sCD40L Release Research Articles

Protective mechanisms of adenosine 5′-monophosphate in platelet activation and thrombus formation

Platelet activation is relevant to a variety of acute thrombotic events. We sought to examine adenosine 5'-monophosphate (AMP) mechanisms of action in preventing platelet activation, thrombus formation and platelet-related inflammatory response. We assessed the effect of AMP on 1) P-selectin expression and GPIIb/IIIa activation by flow cytometry; 2) Platelet aggregation and ATP secretion induced by ADP, collagen, TRAP-6, convulxin and thrombin; 3) Platelet rolling and firm adhesion, and platelet-leukocyte interactions under flow-controlled conditions; and, 4) Platelet cAMP levels, sP-selectin, sCD40L, IL-1β, TGF-β1 and CCL5 release, PDE3A activity and PKA phosphorylation. The effect of AMP on in vivo thrombus formation was also evaluated in a murine model. The AMP docking with respect to A2 adenosine receptor was determined by homology. AMP concentration-dependently (0.1 to 3 mmol/l) inhibited P-selectin expression and GPIIb/IIIa activation, platelet secretion and aggregation induced by ADP, collagen, TRAP-6 and convulxin, and diminished platelet rolling and firm adhesion. Furthermore, AMP induced a marked increase in the rolling speed of leukocytes retained on the platelet surface. At these concentrations AMP significantly decreased inflammatory mediator from platelet, increased intraplatelet cAMP levels and inhibited PDE3A activity. Interestingly, SQ22536, ZM241385 and SCH58261 attenuated the antiplatelet effect of AMP. Docking experiments revealed that AMP had the same orientation that adenosine inside the A2 adenosine receptor binding pocket. These in vitro antithrombotic properties were further supported in an in vivo model of thrombosis. Considering the successful use of combined antiplatelet therapy, AMP may be further developed as a novel antiplatelet agent.

The effects of pneumatic tube transport on fresh and stored platelets in additive solution.

Limited scientific work has been conducted on potential in vitro effects of transport on pneumatic tube systems on blood components, in particular platelets. To evaluate the possible effects of the Swisslog TranspoNet system on the cellular, metabolic, phenotypic and secreting properties of fresh and stored platelets, we set up a four-arm paired study comparing transported and non-transported platelets. Platelets were aliquoted, prepared with the OrbiSac system and suspended in 70% SSP+ (n=8). All in vitro parameters were monitored over a 7-day storage period. Throughout storage, no differences were observed in glucose consumption, lactate production, pH, pCO2, ATP, hypotonic shock response reactivity, CD62P, PAC-1, platelet endothelial cell adhesion molecule-1 or CD42b. The release of sCD40L increased (p<0.01) in all units but without any significant differences between groups. The storage stability of all platelets conveyed by the Swisslog TranspoNet system was not impaired throughout 7 days of storage. The Swisslog TranspoNet system does not, therefore, seem to be a risk for increased metabolic activity, activation or release reactions from the platelets. This lack of effect of the pneumatic tube transport system did not seem to be affected by the age of the platelets or repeated transport.

Phospholipase C-γ2 via p38 and ERK1/2 MAP kinase mediates diperoxovanadate-asparagine induced human platelet aggregation and sCD40L release

ObjectiveRedox imbalance either inside platelets or in their immediate surroundings prove detrimental to their physiologic functions during haemostasis. This study was therefore aimed to assess the effect of peroxide radicals on platelet functions and underlying signalling mechanisms using asparagine-conjugated diperoxovanadate (DPV-Asn).MethodsPlatelet aggregation, ATP secretion, TxB2 release, intra-platelet calcium mobilization, protein tyrosine phosphorylation, GPIIbIIIa activation by PAC1 labelling and sCD40L release (enzyme-linked immunosorbent assay) was monitored using various concentrations of DPV-Asn. Cell viability was assessed by Annexin V labelling, MTT assay, LDH leakage and mitochondrial membrane potential by JC-1.ResultsPlatelet aggregation induced by DPV-Asn was chiefly regulated by dense granule secretion, thromboxane A2 (TxA2) generation, intra-platelet [Ca2+] influx, GPIIbIIIa activation and sCD40L release, which were significantly reduced in presence of U73122 (PLC inhibitor), aspirin (COX), SB203580 (p38 inhibitor), and PD98059 (ERK inhibitor). This was further corroborated by enhanced tyrosine phosphorylation of numerous platelet proteins including PLC-γ2, which apparently played a central role in transducing peroxide signals to regulate [Ca2+] influx and phosphorylation of p38 and ERK1/2 MAP kinase.DiscussionPeroxide radicals critically regulate the thrombo-inflammatory functions of platelets via the PLCγ2-p38-ERK1/2-TxA2 pathway, which closely resembles the clinical scenario of various pathologies like hyperglycemia and atherosclerosis during which oxidative stress disrupts platelet functions.

