adhesion molecules on their surface, including P-selectin, that assist in arresting leukocytes at the site of platelet activation (19). These recruitment functions of platelets aid in repair of vessels, but in the setting of an allogeneic transplant, platelet-induced inflammation can result in increased antigen expression and recognition.
Platelets not only release chemokines but also express functional receptors for chemokines including CCR1, CCR3, CCR4 and CXCR4. Chemokines for CCR1 (MIP-1α), CCR3 (RANTES), CCR4 (TARC and MDC) and CXCR4 (SDF-1), as well as MCP-1 can induce platelet aggregation. This provides multiple pathways for immune cells to modulate platelet activation (32).
When activated, platelets produce microparticles that have many pathophysiologic functions including initiation and exacerbation of inflammation (33). The submicron particles that are shed by activated platelets were originally described by Wolf in 1967 as ‘platelet dust’ (34). Since then, platelet microparticles have been found to express specific pro-inflammatory and pro-coagulation membrane components including proteins, such as P-selectin and GP IIb/IIIa, and lipid components, such as phosphatidylserine and sphingomyelin (33). These platelet-derived microparticles have been shown to increase expression of ICAM-1 on endothelial cells, and CD11a/CD18 and CD11b/CD18 on leukocytes. Expression of CD11b/CD18 allows monocytes to interact with platelets through GPIb, junctional adhesion molecule-C (JAM-C) and ICAM-2. As a result, activated platelets increase cell interactions and leukocyte phagocytic activity (35,36). It has also been demonstrated that P-selectin on platelet microparticles interacts with monocyte PSGL-1 to induce monocyte activation and further microparticle production from monocytes (37). The association of platelet microparticles with the onset and progression of atherosclerosis is consistent with their pro-inflammatory characteristics (33). A logical, but unstudied, extrapolation is that platelet-derived microparticles may also have an important function in transplant vasculopathy.
Involvement of platelets in antibody and complement-mediated immune responses
Intravascular aggregates of platelets have been noted in clinical and experimental models of antibody-mediated rejection of transplants (9–12). In experimental models, the terminal complement components significantly increase the numbers of platelets adherent to arterial endothelium in transplants (10–12). The terminal complement components (C5b-C9) form the membrane attack complex (MAC), which is capable of activating platelets directly or indirectly through activation of endothelial cells. MAC causes several responses in endothelial cells that can activate platelets, including retraction of endothelial cells to expose subendothelial matrix, release of vWF, expression of ICAM-1 and production of platelet-activating factor. Direct activation of platelets occurs when MAC inserts into the platelet membrane. This also stimulates production of microparticles enriched in C5b-9 and deficient in GPIIb/IIIa, suggesting that these are shed from the site of complement complex deposition (38). A logical extrapolation of these data is that complement activation by antibody during transplant rejection would stimulate platelet activation and microparticle production with potential adverse outcomes in accelerated graft arteriosclerosis.
Platelets express receptors for antibodies and early complement components that can result in activation even in the absence of MAC formation (39,40). The FcRγIIA and C1q receptor on platelets have been examined mainly in the context of immune complexes. However, these receptors would be expected to be engaged by antibodies and complement arrayed on the surface of an endothelial cell. This would provide an added mechanism for platelet adhesion to endothelial cells in antibody-mediated rejection.
Besides reacting to complement, platelets store and activate complement. An extensive analysis of platelet releasate demonstrated that platelet granules contain significant quantities of complement proteins including complement C3 and C4 precursors (41). It is therefore easy to envision that platelet activation may enhance inflammation by increasing the local concentration of complement proteins. Platelets also act as a site of C3 activation. A novel study by Del Conde et al. demonstrated that P-selectin expressed on activated platelets serves as a nidus for C3 activation (42). Binding of C3 to P-selectin results in production of the small fluid phase cleavage product C3a that can chemoattract and activate leukocytes, as well as activate endothelial cells through receptors for C3a. The larger cleavage product, C3b, that remains covalently bound to the platelet membrane serves as a ligand for complement receptor 1 (CR1; CD35) that is expressed on neutrophils, monocytes and some T cells.
Activated platelets not only release and activate complement, but can also modify complement components and alter their activity. Upon activation platelets release casein kinase. Casein kinase can phosphorylate C3b delaying its cleavage and increasing its concentration at sites of activation (43,44). Thus the inflammatory potential of C3b is increased. Therefore, activation of complement by low levels of antibodies can be augmented through activated platelets.
