Platelet activation by Streptococcus sanguinis is accompanied by MAP kinase phosphorylation.

There is increasing interest in the role of infections in atherothrombotic conditions. In particular, bacteria, notably those of oral origin, have been shown to activate platelets using a variety of mechanisms. Previous studies have shown that S. sanguinis strain 2017-78 induces platelet aggregation which requires the presence of both vWF and IgG. This aggregation is accompanied by the consecutive phosphorylation/desphosphorylation/rephosphorylation of several signalling proteins. The first two phases are thromboxane-dependent whereas the rephosphorylation phase is mediated by engagement of the αIIbß3 integrin. Here signalling events, specifically the potential role of MAP kinases, associated with S. sanguinis strain 2017-78-induced platelet activation have been further examined using an immunoblotting approach. The addition of S. sanguinis strain 2017-78 caused a similar triphasic phosphorylation profile of the platelet MAP kinase Erk2 to that seen with other phosphoproteins. Pretreatment with aspirin or RGDS did not affect 2017-78-induced Erk2 phosphorylation or desphosphorylation but both inhibited the rephosphorylation phase. In contrast the level of 2017-78-induced platelet MAP kinase p38 phosphorylation remained at an elevated level, and this was unaffected by aspirin. Similarly, 2017-78-induced cPLA(2) phosphorylation remained above basal levels during the aggregation process. The p38 inhibitor SB203580 inhibited S. sanguinis-induced aggregation with no effect on the phosphorylation of either p38 or cPLA(2). Thus the current study demonstrates the activation of both the Erk2 and p38 forms of MAP kinases, and of cPLA(2), in platelets stimulated with S. sanguinis strain 2017-78, and is consistent with a role for Erk2, but not for p38, in the cPLA(2) phosphorylation in response to S. sanguinis.

Human platelets can discriminate between various bacterial LPS isoforms via TLR4 signaling and differential cytokine secretion.

Platelets are currently acknowledged as cells of innate immunity and inflammation and play a complex role in sepsis. We examined whether different types of LPS have different effects on the release of soluble signaling/effective molecules from platelets. We used platelet-rich plasma from healthy volunteers and LPS from two strains of gram-negative bacteria with disparate LPS structures. We combined LPS-stimulated platelet supernatants with reporter cells and measured the PBMC cytokine secretion profiles. Upon stimulation of platelets with both Escherichia coli O111 and Salmonella minnesota LPS, the platelet LPS::TLR4 interaction activated pathways to trigger the production of a large number of molecules. The different platelet supernatants caused differential PBMC secretion of IL-6, TNFα, and IL-8. Our data demonstrate that platelets have the capacity to sense external signals differentially through a single type of pathogen recognition receptor and adjust the innate immune response appropriately for pathogens exhibiting different types of 'danger' signals.

Mechanisms of oral bacteria-induced platelet activation.

The oral cavity is inhabited by over 500 different bacterial species that normally exist in ecological balance both with each other and with the host. When this equilibrium is disturbed, an overgrowth of individual organisms can occur, which, in turn, can lead to the onset of pathological processes, notably dental caries and periodontitis. Generally, bacteraemias occur more frequently in individuals with periodontal disease, and these bacteraemias have been implicated in the development of a range of systemic diseases, including atherothrombotic disorders. The mechanism underlying this relationship remains to be precisely defined, although studies have shown a link between bacteria of oral origin and platelet activation. Several orally derived species of bacteria interact with platelets, including those of the Streptococcus (Streptococcus sanguinis, Streptococcus mutans, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus gordonii, Streptococcus pneumoniae, Streptococcus mitis) and Staphylococcus (Staphylococcus epidermidis, Staphylococcus capitis) genera, as well as Pseudomonas aeruginosa and Porphyromonas gingivalis. In addition, some members of both the Streptococcus and the Staphylococcus genera, as well as Porphyromonas gingivalis, can activate platelets in vitro. The current review describes the heterogeneous mechanisms of platelet activation employed by individual bacterial species. The pathological and clinical implications of platelet activation by orally derived bacteria are discussed.

NF-κB Links TLR2 and PAR1 to Soluble Immunomodulator Factor Secretion in Human Platelets.

