sCD40L Release Research Articles

Synergy by Ristocetin and CXCL12 in Human Platelet Activation: Divergent Regulation by Rho/Rho-Kinase and Rac.

CXCL12, belonging to the CXC chemokine family, is a weak agonist of platelet aggregation. We previously reported that the combination of CXCL12 and collagen at low doses synergistically activates platelets via not CXCR7 but CXCR4, a specific receptor for CXCL12 on the plasma membrane. Recently, we reported that not Rho/Rho kinase, but Rac is involved in the platelet aggregation induced by this combination. Ristocetin is an activator of the von Willebrand factor that interacts with glycoprotein (GP) Ib/IX/V, which generates thromboxane A2 via phospholipase A2 activation, resulting in the release of the soluble CD40 ligand (sCD40L) from human platelets. In the present study, we investigated the effects of a combination of ristocetin and CXCL12 at low doses on human platelet activation and its underlying mechanisms. Simultaneous stimulation with ristocetin and CXCL12 at subthreshold doses synergistically induce platelet aggregation. A monoclonal antibody against not CXCR7 but CXCR4 suppressed platelet aggregation induced by the combination of ristocetin and CXCL12 at low doses. This combination induces a transient increase in the levels of both GTP-binding Rho and Rac, followed by an increase in phosphorylated cofilin. The ristocetin and CXCL12-induced platelet aggregation as well as the sCD40L release were remarkably enhanced by Y27362, an inhibitor of Rho-kinase, but reduced by NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction. These results strongly suggest that the combination of ristocetin and CXCL12 at low doses synergistically induces human platelet activation via Rac and that this activation is negatively regulated by the simultaneous activation of Rho/Rho-kinase.

Synergistic effect of collagen and CXCL12 in the low doses on human platelet activation.

CXCL12, also known as stromal cell-derived factor-1, is a chemokine classified into CXC families, which exerts its function by binding to specific receptors called CXCR4 and CXCR7. Human platelets express CXCR4 and CXCR7 on the plasma membrane. It has been reported that CXCL12 potentiates to induce platelet aggregation in cooperation with agonists including collagen. However, the precise roles and mechanisms of CXCL12 in human platelet activation are not fully elucidated. In the present study, we investigated the effect of simultaneous stimulation with low doses of collagen and CXCL12 on the activation of human platelets. The simultaneous stimulation with collagen and CXCL12 induced the secretion of platelet-derived growth factor (PDGF)-AB and the release of soluble CD40 ligand (sCD40L) from human platelets in addition to their aggregation, despite the fact that the simultaneous stimulation with thrombin receptor-activating peptide (TRAP) or adenosine diphosphate (ADP), and CXCL12 had little effects on the platelet aggregation. The agonist of Glycoprotein (GP) Ⅵ convulxin and CXCL12 also induced platelet aggregation synergistically. The monoclonal antibody against CXCR4 but not CXCR7 suppressed the platelet aggregation induced by simultaneous stimulation with collagen and CXCL12. The phosphorylation of p38 mitogen-activated protein kinase (MAPK), but not p44/p42 MAPK, was induced by the simultaneous stimulation. In addition, the simultaneous stimulation with collagen and CXCL12 induced the phosphorylation of HSP27 and the subsequent release of phosphorylated-HSP27 from human platelets. SB203580, a specific inhibitor of p38 MAPK, attenuated the platelet aggregation, the phosphorylation of p38 MAPK and HSP27, the PDGF-AB secretion, the sCD40L release and the phosphorylated-HSP27 release induced by the simultaneous stimulation with collagen and CXCL12. These results strongly suggest that collagen and CXCL12 in low doses synergistically act to induce PDGF-AB secretion, sCD40L release and phosphorylated-HSP27 release from activated human platelets via p38 MAPK activation.

Olive polyphenol reduces the collagen-elicited release of phosphorylated HSP27 from human platelets.

