Platelet Secretion in Inflammatory and Infectious Diseases

Mechanisms of Platelet Exocytosis

Platelet granular secretion, or exocytosis, is required for normal platelet function and in health and disease. The exocytosis mechanisms of platelet α- and dense granules are similar to those used by other specialized secretory cells such as neurons and neutrophils. Platelet granule exocytosis is initiated and regulated through activation of cell surface receptors. Upon platelet activation, exocytosis of granules is initiated by the movement of granules into the plasma membrane, which then leads to granule-plasma membrane fusion and release of intracellular contents (Figure 1). The mechanisms of platelet secretion and the proteins that regulate these events have been elaborately studied using murine models and human patients and some aspects are briefly highlighted below.

SNAREs play crucial roles in platelet granule exocytosis

Platelet secretion occurs primarily through a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent mechanism[23]. SNAREs play a crucial role in the fusion of the granular and plasma membrane. t-SNAREs and v-SNAREs, which are present on different membranes (t-target and v-vesicle), can form a tight trans-complex that prompts membrane fusion to facilitate secretion[24]. The SNARE proteins are derived from syntaxins, VAMP, and SNAP-25-related genes that orchestrate granule membrane fusion to the plasma membrane or open canalicular system[25]. The precise role(s) of some of the individual proteins mediating these process remains incompletely understood; in part due to the close structural similarities and compensatory mechanisms. The discovery of naturally occurring human variants in SNAREs and the development of knockout murine models have helped us to better understand platelet granular exocytosis mechanisms. For example, VAMP-8, a vesicle-membrane SNARE (v-SNARE), is thought to be the primary regulator of platelet granule release although redundant mechanisms exist. In VAMP-8 knockout mice, platelet granular secretion is impaired with mild agonist stimulation. With stronger stimulation, secretory responses are preserved, albeit in a less efficient manner. This secondary system, which is slower, involves VAMP-2 and VAMP-3[26]. Consistent with this, thrombus formation to laser-induced injury is delayed and reduced, but not completely abrogated, in the absence of VAMP-8[27].

Syntaxin-11 (STX-11) is one of the major t-SNARE expressed in platelets plasma membrane. The significance of STX-11 in secretion was first discovered in patients with Familial Hemophagocytic Lymphohistiocytosis type 4 (FHL-4), a rare genetic disease characterized by cytotoxic T lymphocytes and NK cell dysfunction. Patients with FHL-4 have mutations in STX-11, which makes this protein functionally inactive. Platelet α- and dense granule secretion is substantially impaired in patients with FHL-4[28]. In contrast, genetically ablating either STX-2 or STX-4 in mice did not inhibit platelet secretion, demonstrating that STX-11 is required for platelet secretion[28].

Munc and Rab proteins regulate SNARE complexes

The Sec1/Munc18 and Rab proteins regulate SNARE interactions. There are three Munc18 (mammalian uncoordinated-18) isoforms in platelets: Munc18a, Munc18b, and Munc18c. Of these three isoforms, Munc18b (also called STXBP2 or Munc18-2) is thought to be the primary isoform in platelets and necessary for platelet secretion. In humans with FHL-3, mutations occur in munc13-4 (UNC13-D). Platelets from FHL-3 patients and munc-13-4 knockout mice exhibit severe impairments in dense granule, α-granule, and lysosome exocytosis[29–31]. In patients with FHL-5, mutations occur in munc18b[32–35]. Patients with FHL-5 also have defects in platelet dense and α-granule secretion, but lysosome exocytosis appears to be only affected in FHL-5 biallelic patients[35]. These findings suggest that munc18b may be a more specific regulator of dense and α-granule secretion machinery. The Rab proteins, which may mediate vesicle docking and participate in secretory events, regulate Munc13 proteins[35–37]. As one example, Rab27b is a small GTPase that is highly expressed in platelets that, when absent, results in defects in dense granule secretion.