The primary toll-like receptor (TLR)-mediated immune cell response pathway common for all TLRs is MyD88-dependent activation of NF-κB, a seminal transcription factor for many chemokines and cytokines. Remarkably, anucleate platelets express the NF-κB machinery, whose role in platelets remains poorly understood. Here, we investigated the contribution of NF-κB in the release of cytokines and serotonin by human platelets, following selective stimulation of TLR2 and protease activated receptor 1 (PAR1), a classical and non-classical pattern-recognition receptor, respectively, able to participate to the innate immune system. We discovered that platelet PAR1 activation drives the process of NF-κB phosphorylation, in contrast to TLR2 activation, which induces a slower phosphorylation process. Conversely, platelet PAR1 and TLR2 activation induces similar ERK1/2, p38, and AKT phosphorylation. Moreover, we found that engagement of platelet TLR2 with its ligand, Pam3CSK4, significantly increases the release of sCD62P, RANTES, and sCD40L; this effect was attenuated by incubating platelets with a blocking anti-TLR2 antibody. This effect appeared selective since no modulation of serotonin secretion was observed following platelet TLR2 activation. Platelet release of sCD62P, RANTES, and sCD40L following TLR2 or PAR1 triggering was abolished in the presence of the NF-κB inhibitor Bay11-7082, while serotonin release following PAR1 activation was significantly decreased. These new findings support the concept that NF-κB is an important player in platelet immunoregulations and functions.

A role for immunoglobulin G in donor-specific Streptococcus sanguis-induced platelet aggregation.

There is increasing evidence for a relationship between bacterial infections and several cardiovascular disorders. Although the precise mechanism(s) underlying this association is unknown, the direct activation of platelets by bacteria is one possibility. Individual strains of S. sanguis activate platelets in a non-uniform, donor-dependent manner. In the current study, platelet aggregation profiles were obtained for fourteen donors in response to four strains of S. sanguis (2017-78, 133-79, SK112, SK108a) and one of S. gordonii (SK8) . The platelets from all donors responded to strains 2017-78 and 133-79, whereas strains SK112, SK8 and SK108a caused aggregation in one, five and twelve donors, respectively. Immunoglobulin G (IgG) binding to strains 2017-78, 133-79 and SK108a were significantly greater than to strains SK112 and SK8. Absorption of IgG by strain 2017-78 caused significant decreases in IgG binding, and platelet aggregation in response, to all strains. Single-strand conformational polymorphisms were observed in the Fcgamma RIIA gene from four donors. Sequencing revealed two known and two novel point mutations, none of which correlated with the aggregation profile. Thus, platelet activation to the various strains depends on a common IgG and, while in most cases the level of IgG binding to S. sanguis determines platelet responsiveness, neither the levels of IgG nor FcgammaRIIA polymorphisms can fully account for donor variability.

Streptococcus sanguis-induced platelet activation involves two waves of tyrosine phosphorylation mediated by FcgammaRIIA and alphaIIbbeta3.

The low-affinity IgG receptor, FcgammaRIIA, has been implicated in Streptococcus sanguis-induced platelet aggregation. Therefore, it is likely that signal transduction is at least partly mediated by FcgammaRIIA activation and a tyrosine kinase-dependent pathway. In this study the signal transduction mechanisms associated with platelet activation in response to the oral bacterium, S. sanguis were characterised. In the presence of IgG, S. sanguis strain 2017-78 caused the tyrosine phosphorylation of FcgammaRIIA 30s following stimulation, which led to the phosphorylation of Syk, LAT, and PLCgamma2. These early events were dependent on Src family kinases but independent of either TxA(2) or the engagement of the alpha(IIb)beta(3) integrin. During the lag phase prior to platelet aggregation, FcgammaRIIA, Syk, LAT, and PLCgamma2 were each dephosphorylated, but were re-phosphorylated as aggregation occurred. Platelet stimulation by 2017-78 also induced the tyrosine phosphorylation of PECAM-1, an ITIM-containing receptor that recruits protein tyrosine phosphatases. PECAM-1 co-precipitated with the protein tyrosine phosphatase SHP-1 in the lag phase. SHP-1 was also maximally tyrosine phosphorylated during this phase, suggesting a possible role for SHP-1 in the observed dephosphorylation events. As aggregation occurred, SHP-1 was dephosphorylated, while FcgammaRIIA, Syk, LAT, and PLCgamma2 were rephosphorylated in an RGDS-sensitive, and therefore alpha(IIb)beta(3)-dependent, manner. Additionally, TxA(2) release, 5-hydroxytryptamine secretion and phosphatidic acid formation were all blocked by RGDS. Aspirin also abolished these events, but only partially inhibited alpha(IIb)beta(3) -mediated re-phosphorylation. Therefore, S. sanguis -bound IgG cross links FcgammaRIIA and initiates a signaling pathway that is down-regulated by PECAM-1-bound SHP-1. Subsequent engagement of alpha(IIb)beta(3) leads to SHP-1 dephosphorylation permiting a second wave of signaling leading to TxA(2) release and consequent platelet aggregation.