Hydroxytyrosol (HT) and oleuropein (OLE) are natural polyphenols found in extra virgin olive oil. Accumulating evidence indicates that ingestion of olive oil contributes to reduce the risk of cardiovascular diseases and stroke. It has been reported that HT and OLE inhibit human platelet aggregation. We have shown that collagen induces the phosphorylation of heat shock protein 27 (HSP27) in human platelets, resulting in the release of HSP27, an extracellular pro-inflammatory agent. In this study, we investigated the effects of HT and OLE on the collagen-stimulated human platelet activation. The PDGF-AB secretion and the soluble CD40 ligand (sCD40L) release by collagen were reduced by HT or OLE. HT and OLE significantly suppressed the phosphorylation of HSP27 and the release of phosphorylated-HSP27. These findings suggest that olive polyphenol reduces the collagen-stimulated phosphorylation of HSP27 in human platelets and the release. Our results may provide a novel anti- inflammatory effect of olive polyphenol.

The Role of Human Platelet Preparation for Toll-Like Receptors 2 and 4 Related Platelet Responsiveness.

Background Like immune cells, platelets express the repertoire of toll-like receptors (TLR), among them TLR2 and TLR4, which are important for the recognition of bacterial patterns. Receptor-mediated functional effects in platelets have been investigated, but reliable conclusions are tampered due to heterogeneous study designs with variable platelet preparation methods. This study compares TLR2- and TLR4-dependent platelet responsiveness in platelet-rich plasma (PRP) and in washed platelets (WPs). Material and Methods Fresh peripheral blood samples from healthy donors served for the preparation of PRP and WP. Basal and agonist-stimulated TLR2 and TLR4 expression levels were evaluated by flow cytometry. Light transmission aggregometry was used to investigate functional effects of TLR2 and TLR4 stimulation with Pam3CSK4 or LPS (lipopolysaccharides from Escherichia coli ) as ligands. The capacity of chemokine release was determined by immunoassays. Results Pam3CSK4 and LPS (in combination with thrombin) were able to induce aggregation in WP, but not in PRP, with threshold concentrations of 15 µg/mL. Basal expression levels of TLR2 and TLR4 were higher in WP than in PRP, increasing several-fold rapidly and persistently upon platelet activation with potent agonists. Pam3CSK4 (15 µg/mL) or LPS led to the submaximal release of RANTES, PF4, PDGF, NAP-2, and sCD40L from WP. In PRP, secretory effects are less pronounced for RANTES, PDGF, or PF4, and not detectable for NAP-2 or sCD40L. Conclusion The effects mediated by TLR2 and TLR4 stimulation are dependent on platelet preparation, an important issue for experimental designs and for manufacturing of platelet concentrates in transfusion medicine.

SHARPIN at the nexus of integrin, immune, and inflammatory signaling in human platelets

Platelets mediate primary hemostasis, and recent work has emphasized platelet participation in immunity and inflammation. The function of the platelet-specific integrin αIIbβ3 as a fibrinogen receptor in hemostasis is well defined, but the roles of αIIbβ3 or integrin-associated proteins in nonhemostatic platelet functions are poorly understood. Here we show that human platelets express the integrin-associated protein SHARPIN with functional consequences. In leukocytes, SHARPIN interacts with integrin α cytoplasmic tails, and it is also an obligate member of the linear ubiquitin chain assembly complex (LUBAC), which mediates Met1 linear ubiquitination of proteins leading to canonical NF-κB activation. SHARPIN interacted with αIIb in pull-down and coimmunoprecipitation assays. SHARPIN was partially localized, as was αIIbβ3, at platelet edges, and thrombin stimulation induced more central SHARPIN localization. SHARPIN also coimmunoprecipitated from platelets with the two other proteins comprising LUBAC, the E3 ligase HOIP and HOIL-1. Platelet stimulation with thrombin or inflammatory agonists, including lipopolysaccharide or soluble CD40 ligand (sCD40L), induced Met1 linear ubiquitination of the NF-κB pathway protein NEMO and serine-536 phosphorylation of the p65 RelA subunit of NF-κB. In human megakaryocytes and/or platelets derived from induced pluripotent stem (iPS) cells, SHARPIN knockdown caused increased basal and agonist-induced fibrinogen binding to αIIbβ3 as well as reduced Met1 ubiquitination and RelA phosphorylation. Moreover, these SHARPIN knockdown cells exhibited increased surface expression of MHC class I molecules and increased release of sCD40L. These results establish that SHARPIN functions in the human megakaryocyte/platelet lineage through protein interactions at the nexus of integrin and immune/inflammatory signaling.