Rac regulates collagen-induced HSP27 phosphorylation via p44/p42 MAP kinase in human platelets

We previously reported that the collagen-induced phosphorylation of heat shock protein (HSP) 27 via p44/p42 mitogen-activated protein (MAP) kinase is sufficient to induce the secretion of platelet-derived growth factor (PDGF)-AB and the release of soluble CD40 ligand (sCD40L) from human platelets. It has been shown that Rac, which belongs to the Rho family of small GTPases, is involved in the collagen-induced platelet aggregation. In this study, we investigated the role of Rac in the collagen-stimulated release of PDGF-AB and sCD40L in human platelets. Human blood was donated from healthy volunteers and platelet-rich plasma was obtained from the blood samples. The samples were then treated with 1.0 µg/ml collagen for 0, 1, 3, or 5 min and Rac1 activity was determined using the Rac1 Activation Assay kit. We found that collagen stimulated the activation of Rac in human platelets in a time-dependent manner. However, pre-treatment with NSC23766, a selective inhibitor of Rac-guanine nucleotide exchange factor interaction, reduced the collagen-induced platelet aggregation. NSC23766 markedly attenuated not only the collagen-induced p44/p42 MAP kinase phosphorylation, but also the phosphorylation of HSP27 at three serine residues (Ser-15, Ser-78 and Ser-82). In addition, the collagen‑induced release of PDGF-AB and sCD40L was significantly suppressed by NSC23766 in a dose-dependent manner. These results strongly suggest that Rac regulates the collagen-induced HSP27 phosphorylation via p44/p42 MAP kinase in human platelets, resulting in the stimulation of PDGF-AB secretion and the release of sCD40L.

Intracoronary abciximab and local anti-inflammatory effects

To the Editor:We have read with great interest the article by De Rosa et al., in re-lation to a meta-analysis of clinical trials on the use of intracoronaryabciximab [1]. In our opinion, the author's state in the discussiononly one possible potential mechanism by which the intracoronaryabciximab obtains better results compared to intravenously, a higherdegree of GP IIb/IIIa receptor occupancy and a more pronounced plate-let inhibition. We would like to point out another possible mechanismthat can account for the potential clinical benefits observed in pa-tients receiving intracoronary abciximab, the local anti-inflammatoryproperties.Platelets are a central cellular component in both thrombotic andinflammatory processes characteristic of coronary atherosclerosis.Platelets modulate inflammatory processes by binding to and alteringleukocyte function [2]. In addition to forming circulating aggregateswith leukocytes, platelets regulate inflammation by secreting a va-riety of inflammatory modulators, including CD40 ligand (CD40L).CD40L is an essential component in the pathophysiology of athero-sclerosis and platelets are the predominant source of circulating solu-ble CD40L (sCD40L) [3].Glycoprotein IIb/IIIa antagonists (abciximab, eptifibatide, tirofiban)have been reported to inhibit the release of sCD40L and plateletaggregation initially in vitro and, afterward, in clinical studies [4].Morespecifically, abciximab reduced circulating sCD40L and leukocyte–platelet aggregation in patients with acute coronary syndrome under-going percutaneous coronary intervention [5]. Moreover, in patientswith ST-elevation myocardial infarction undergoing primary percuta-neous coronary intervention, results from our group have shown thatintracoronary bolus administration of abciximab was associated withalargerdecreaseinsCD40Llevelscomparedwithstandardintravenousbolus [6]. Therefore, we believe that the rationale of intracoronaryabciximab use is based not only on its dose-dependent antiplatelet,antithromboticeffectbutalsoonitslocalanti-inflammatoryproperties.AcknowledgmentsThe authors of this manuscript have certified that they complywith the Principles of Ethical Publishing in the International Journalof Cardiology.References