The Inflammatory Role of Platelets via Their TLRs and Siglec Receptors.

Platelets are non-nucleated cells that play central roles in the processes of hemostasis, innate immunity, and inflammation; however, several reports show that these distinct functions are more closely linked than initially thought. Platelets express numerous receptors and contain hundreds of secretory products. These receptors and secretory products are instrumental to the platelet functional responses. The capacity of platelets to secrete copious amounts of cytokines, chemokines, and related molecules appears intimately related to the role of the platelet in inflammation. Platelets exhibit non-self-infectious danger detection molecules on their surfaces, including those belonging to the "toll-like receptor" family, as well as pathogen sensors of other natures (Ig- or complement receptors, etc.). These receptors permit platelets to both bind infectious agents and deliver differential signals leading to the secretion of cytokines/chemokines, under the control of specific intracellular regulatory pathways. In contrast, dysfunctional receptors or dysregulation of the intracellular pathway may increase the susceptibility to pathological inflammation. Physiological vs. pathological inflammation is tightly controlled by the sensors of danger expressed in resting, as well as in activated, platelets. These sensors, referred to as pathogen recognition receptors, primarily sense danger signals termed pathogen associated molecular patterns. As platelets are found in inflamed tissues and are involved in auto-immune disorders, it is possible that they can also be stimulated by internal pathogens. In such cases, platelets can also sense danger signals using damage associated molecular patterns (DAMPs). Some of the most significant DAMP family members are the alarmins, to which the Siglec family of molecules belongs. This review examines the role of platelets in anti-infection immunity via their TLRs and Siglec receptors.

Inhibition of the MEK/ERK pathway has no effect on agonist-induced aggregation of human platelets.

The activation of human platelets by a variety of agonists is accompanied by the phosphorylation of the extracellular signal-regulated kinase (ERK) isoforms of mitogen-activated protein (MAP) kinases. However, the role(s) of, and the substrate(s) for, these enzymes in platelet function remain unclear. Studies on ERKs in platelets have relied on pharmacological tools, including an inhibitor of ERK activation, U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene]. In the present study, the effects of U0126 and its "inactive" analogue, U0125 [1,4-diamino-2,3-dicyano-1,4-bis(phenylthio)butadiene], on human platelet aggregation and MAP kinase activity were examined. Several agonists with a variety of signaling pathways were studied including thrombin, a thromboxane analogue, arachidonic acid, collagen, calcium ionophores, and the phorbol ester phorbol myristate acetate (PMA). U0126, at concentrations consistent with inhibition of the isolated enzyme, inhibited ERK phosphorylation, and therefore MEK activation, in response to each agonist. Under such conditions, U0126 did not affect the phosphorylation of a second MAP kinase, p38(MAPK); however, platelet aggregation was also unaffected. Higher concentrations of U0126, and of U0125, inhibited platelet aggregation in response to collagen and PMA with no effect on that induced by the other agonists. These results dissociate ERK activation from platelet aggregation, suggesting an alternative role for ERKs in platelet function. In addition, the effects of higher concentrations of U0126 are likely due to an action on protein kinase C, likely unrelated to ERK inhibition, suggesting that the inhibitor concentration is crucial to the interpretation of such studies.

Evidence for a role of mitogen-activated protein kinases in proliferating and differentiating odontogenic epithelia of inflammatory and developmental cysts.