Soluble CD40L and CD62P levels differ in single-donor apheresis platelet concentrates and buffy coat-derived pooled platelet concentrates.

Platelet storage lesions are structural and biochemical changes in platelet concentrates (PCs), and depend on variables in collection and processing, as well as secondary procedures and storage conditions; such lesions can be mitigated by the use of platelet additive solutions (PASs). This study investigated release of the inflammatory markers sCD40L and sCD62P by single-donor apheresis platelet concentrates (SDA-PCs) and buffy coat-derived pooled platelet concentrates (PPCs) before and after storage. SDA-PC and PPC samples (n = 9089) processed by various methods and stored for different durations were obtained following production in one regional setting, the French National Blood Service. Soluble factors were quantified in PC supernatants immediately after processing and at the time of delivery, using biological testing technology (Luminex). SDA-PCs appeared more activated than PPCs at the end of the production step (i.e., prior to storage); however, proinflammatory soluble factors exhibited greater increases in PPCs than in SDA-PCs during storage. In SDA-PCs, PAS-D (65%) led to reduced secretion of sCD62P, but favored secretion of sCD40L, compared with the alternative PAS-E. These data stress the importance of the production (processing) steps of PC manufacture and of storage. The extent to which they affect patient outcomes awaits further investigation in clinical studies.

Dengue Virus Induces the Release of sCD40L and Changes in Levels of Membranal CD42b and CD40L Molecules in Human Platelets.

Platelets are considered as significant players in innate and adaptive immune responses. The adhesion molecules they express, including P-selectin, CD40L, and CD42b, facilitate interactions with many cellular effectors. Upon interacting with a pathogen, platelets rapidly express and enhance their adhesion molecules, and secrete cytokines and chemokines. A similar phenomenon occurs after exposure of platelets to thrombin, an agonist extensively used for in vitro activation of these cells. It was recently reported that the dengue virus not only interacts with platelets but possibly infects them, which triggers an increased expression of adhesion molecule P-selectin as well as secretion of IL-1β. In the present study, surface molecules of platelets like CD40L, CD42b, CD62P, and MHC class I were evaluated at 4 h of interaction with dengue virus serotype 2 (DENV-2), finding that DENV-2 induced a sharp rise in the membrane expression of all these molecules. At 2 and 4 h of DENV-2 stimulation of platelets, a significantly greater secretion of soluble CD40L (sCD40L) was found (versus basal levels) as well as cytokines such as GM-CSF, IL-6, IL-8, IL-10, and TNF-α. Compared to basal, DENV-2 elicited more than two-fold increase in these cytokines. Compared to the thrombin-induced response, the level generated by DENV-2 was much higher for GM-CSF, IL-6, and TNF-α. All these events induced by DENV end up in conspicuous morphological changes observed in platelets by confocal microscopy and transmission electron microscopy, very different from those elicited by thrombin in a more physiological scenery.

Soluble CD40 Ligand and Oxidative Response Are Reciprocally Stimulated during Shiga Toxin-Associated Hemolytic Uremic Syndrome.

Shiga toxin (Stx), produced by Escherichia coli, is the main pathogenic factor of diarrhea-associated hemolytic uremic syndrome (HUS), which is characterized by the obstruction of renal microvasculature by platelet-fibrin thrombi. It is well known that the oxidative imbalance generated by Stx induces platelet activation, contributing to thrombus formation. Moreover, activated platelets release soluble CD40 ligand (sCD40L), which in turn contributes to oxidative imbalance, triggering the release of reactive oxidative species (ROS) on various cellular types. The aim of this work was to determine if the interaction between the oxidative response and platelet-derived sCD40L, as consequence of Stx-induced endothelium damage, participates in the pathogenic mechanism during HUS. Activated human glomerular endothelial cells (HGEC) by Stx2 induced platelets to adhere to them. Although platelet adhesion did not contribute to endothelial damage, high levels of sCD40L were released to the medium. The release of sCD40L by activated platelets was inhibited by antioxidant treatment. Furthermore, we found increased levels of sCD40L in plasma from HUS patients, which were also able to trigger the respiratory burst in monocytes in a sCD40L-dependent manner. Thus, we concluded that platelet-derived sCD40L and the oxidative response are reciprocally stimulated during Stx2-associated HUS. This process may contribute to the evolution of glomerular occlusion and the microangiopathic lesions.