Thrombopoietin amplifies ADP-induced HSP27 phosphorylation in human platelets: Importance of pre-treatment

It has been shown that thrombopoietin (TPO) amplifies agonist-induced platelet activation. However, the precise mechanism of action of TPO has not yet been fully elucidated. We have previously reported that the adenosine diphosphate (ADP)‑induced phosphorylation of heat shock protein 27 (HSP27) via the p38 mitogen-activated protein (MAP) kinase pathway correlates with the ADP-induced platelet-derived growth factor (PDGF)-AB secretion and the release of soluble CD40 ligand (sCD40L) from human platelets. In the present study, we investigated the effects of TPO on platelet activation induced by ADP. We examined the effects of TPO on ADP-induced platelet activation under different treatments: TPO was administered 15 min prior to stimulation with ADP (pre-treatment); TPO and ADP were simultaneously administered (simultaneous treatment); and TPO was administered 2 min following stimulation with ADP (post-treatment). TPO, which alone had no effect on platelet aggregation, synergistically enhanced the ADP (1 mM)-induced platelet aggregation only when it was administered prior to stimulation with ADP. Pre-treatment with TPO significantly increased the secretion of PDGF-AB and the release of sCD40L, and markedly enhanced the ADP-induced phosphorylation of p38 MAP kinase and HSP27 in the platelets. However, simultaneous treatment with TPO or TPO post-treatment failed to affect the ADP-induced platelet aggregation, the secretion of PDGF-AB, the release of sCD40L and the phosphorylation p38 MAP kinase or HSP27. These results strongly suggest that pre-treatment with TPO significantly amplifies ADP-induced HSP27 phosphorylation via the p38 MAP kinase pathway in human platelets.

Valproic Acid Inhibits the Release of Soluble CD40L Induced by Non-Nucleoside Reverse Transcriptase Inhibitors in Human Immunodeficiency Virus Infected Individuals

Despite the use of highly active antiretroviral therapies (HAART), a majority of Human Immunodeficiency Virus Type 1 (HIV) infected individuals continually develop HIV – Associated Neurocognitive Disorders (HAND), indicating that host inflammatory mediators, in addition to viral proteins, may be contributing to these disorders. Consistent with this notion, we have previously shown that levels of the inflammatory mediator soluble CD40 ligand (sCD40L) are elevated in the plasma and cerebrospinal fluid (CSF) of HIV infected, cognitively impaired individuals, and that excess sCD40L can contribute to blood brain barrier (BBB) permeability in vivo, thereby signifying the importance of this inflammatory mediator in the pathogenesis of HAND. Here we demonstrate that the non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV) induces the release of circulating sCD40L in both HIV infected individuals and in an in vitro suspension of washed human platelets, which are the main source of circulating sCD40L. Additionally, EFV was found to activate glycogen synthase kinase 3 beta (GSK3β) in platelets, and we now show that valproic acid (VPA), a known GSK3β inhibitor, was able to attenuate the release of sCD40L in HIV infected individuals receiving EFV, and in isolated human platelets. Collectively these results have important implications in determining the pro-inflammatory role that some antiretroviral regimens may have. The use of antiretrovirals remains the best strategy to prevent HIV-associated illnesses, including HAND, however these drugs have clear limitations to this end, and thus, these results underscore the need to develop adjunctive therapies for HAND that can also minimize the undesired negative effects of the antiretrovirals.