OBJECTIVE: The activation of intracellular signaling cascades involving serine/threonine kinases ERK1/2 has been variably reported either to stimulate or inhibit epithelial cell differentiation in response to extracellular signals. The purpose of our study was to determine the distribution of the signaling molecule ERK1 and its activated form pERK1/2 in the epithelial components of developmental and inflammatory odontogenic cysts in relation to parameters of differentiation and proliferation. STUDY DESIGN: Thirty samples of dental follicles, dentigerous cysts, and radicular cysts were immunostained with antibodies to ERK1, pERK1/2, and proliferating cell nuclear antigen (a marker for proliferation). The tissues were subclassified according to the pattern of histomorphological differentiation (ie, squamous differentiation) and the proliferation rate of their epithelial components. The significance of differences in the proportion of ERK1- and pERK1/2-expressing cells among the tissue groups was determined by chi-square analysis or Fisher's exact test. RESULTS: ERK1 and pERK1/2 were found to be expressed in a significantly higher proportion of cells with differentiated and highly proliferating epithelial components, as compared with those of nondifferentiated, quiescent epithelial rests. The epithelium of radicular cysts exhibited the highest proportion of pERK1/2-positive cells. In both dentigerous and radicular cyst samples, pERK1/2 expression was significantly higher in the inflamed tissues. CONCLUSIONS: These data demonstrate that ERK1 and its active form pERK1/2 are associated with differentiating and actively proliferating epithelia of odontogenic cysts, and are consistent with pERK1/2 involvement in the activation of odontogenic epithelia in response to inflammation.

Markers of platelet activation are increased in adolescents with type 2 diabetes.

OBJECTIVE: In adults with diabetes, in vivo platelet activation is a marker for atherosclerosis and cardiovascular disease (CVD). This pilot study investigated whether adolescents with diabetes had evidence of increased in vivo platelet activation. RESEARCH DESIGN AND METHODS: In vivo platelet activation was compared in four groups of age-matched adolescents: type 1 diabetes (T1D, n = 15), type 2 diabetes (T2D; n = 15), control subjects with normal BMI (n = 14), and overweight/obese control subjects (n = 13). Platelet surface activation markers and plasma levels of soluble activation markers were measured and compared among groups. RESULTS: Increased expression of all activation markers was observed in T2D compared with either control group (P < 0.05); levels of soluble markers were also higher in T2D than in T1D (P < 0.05). There were no differences in marker expression between the nondiabetic control groups. CONCLUSIONS: Platelet activation in adolescents with T2D may be a marker for the risk of CVD development in early adulthood.

Cyclic nucleotides inhibit MAP kinase activity in low-dose collagen-stimulated platelets.

Collagen-induced platelet activation is a complex process involving multiple signaling pathways. The role(s) of MAP kinases (ERKs and p38(MAPK)) are unclear, although at high, but not low, collagen concentrations p38(MAPK) is involved in cPLA(2)-mediated arachidonic acid release, prior to thromboxane generation. Cyclic nucleotides are conventionally regarded as mediators of platelet inhibition. However recent studies suggested a role for cGMP early in a MAP kinase pathway in platelet activation. In the current study the roles and relationships of MAP kinases, cyclic nucleotides and cPLA(2) in platelet activation by low-dose collagen and a thromboxane analogue (U46619) have been evaluated. Stimulants of neither adenylate cyclase (PGI(2)) nor guanylate cyclase (NaNP) alone had any effect on the basal phosphorylation of either MAP kinase. PGI(2) inhibited ERK/p38(MAPK) phosphorylation in response to both agonists which was unaffected by a cPLA(2) inhibitor (AACOCF(3)). NaNP inhibited collagen-induced ERK/p38(MAPK) phosphorylation, which was enhanced by AACOCF(3) and reversed by a guanylate cyclase inhibitor (ODQ). However NaNP had no effect on U46619-induced p38(MAPK) phosphorylation. Thus adenylate cyclase activation inhibits low-dose collagen-induced MAP kinase phosphorylation both prior, and distal, to thromboxane release. The study also supports an inhibitory, rather than stimulatory, role for guanylate cyclase in platelet signaling.

Beyond hemostasis: the role of platelets in inflammation, malignancy and infection.

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 alphaIIbbeta3 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.

Expression of functional recombinant mosquito salivary apyrase: a potential therapeutic platelet aggregation inhibitor.

Excessive platelet activation and accumulation can lead to vessel occlusion and thus present major therapeutic challenges in cardiovascular medicine. Apyrase, an ecto-enzyme with ADPase and ATPase activities, rapidly metabolizes ADP and ATP released from platelets and endothelial cells, thereby reducing platelet activation and recruitment. In the present study, we expressed a 68-kDa recombinant mosquito (Aedes aegypti) salivary apyrase using a baculovirus/insect cell expression system and purified it to homogeneity using anion-exchange chromatography on a large scale. A yield of 18 mg of purified recombinant apyrase was obtained from 1 litre of the medium. Kinetic analysis indicated that the recombinant apyrase had a K(m) of 12.5 microM for ADP and a K(m) of 15.0 microM for ATP. The recombinant apyrase inhibited ADP-, collagen- and thrombin-induced human platelet aggregation in a dose-dependent manner, indicating that the recombinant protein retained nucleotidase activity in a whole cell system, which suggests that it may serve as a therapeutic agent for inhibition of platelet-mediated thrombosis.