Sphingosine 1-phosphate (S1P) suppresses the collagen-induced activation of human platelets via S1P4 receptor

Sphingosine 1-phosphate (S1P) is as an extracellular factor that acts as a potent lipid mediator by binding to specific receptors, S1P receptors (S1PRs). However, the precise role of S1P in human platelets that express S1PRs has not yet been fully clarified. We previously reported that heat shock protein 27 (HSP27) is released from human platelets accompanied by its phosphorylation stimulated by collagen. In the present study, we investigated the effect of S1P on the collagen-induced platelet activation. S1P pretreatment markedly attenuated the collagen-induced aggregation. Co-stimulation with S1P and collagen suppressed collagen-induced platelet activation, but the effect was weaker than that of S1P-pretreatment. The collagen-stimulated secretion of platelet-derived growth factor (PDGF)-AB and the soluble CD40 ligand (sCD40L) release were significantly reduced by S1P. In addition, S1P suppressed the collagen-induced release of HSP27 as well as the phosphorylation of HSP27. S1P significantly suppressed the collagen-induced phosphorylation of p38 mitogen-activated protein kinase. S1P increased the levels of GTP-bound Gαi and GTP-bound Gα13 coupled to S1PPR1 and/or S1PR4. CYM50260, a selective S1PR4 agonist, but not SEW2871, a selective S1PR1 agonist, suppressed the collagen-stimulated platelet aggregation, PDGF-AB secretion and sCD40L release. In addition, CYM50260 reduced the release of phosphorylated-HSP27 by collagen as well as the phosphorylation of HSP27. The selective S1PR4 antagonist CYM50358, which failed to affect collagen-induced HSP27 phosphorylation, reversed the S1P-induced attenuation of HSP27 phosphorylation by collagen. These results strongly suggest that S1P inhibits the collagen-induced human platelet activation through S1PR4 but not S1PR1.

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.

Influence of GCSF stimulation on sCD40L release kinetic

It has been demonstrated by in vitro experiments using inhibitors that CD40L is cleaved from the surface of activated platelets by matrix-metalloproteinases (MMP). Additionally, after granulocyte colony stimulating factor (GCSF) activation of peripheral stem cell donors a MMP elevation is induced, and stem cell products contain a considerable amount of platelets. Therefore stem cell donations (peripheral stem cell apheresis (PBSC) as well as bone marrow (BM) donation) were used as an in vivo model to observe alterations of the sCD40L release kinetic and its effects on the respective stem cell products. AND METHODS: The study included twelve healthy stem cell donors (PBSC n =6 , BMn = 6). sCD40L concentrations, relative MMP activities and blood cell compositions were determined in peripheral blood as well as in the respective stem cell products before, during and after donation and/or during cell product storage. RESULTS: In stem cell products, sCD40L concentrations were manifold elevated (range from 1121 to 4123 pg/mL) in comparison to concentrations of peripheral blood samples (range from 39 to 334 pg/mL). Dependent on storage duration a sCD40L accumulation in the products could be observed. MMP concentrations were elevated by GCSF stimulation and resulted in an about 2-fold increase of sCD40L release kinetic. In parallel, MMP concentrations were increased in the stem cell products (PBSC as well as BM products). In the peripheral blood of PBSC donors, the sCD40L concentration decreased after apheresis in correlation to a decrease in platelet count. CONCLUSION: As known from platelet concentrates, an accumulation of sCD40L could also be observed in stem cell products pointing out the importance of sCD40L release by platelets. Stem cell products showed elevated MMP concentrations compared to peripheral blood and additionally during stem cell apheresis, sCD40L release kinetic is accelerated by MMP elevation.