Calcium and PKC regulate platelet secretion by phosphorylation of SNAREs

Platelet secretion occurs upon activation by specific agonists (ADP, thrombin, collagen, thromboxane A2, etc.) through Gq-protein-coupled receptors or other mechanisms (e.g., collagen, fucoidan, rhodocytin)[38–41]. While increases in intracellular [Ca2+] are sufficient to induce platelet exocytosis, phosphokinase C (PKC) also appears to act synergistically with calcium to amplify platelet secretion. PKC regulated platelet secretion acts through phosphorylation of SNARE proteins. For example, upon stimulation by thrombin, Munc18c, syntaxin 4, and SNAP-23 undergo PKC-dependent phosphorylation in platelets[33, 42]. In addition, PKC-dependent phosphorylation also mediates the binding of syntaxin 4 to SNAP-23. Recent studies have shown that SNAP-23 at serine residue (Ser95) is phosphorylated by IkB Kinase 2 is crucial for granular secretion by regulating SNARE complex formation and membrane fusion[43]. Furthermore, Reed and colleagues also used SNAP-23 mutants (Ser²³ → Asp/Thr²⁴ → Asp), which mimic the phosphorylation at Ser23/Thr24 and block syntaxin 4 interactions with SNAP-23. This demonstrates that phosphorylation events at Ser23 also regulate SNARE-complex interactions during membrane trafficking and fusion [44]. These studies suggest that phosphorylation sites on SNARE proteins regulate platelet granular secretion.

Mechanisms of platelet endocytosis

Endocytosis is a cellular process where cells absorb molecular substances from outside the cell by engulfing it with the cell membrane. Platelets not only uptake, or endocytose, particles and solutes from the surrounding environment, but also retain and transport these substances for prolonged period of time[45]. Endocytosis is an important process in platelets and megakaryocytes for loading certain granule cargo, such as fibrinogen (Fg) and vascular endothelial growth factor (VEGF)[46–48]. While the mechanisms of platelet endocytosis remain incompletely understood, recent studies demonstrate that adenosine 5′-diphosphate–ribosylation factor 6 (Arf6), a small guanosine triphosphate-binding protein, regulates integrin α2bβ3 trafficking and fibrinogen uptake[49]. Genetic ablation of Arf6 in platelets enhanced clot retraction and spreading, indicating that endocytosis contributes to rapid platelet functions. These findings are consistent with Kanamarlapudi and colleagues who showed that Arf6 regulates P2Y trafficking in platelets as well as dynamin-dependent fission of coated vesicles during endocytosis[50]. Additionally, two Rab proteins (Rab4 and Rab11) are also involved in integrin-mediated fibrinogen uptake and trafficking in platelets[49, 51].

P-selectin

Platelet surface p-selectin, also known as CD62P (and previously known as PADGEM or GM-140) is a membrane protein stored within α-granules and, upon secretion, exposed at the cell surface where it mediates platelet interactions with p-selectin glycoprotein ligand (PSGL)-1 expressing cells[12, 52–54]. P-selectin is also stored within Weibel-Palade bodies in endothelial cells and thus is not absolutely specific to platelets. Through expression of p-selectin on their surface, activated platelets bind to circulating immune cells in the blood stream. In this manner, surface-adherent platelets facilitate the recruitment, rolling, and arrest of monocytes, neutrophils, and lymphocytes to the activated endothelium. Platelets also express PSGL-1 and employ this ligand to bind p-selectin on endothelial cells. P-selectin and PSGL-1 are the primary receptor-ligand pair facilitating the formation of heterotypic platelet-leukocyte aggregates (PLAs). The formation of PLAs is a very sensitive marker of in vivo platelet activation during human thrombotic and infectious disease[55–58]. Platelet-leukocyte aggregate formation also induces fibrin clot formation via interactions of PSGL-1 on leukocyte-derived microparticles with tissue factor on the platelet surface[59–62].

Through p-selectin, platelets have been shown to play a critical role in the recruitment of neutrophils to damaged lung capillaries. Platelet depletion, p-selectin neutralization, or selective knockout of p-selectin in the hematopoietic compartment reduces neutrophil recruitment and inhibits the development of acute lung injury[63, 64]. Adherence to platelets induces a host of pro-inflammatory responses in immune cells, including activation of pro-coagulant molecules, and promotion of cellular differentiation. These interactions may drive further platelet activation and secretion of granule contents. Below, we briefly review in more detail several cargo proteins secreted by platelets and their roles in human inflammatory and infectious diseases.

RANTES

RANTES (regulated on activation, normal T-cell expressed and secreted, also known as CCL5) contributes to the vascular inflammation associated with atherosclerosis[65, 66]. RANTES is stored in platelet α-granules and released following activation as a component of microparticles. RANTES is a key chemoattractant for eosinophils as well as triggering monocyte and T lymphocyte adhesion and transmigration[12, 67–69]. The packaging and release of RANTES within platelet-derived microparticles (PMPs) provides a mechanism whereby RANTES is transported and then deposited onto areas of injured or activated endothelium[70]. Binding of RANTES to injured endothelium enhances monocyte recruitment and activates monocyte integrins, consolidating their attachment and rolling on the endothelium and, in some settings, incorporation into atherosclerotic lesions[70–72], as further discussed below.