Cellular prion protein is released on exosomes from activated platelets.

Cellular prion protein (PrP(C)) is a glycophosphatidylinositol (GPI)-anchored protein, of unknown function, found in a number of tissues throughout the body, including several blood components of which platelets constitute the largest reservoir in humans. It is widely believed that a misfolded, protease-resistant form of PrP(C), PrP(Sc), is responsible for the transmissible spongiform encephalopathy (TSE) group of fatal neurodegenerative diseases. Although the pathogenesis of TSEs is poorly understood, it is known that PrP(C) must be present in order for the disease to progress; thus, it is important to determine the physiologic function of PrP(C). Resolving the location of PrP(C) in blood will provide valuable clues as to its function. PrP(C) was previously shown to be on the alpha granule membrane of resting platelets. In the current study platelet activation led to the transient expression of PrP(C) on the platelet surface and its subsequent release on both microvesicles and exosomes. The presence of PrP(C) on platelet-derived exosomes suggests a possible mechanism for PrP(C) transport in blood and for cell-to-cell transmission.

Platelets and anti-platelet therapy.

Platelets play a central role in the hemostatic process and consequently are similarly involved in the pathological counterpart, thrombosis. They adhere to various subendothelial proteins, exposed either by injury or disease, and subsequently become activated by the thrombogenic surface or locally produced agonists. These activated platelets aggregate to form a platelet plug, release agonists which recruit more platelets to the growing thrombus, and provide a catalytic surface for thrombin generation and fibrin formation. These platelet-rich thrombi are responsible for the acute occlusion of stenotic vessels and ischemic injury to heart and brain. A range of anti-platelet drugs are currently used, both prophylactically and therapeutically, in regimens to manage thrombo-embolic disorders. These include inhibitors of the generation, or effects, of locally produced agonists; several large clinical trials have supported roles for cyclooxygenase inhibitors, which prevent thromboxane generation, and thienopyridine derivatives, which antagonize ADP receptors. Similarly intravenous alpha IIb beta 3 antagonists have been shown to be effective anti-thrombotics, albeit in highly selective situations; in contrast, to date studies with their oral counterparts have been disappointing. Recent advances in understanding of platelet physiology have suggested several novel, if yet untested, targets for anti-platelet therapy. These include the thrombin receptor, the serotonin handling system, and the leptin receptor.

Mechanism of collagen activation in human platelets.

The mechanism of collagen-induced human platelet activation was examined using Ca2+, Na+, and the pH-sensitive fluorescent dyes calcium green/fura red, sodium-binding benzofuran isophthalate, and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Administration of a moderate dose of collagen (10 microg/ml) to human platelets resulted in an increase in [Ca2+](i) and platelet aggregation. The majority of this increase in [Ca2+](i) resulted from the influx of calcium from the extracellular milieu via the Na+/Ca2+ exchanger (NCX) functioning in the reverse mode and was reduced in a dose-dependent manner by the NCX inhibitors 5-(4-chlorobenzyl)-2',4'-dimethylbenzamil (KD(50) = 4.7 +/- 1.1 microm) and KB-R7943 (KD(50) = 35.1 +/- 4.8 microm). Collagen-induced platelet aggregation was dependent on an increase in [Ca2+](i) and could be inhibited by chelation of intra- and extracellular calcium through the administration of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) and EGTA, respectively, or via the administration of BAPTA-AM to platelets suspended in no-Na+/HEPES buffer. Collagen induced an increase in [Ca2+](i) (23.2 +/- 7.6 mm) via the actions of thromboxane A(2) and, to a lesser extent, of the Na+/H+ exchanger. This study demonstrates that the collagen-induced increase in [Ca2+](i) is dependent on the concentration of Na+ in the extracellular milieu, indicating that the collagen-induced increase in [Ca2+](i) causes the reversal of the NCX, ultimately resulting in an increase in [Ca2+](i) and platelet aggregation.