Modeling HIV-1 Induced Neuroinflammation in Mice: Role of Platelets in Mediating Blood-Brain Barrier Dysfunction

The number of HIV-1 positive individuals developing some form of HIV-associated neurocognitive disorder (HAND) is increasing. In these individuals, the integrity of the blood-brain barrier (BBB) is compromised due to an increase in exposure to pro-inflammatory mediators, viral proteins, and virus released from infected cells. It has been shown that soluble CD40L (sCD40L) is released upon platelet activation and is an important mediator of the pathogenesis of HAND but the underlying mechanisms are unclear, emphasizing the need of an effective animal model. Here, we have utilized a novel animal model in which wild-type (WT) mice were infected with EcoHIV; a derivative of HIV-1 that contains a substitution of envelope protein gp120 with that of gp80 derived from murine leukemia virus-1 (MuLV-1). As early as two-weeks post-infection, EcoHIV led to increased permeability of the BBB associated with decreased expression of tight junction protein claudin-5, in CD40L and platelet activation-dependent manner. Treatment with an antiplatelet drug, eptifibatide, in EcoHIV-infected mice normalized BBB function, sCD40L release and platelet activity, thus implicating platelet activation and platelet-derived CD40L in virally induced BBB dysfunction. Our results also validate and underscore the importance of EcoHIV infection mouse model as a tool to explore therapeutic targets for HAND.

Anti-citrullinated protein antibodies contribute to platelet activation in rheumatoid arthritis.

IntroductionAlthough the role of platelets in rheumatoid arthritis (RA) is relatively unexplored, recent studies point towards a contribution of platelets in arthritis. We set out to determine platelet phenotype in RA and studied whether this could be influenced by the presence of anti-citrullinated protein antibodies (ACPA).MethodsPlatelets from healthy controls were incubated in the presence of plasma of patients with RA or age- and sex-matched healthy controls and plasma from ACPAneg or ACPApos patients or in the presence of plate-bound ACPA. Characteristics of platelets isolated from patients with RA were correlated to disease activity.ResultsPlatelets isolated from healthy controls displayed markers of platelet activation in the presence of plasma derived from RA patients, as determined by P-selectin expression, formation of aggregates and secretion of soluble CD40 ligand (sCD40L). Furthermore, levels of P-selectin expression and sCD40L release correlated with high ACPA titres. In accordance with these findings, enhanced platelet activation was observed after incubation with ACPApos plasma versus ACPAneg plasma. Pre-incubation of platelets with blocking antibodies directed against low-affinity immunoglobulin G receptor (FcγRIIa) completely inhibited the ACPA-mediated activation. In addition, expression of P-selectin measured as number of platelets correlated with Disease Activity Score in 44 joints, C-reactive protein level, ACPA status and ACPA level.ConclusionsWe show for the first time that ACPA can mediate an FcγRIIa-dependent activation of platelets. As ACPA can be detected several years before RA disease onset and activated platelets contribute to vascular permeability, these data implicate a possible role for ACPA-mediated activation of platelets in arthritis onset.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0665-7) contains supplementary material, which is available to authorized users.

Mechanisms of Nifedipine-Downregulated CD40L/sCD40L Signaling in Collagen Stimulated Human Platelets.

The platelet-derived soluble CD40L (sCD40L) release plays a critical role in the development of atherosclerosis. Nifedipine, a dihydropyridine-based L-type calcium channel blocker (CCB), has been reported to have an anti-atherosclerotic effect beyond its blood pressure-lowering effect, but the molecular mechanisms remain unclear. The present study was designed to investigate whether nifedipine affects sCD40L release from collagen-stimulated human platelets and to determine the potential role of peroxisome proliferator-activated receptor-β/-γ (PPAR-β/-γ). We found that treatment with nifedipine significantly inhibited the platelet surface CD40L expression and sCD40L release in response to collagen, while the inhibition was markedly reversed by blocking PPAR-β/-γ activity with specific antagonist such as GSK0660 and GW9662. Meanwhile, nifedipine also enhanced nitric oxide (NO) and cyclic GMP formation in a PPAR-β/-γ-dependent manner. When the NO/cyclic GMP pathway was suppressed, nifedipine-mediated inhibition of sCD40L release was abolished significantly. Collagen-induced phosphorylation of p38MAPK, ERK1/2 and HSP27, matrix metalloproteinase-2 (MMP-2) expression/activity and reactive oxygen species (ROS) formation were significantly inhibited by nifedipine, whereas these alterations were all attenuated by co-treatment with PPAR-β/-γ antagonists. Collectively, these results demonstrate that PPAR-β/-γ-dependent pathways contribute to nifedipine-mediated downregulation of CD40L/sCD40L signaling in activated platelets through regulation of NO/ p38MAPK/ERK1/2/HSP27/MMP-2 signalings and provide a novel mechanism regarding the anti-atherosclerotic effect of nifedipine.