Platelets and T-cell mediated immune responses
Activated platelets can recruit T and B cells to the site of the lesion development. As noted before, activated platelets secrete RANTES, which in addition to recruiting monocytes, also recruits T cells. Danese et al. have noted a platelet-mediated T-cell feedback loop in which platelets secrete RANTES to recruit T cells, and then activated T cells stimulate platelets through CD40-CD154 interactions to secrete RANTES thereby recruiting more T cells to the site of interaction (45,46). In experiments of ischemia/reperfusion injury that are of direct relevance to organ procurement for transplantation, Khandoga et al. reported that platelets augment the recruitment of CD4+ T cells to post-ischemic liver sinusoids (47). These authors suggest that CD154 on both platelets and CD4+ cells adhere to CD40 on endothelial cells. The adherent platelets and T cells then interact with each other through CD40/CD154 and more effectively through P-selectin/PSGL-1 stimulation. The stimulated platelets release RANTES that recruits more T cells into the sinusoid.
In studies of antibody responses to adenovirus, Elzey et al. established that CD154 on platelets could augment the help delivered by low levels of CD4+ T cells for germinal center development and isotype switching to IgG antibody production (3). Passive transfer of CD154+ platelets and T cells to CD154 deficient recipients identified costimulation as the mechanism by which platelets supplemented helper T-cell function.
Platelets have been shown to interact with cytotoxic as well as helper T cells. Iannacone et al. demonstrated that activated platelets enhance intrahepatic accumulation of cytotoxic T-lymphocytes in murine models of viral hepatitis and that platelet depletion ameliorates disease severity (48). Again using an adenovirus model, Elzey et al. found that augmentation of the primary CD8+ T-cell response was dependent on the expression of CD154 by platelets. They hypothesized that passively transferred CD154+ platelets from wild-type mice caused maturation of dendritic cells in CD154-deficient mice (3,49).
Studies examining the influence of platelets on dendritic cells (DC) have yielded varied results depending upon the progenitor cell and the conditions. Coincubation of active platelets with bone marrow dendritic cells induces DC maturation and secretion of IL-6 and IL-12 (3). Other studies have demonstrated that platelets express heat shock protein gp96, which can initiate the induction of a pro-inflammatory response by macrophages and DC (50). However, Katoh et al. found that macrophages and DCs stimulated by platelet-derived serotonin have significantly reduced stimulatory activity toward T cells (51).
These studies of platelet interactions with dendritic cells, T cells and B cells provide a provocative background to consider mechanism by which platelets can alter adaptive immune responses to transplants. For example, the soluble CD154 that Kirk and colleagues found to induce rejection of murine cardiac allografts may alter antigen presentation, T-cell helper or cytotoxic responses (13,52).
Regulatory T cells may counterbalance some of the pro-inflammatory effects of platelets on endothelial cells and leukocytes. CD39 is expressed on the surface of regulatory T cells (53,54). As discussed before, CD39 is an ectonucleotidase that causes hydrolysis of extracellular ATP and ADP to AMP. This inhibits ADP-mediated platelet activation. Then another ectonucleotidase, CD73, which is also expressed regulatory T cells, converts extracellular AMP to adenosine, and adenosine suppresses proliferation and cytokine secretion by helper T cells (53).
Platelets and stem cell migration
With improved transplant survival, accelerated graft arteriosclerosis (AGA) has become an increasingly greater cause of graft loss. AGA is characterized by neointimal thickening mainly due to a proliferation of vascular smooth muscle cells (VSMCs). The origin of VSMC in AGA is controversial, but the predominant current view is that the VSMC originate from at least two sources: (1) proliferation of donor VSMC and (2) localization of progenitor cells from recipient marrow (55,56).
Studies in the past few years have clearly demonstrated a role for platelets in localizing circulating progenitor cells to a site of vascular damage. For example, Massberg et al. have demonstrated that platelets secrete stromal cell-derived factor α (SDF-1α) and recruit bone marrow-derived progenitor cells to arterial thrombi in vivo (5). In addition, Langer et al. demonstrated in vitro that adherent platelets can immobilize and promote the differentiation of endothelial progenitor cells to endothelial cells (4,5). These and other studies indicate that a graft endothelial cell dysfunction may lead to platelet localization, promotion of progenitor cell migration and acceleration of intimal thickening.