Platelet Factor 4

Platelet factor 4 (PF4, also known as CXCL4) was the first member of the chemokine family identified in platelets and is one of the most abundant proteins contained within platelet α-granules [73]. PF4 is released from activated platelets in a p-selectin dependent manner and exerts effects on numerous other cells. Secreted PF4 can trigger activation and adhesion of neutrophils to endothelial cells, phagocytosis and respiratory burst in monocytes, and chemotaxis of T lymphocytes [74–77]. Other secreted chemokines, including tumor necrosis factor and RANTES, may act synergistically with PF4 to signal to neutrophils and monocytes. For example, PF4 and RANTES may heterodimerize and arrest circulating monocytes on the endothelium[78].

Interleukin-1β

Several cytokines are also secreted by activated platelets, of which IL-1ß has been the most widely studied[79, 80]. Platelets store IL-1ß in its inactive, precursor pro-IL-1ß form. Upon platelet activation, pro-IL-1ß is rapidly processed to the mature, active form of IL-1ß. Platelets then secrete active IL-1ß in both a soluble form and in association with PMPs[81]. Platelets also possess the ability to synthesis de novo IL-1ß from mRNA that is present basally within resting human platelets[82]. Although both forms of IL-1ß enhance endothelial cell adhesiveness for neutrophils, microparticle-associated IL-1ß is significantly more effective at promoting adhesion. While still incompletely understood, this increased adhesive potency may be due to the presence of additional pro-inflammatory factors, such as p-selectin, on microparticles. 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.

Serotonin

Serotonin, which accumulates in dense granules as platelets circulate, is secreted by platelets as a highly sensitive and specific diagnostic test for heparin-induced thrombocytopenia[83]. Plasma and platelet serotonin levels are decreased in patients with systemic lupus erythematosis (SLE), an autoimmune disorder characterized by systemic inflammation and thrombosis[84]. Serotonin secreted from the dense granules of activated platelets influences vascular tone and promotes platelet aggregation. This secreted amino acid has also been targeted for clinical therapeutics. Sarpogrelate, an inhibitor of the 5-HT_2A serotonin receptor, was shown to inhibit platelet aggregation and reduce the risk of cerebral infarction. Interestingly, bleeding events in this phase III study were lower in patients receiving sarpogrelate than aspirin[85].

Atherosclerosis

In atherosclerosis, activated platelets facilitate recruitment of inflammatory cells, such as leukocytes and progenitor cells, to areas of injured endothelium or at sites of acute plaque rupture (Figure 2) via secretion of cytokines and chemokines from α-granules[86]. For example, the translocation of p-selectin from α-granules to the platelet surface allows platelets to interact with and bind to PSGL-1 on endothelial cells and leukocytes[53, 87, 88]. The canonical role of p-selectin in atherosclerosis is evident in murine models utilizing Apoe^−/− mice, where the genetic loss of p-selectin results in smaller lesions [89]. These results suggest that p-selectin expressed upon platelet activation from α-granules is at least partly necessary for platelet-endothelial cell interactions. In murine models, atherosclerosis progression may also depend on CD40L expression by platelets[90]. P-selectin also mediates the formation of platelet-leucocyte aggregates when other chemokines, including SDF-1, RANTES, PF4 and IL-1β, are concurrently secreted by activated platelets[91]. For example, disruption of PF4-RANTES heterodimers attenuates atherosclerotic lesions in some murine models[92]. RANTES is also deposited within atherosclerotic plaques, driving further monocyte recruitment and plaque progression.

SDF-1 (stromal cell-derived factor 1, also known as CXCL12) is also expressed within atherosclerosis lesions. SDF-1 plays an essential role in neointima formation after arterial injury in murine models[93, 94]. The mechanism involves regulation of neointimal smooth muscle content and expansion of circulating Sca-1⁺ lineage-progenitor cells. In human investigations, recent genomic studies have found an association between genetic variants in SDF and stroke[95].

PF4, a platelet specific chemokine, also localizes in atherosclerotic lesions and its expression within the lesion correlates with disease severity. Mechanistically, PF4 in the presence of TNF-α induces exocytosis and firm neutrophil interaction with endothelium there by enhancing inflammation in the lesion[75]. The secretion of PF4 and RANTES together in concert may synergistically promote atherosclerosis development and progression. Conversely antagonism of RANTES receptors significantly decreases the size of atherosclerotic lesions[96]. The effects of secreted PF4 are not limited to direct interactions with leukocytes. PF4 has also been shown to inhibit the degradation of the LDL receptor, thereby limiting lipoprotein removal with associated pro-atherogenic consequences[97]. PF4 also enhances the uptake of oxidized LDL by macrophages, which increased the foam cells in late atherosclerotic lesions[98].