<i>In Vitro</i> Effect of Soy Isoflavone and Equol on Soluble CD40L Release Stimulated by Ristocetin in Platelets from Postmenopause Women

The inhibition of specific flavonoid on the in vitro platelet aggregation induced by collagen, arachidonic acid and thromboxane A2 (TxA2) agonist, seems to be related mostly to their ability to compete for binding to the TxA2 receptor (TP). The aim of this study was to analyze the effect of soy isoflavone and equol in terms of inhibiting the platelet aggregation and sCD40L release stimulated by ristocetin, an in vitro-activator of glycoprotein Ib/IX/V, in platelets from postmenopausal women. When platelets were stimulated by 0.75 mg/ml ristocetin, equol (10 μM) exhibited a greater inhibitory activity on platelet aggregation (~68%) than genistein or daidzein. The effect of equol was dependent on the concentration of platelet aggregation agonist. In the presence of ristocetin (0.75 mg/ml, 1.125 mg/ml and 1.5 mg/ml), the inhibitory effect of 10 μM equol was 68% ± 5%, 54% ± 4% and 31% ± 5%, respectively. Equol (10 μM) was a potent inhibitor (~35%) of sCD40L release when stimulated with 1.5 mg/ml ristocetin. However, no significant differences were noted in platelets incubated in the presence of genistein or daidzein and stimulated by ristocetin. On the other hand, SQ29548, a high TP antagonist, also inhibited the sCD40L release stimulated by ristocetin. Finally, 10 μM of genistein, daidzein or equol did not significantly affect the thromboxane B2 production when platelets were incubated with 1.5 mg/ml ristocetin. The relevance of this study was to find that equol exhibits a potent activity by inhibiting ristocetin-induced sCD40L release, suggesting that soy isoflavone has important biological effects on the hemostatic system. However, clinical trials will be necessary to assess the effect of equol on platelet and their impact on inflammatory markers.

Inhibitory effects of Cyperus digitatus extract on human platelet function in vitro

The purpose of this research was to investigate the mechanisms of antiplatelet action of Cyperus digitatus. The antiplatelet action of C. digitatus was studied on platelet function: secretion, adhesion, aggregation, and sCD40L release. The platelet ATP secretion and aggregation were significantly inhibited by CDA (ethyl acetate extract) at 0.1 mg/ml and after the incubation of whole blood with CDA, the platelet coverage was inhibited by 96 ± 3% (p < 0.001). At the same concentration, CDA significantly decreased sCD40L levels. The mechanism of antiplatelet action of CDA could be by NF-κB inhibition and that is cAMP independent. In conclusion, C. digitatus extract may serve as a new source of antiplatelet agents for food and nutraceutical applications.

Pathogen inactivation of double-dose buffy-coat platelet concentrates photochemically treated with amotosalen and UVA light: preservation of in vitro function.

The INTERCEPT Blood System for Platelets (PLT) utilizes amotosalen (S-59) in combination with ultraviolet A (UVA) light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate PLT concentrates. However, limited data are available on the quality of INTERCEPT-treated double-dose (DD) buffy-coat (BC) PLT units allowing a single treatment procedure to produce two pathogen-inactivated PLT units for transfusion. The objective of this study was to evaluate potential in vitro effects of the INTERCEPT treatment on pools of 7 BCs as compared to untreated units. Functional, phenotypic and mitochondrial properties of DD BC PLTs during storage over 7days were studied. For some parameters measured, small yet significant differences were observed including PLT count (P<0·05), pH, pCO2 and glucose concentration. Throughout storage, no significant differences were observed in ATP levels, ESC, HSR reactivity and CD62P expression. Similarly, no differences were observed in the expression of PAC-1, CD42b and PECAM-1 at any time-points. The mitochondrial membrane potential (MMP) determined by JC-1-labelling was well maintained until day 7 in all treated and untreated units (>90%). The release of sCD40L increased over time (P<0·01) in all units but without any significant differences between treated and untreated PLTs. Our data demonstrate that photochemical pathogen inactivation of DD-BC PLT concentrates with the INTERCEPT Blood System had no influence on the PLT in vitro quality over the 7day of storage. However, whether in vivo efficacy of INTERCEPT-treated PLTs is affected may require clinical evaluation.