Tumor Development and Progression

Platelet secretion is a critical driver of tumor biology and progression (Figure 3). Secreted α-granule contents, including vascular endothelial growth factor (VEGF) and p-selectin, stimulate endothelial migration and proliferation, a crucial step for tumor cells metastasis and survival[99]. An interesting early observation was the finding that thrombocytopenia may reduce tumor metastasis[100, 101]. While the exact mechanisms remain only partially understood, tumor cells aggregate and activate platelets - a process which is known as tumor cell-induced platelet aggregation (TCIPA)[102]. TCIPA is correlated with both thrombosis caused by tumor cells and also to the tumor cells’ metastatic potential[103]. Published studies demonstrate that platelets protect metastatic tumor cells from destruction by immune cells, as they travel in the blood circulation, and help tumor cells to attach to endothelium at metastatic sites[104–106]. Platelet and endothelial cell adhesion proteins may also transport tumor cells from blood circulation to distant sites of metastasis[107]. The mechanism may be at least partly p-selectin mediated as mice lacking p-selectin have reduced tumor metastasis.

In addition, when platelets come in contact with cancer cells they secrete transforming growth factor β (TGF-β), a potent immunoregulatory molecule which also promotes extravasation of colon and breast cancer cells[108]. The amount of TGF-β stored ωιτηιν α-granules appears to have a regulatory effect on systemic levels of TGF-β ωιτηιν the circulation[109]. Transforming growth factor-β from platelets also diminishes NK granule mobilization, cytotoxicity and interferon-γ secretion[110], key host defense mechanisms critical for detection and clearance of tumor cells. Other soluble mediators, including prostaglandin E2 (PGE2), may have similar activity. The ability of platelets to form heterotypic aggregates with tumor cells may also play a pivotal part in determining tumor cell survival within the microvasculature of affected organs and of tumor metastasis.

Platelet Secretion during Infectious Syndromes

In response to infectious cues, platelets secrete many factors, which activate classical immune cells (Table 1). In addition, platelets also secrete molecules that have direct antimicrobial properties[111]. For example, in experimental models and human studies of acute infectious settings, platelets circulate with increased surface expression of p-selection. Increased p-selectin, translocated from α-granules to the platelet surface, promotes the formation of circulating platelet-leukocyte aggregates and may contribute to adverse outcomes in septic syndromes[56, 57, 112, 113]. Platelet surface p-selectin (as well as endothelial cell p-selectin) mediate host responses to Klepsiella pneumoniae sepsis as mice deficient in p-selectin had increased bacterial loads within the pulmonary system, the blood, and organs[114]. P-selectin deficiency was also associated with decreased platelet-monocyte aggregates and, interestingly, increased cytokine release suggesting that p-selectin may attenuate injurious cytokine production and/or secretion during septic syndromes. P-selectin also serves as a trafficking molecule directing neutrophils to the lung during septic conditions[64, 115].

Platelet factor 4 (PF4) and CD40L, two key α-granule cargo proteins secreted upon activation, mediate key aspects of host responses to HIV infection. PF4 is able to bind the HIV-1 envelop protein, inhibiting the attachment of HIV-1 virus to the cell surface in a concentration-dependent effect[116, 117]. The inhibitory activity of PF4 identifies that platelet secretion augments defense mechanisms to HIV-1 and may extend to other pathogens. In contrast, soluble CD40L, which is increased during HIV-1 infection[118], impairs dendritic cell function and contributes to immune dysfunction in an IL-12 dependent mechanism[119].

Intriguingly, emerging studies suggest that platelets may be immune effector cells during malaria infection. Malaria is a mosquito-borne infectious disease that is widespread in tropical and subtropical regions. Thrombocytopenia commonly occurs in patients with malaria and correlates with adverse outcomes, particularly in settings where plasmodium falciparum is the causative strain[120, 121]. Recent evidence suggests that the primary platelet secretion component responsible for cytocidal action during malaria infections is PF4[122, 123]. PF4 is released in large quantities by activated platelets when they come in contact with the parasite-infected red blood cells (Figure 4). A recent study has shown that PF4 is internalized by parasite, which then lyses the parasite organ called the digestive vesicle (DV)[124, 125]. Platelet PF4 has also been shown to mediate inflammatory responses in cerebral malaria[126]. PF4 secreted from platelets also orchestrates leucocyte cerebral vascular trafficking in murine models of malaria. Interestingly, in human patients with cerebral malaria, PF4 may be a useful predictive biomarker.