Rho-kinase regulates human platelet activation induced by thromboxane A2 independently of p38 MAP kinase

We have previously demonstrated that ristocetin, an activator of GPIb/IX/V, induces the release of soluble CD40 ligand (sCD40L) via thromboxane A2 production in human platelets. It has been shown that thromboxane A2 induces the activation of Rho-kinase, a downstream effector of Rho, in human platelets. In the present study, we investigated the exact roles of Rho-kinase in thromboxane A2-induced platelet activation. We found that U46619, a thromboxane receptor (TP) agonist, induced the phosphorylation of cofilin, a target of Rho-kinase signaling, and that the cofilin phosphorylation by U46619 was suppressed by Y27632 or fasudil, specific inhibitors of Rho-kinase. Y27632 and fasudil markedly decreased large platelet aggregate formation by U46619. The release of sCD40L and secretion of platelet-derived growth factor (PDGF)-AB stimulated by U46619 were inhibited by Y27632 and fasudil. SB203580, a specific inhibitor of p38 mitogen-activated protein (MAP) kinase, reduced the sCD40L release and PDGF-AB secretion. Y27632 and fasudil failed to affect the phosphorylation of p38 MAP kinase whereas SB203580 had little effect on the phosphorylation of cofilin induced by U46619. In conclusion, our results strongly suggest that Rho-kinase regulates thromboxane A2-induced human platelet activation independently of p38 MAP kinase.

Streptococcus sanguinis-induced cytokine and matrix metalloproteinase-1 release from platelets.

BackgroundStreptococcus sanguinis (S.sanguinis), a predominant bacterium in the human oral cavity, has been widely associated with the development of infective endocarditis. Platelets play both a haemostatic function and can influence both innate and adaptive immune responses. Previous studies have shown that S.sanguinis can interact with, and activate, platelets.ResultsThe aim of this study was to determine whether S.sanguinis stimulates the release of matrix metalloproteinases (MMPs) 1, 2 and 9 and the pro-inflammatory mediators SDF-1, VEGF and sCD40L, from platelets and to subsequently pharmacologically address the release mechanism (s). S.sanguinis stimulated the release of MMP-1, SDF-1, VEGF and sCD40L from platelets and inhibitors of cyclooxygenase and phosphatidylinositol 3-kinase, and antagonists of the αIIbβ3 integrin and glycoprotein Ib, each inhibited the secretion of all factors.ConclusionsTherefore the release of MMP-1, SDF-1, VEGF and sCD40L occurs late in the platelet response to S.sanguinis and highlights the complex intracellular signalling pathways stimulated in response to S.sanguinis which lead to haemostasis, MMP and pro-inflammatory mediator secretion.

Involvement of Rac in thromboxane A2‑induced human platelet activation: regulation of sCD40 ligand release and PDGF‑AB secretion.

We have previously shown that glycoproteinIb/IX/V activation stimulates the release of the soluble CD40ligand(sCD40L) via the generation of thromboxaneA2 from human platelets. In the present study, the role of Rac, which is a member of the Rho family, was investigated in the thromboxane A2‑stimulated release of platelet‑derived growth factor (PDGF)‑AB and sCD40L in human platelets. U46619, a thromboxane receptor agonist, stimulated the activation of Rac time‑dependently in human platelets, and NSC23766, a selective inhibitor of the Rac‑guanine nucleotide exchange factor interaction, reduced the U46619‑induced platelet aggregation. NSC23766 markedly suppressed the U46619‑induced p38mitogen-activated protein (MAP) kinase phosphorylation. The thromboxane A2‑induced release of PDGF‑AB and sCD40L was significantly suppressed by NSC23766 in a dose‑dependent manner. In addition, NSC23766 reduced the sCD40L release stimulated by ristocetin, a glycoproteinIb/IX/V activator. These results indicate that Rac regulates the thromboxane A2‑induced stimulation of PDGF‑AB secretion and sCD40L release via the p38 MAP kinase in human platelets.