Soluble CD40 ligand release in patients with stable coronary artery disease during elective stent implantation: effect of drug-eluting stent over bare metal stent

We aimed to determine the effect of drug-eluting stent (DES) implantation on soluble CD40 ligand (sCD40L) levels in patients with stable coronary artery disease undergoing stent replacement. Eighty-nine consecutive patients (33 women, 56 men; mean age 61±10 years) with stable coronary artery disease undergoing stent replacement were recruited. Pre- and post-procedural blood samples were collected for sCD40L analysis, and differences in plasma levels were calculated and expressed as delta sCD40L. Total size and length of implanted stents and pre- and post-dilatation procedures were recorded for each patient, for possible impact on sCD40L release. Patients were followed for one year following procedures for possible adverse cardiac events such as death, myocardial infarction and revascularization. Forty-nine patients received bare metal stent (BMS) and 40 patients received DES. There were no differences between BMS- and DES-implanted patients in terms of age, stent size and length, and delta sCD40L plasma levels. Delta sCD40L was correlated only with total implanted stent length (r=0.374, p<0.001). Delta sCD40L levels were divided into quartiles for better determination of the procedural parameters that are effective on biomarker release. Total stent length (p=0.008), stent size (p=0.038) and pre-dilatation procedure (p=0.034) were the statistically differing parameters between delta sCD40L quartiles. Although statistically non-significant, all three adverse events were observed in patients with the highest quartile (p=0.179). Procedural sCD40L release did not differ between DES- and BMS-implanted stable coronary artery disease patients. Total implanted stent length, stent size and pre-dilatation procedure were the influential parameters on procedural sCD40L release.

Is Delayed Pressure Urticaria Associated with Increased Systemic Release of sCD40L?

Background. Elevated levels of soluble CD40 Ligand (sCD40L) were found in serum but not in plasma of patients with chronic spontaneous urticaria (CU). What is important is that sCD40L has proinflammatory properties, and its elevated plasma level may indicate increased risk of cardiovascular events. These observations should stimulate further evaluation of sCD40L in different forms of urticaria. Aim. In the present study, sCD40L plasma level was investigated in delayed pressure urticaria (DPU). Methods. As platelets are predominant and variable sources of sCD40L, we investigated sCD40L concentration in platelet-poor plasma (PPP), which seems the best way to minimize the potential contribution of these cells to the ligand level. Results. Plasma sCD40L concentration was significantly increased in the DPU group compared to the healthy controls. Conclusions. It seems that DPU is associated with increased systemic release of sCD40L, which is believed to derive predominantly from activated platelets. The present study as well as the earlier contributions suggest that distinct cells activity, including platelets, may be identified in different types of urticaria.

Significant correlation between the acceleration of platelet aggregation and phosphorylation of HSP27 at Ser-78 in diabetic patients

To clarify the mechanism underlying a high risk of thrombotic complications in diabetic patients, we investigated the relationship between HSP27 phosphorylation and the platelet activation induced by adenosine diphosphate (ADP) in diabetic patients. Platelet-rich plasma was prepared from the blood of type 2 diabetes mellitus (DM) patients. By measuring the dose response of platelet aggregation to ADP, an individual ED50 was determined. Based on the normal range identified in non-DM controls, the subjects were divided into a hyper-aggregate (Group 1) and a normo- or hypo-aggregate group (Group 2). The protein phosphorylation was analyzed by western blotting. The release of PDGF-AB and sCD40 ligand (sCD40L) was measured by ELISA. In both groups, ADP induced HSP27 phosphorylation at Ser-78 and Ser-82. The phosphorylation at Ser-78 and the release of both PDGF-AB and sCD40L induced by a low dose of ADP (1 µM) in Group 1 were significantly higher than these values in Group 2. There was a significant relationship between the ADP-induced HSP27 phosphorylation level at Ser-78 and the ADP ED50 value of platelet aggregation. The ADP (1 µM)-induced phosphorylation of HSP at Ser-78 observed in the platelets from Group 1 was inhibited by PD98059 or SB203580. The use of aspirin ameliorated the accelerated microaggregation of platelets in Group 1, and the low-dose ADP-induced phosphorylation of HSP27 at Ser-78 was no longer observed. These results strongly suggest that the phosphorylation of HSP27 at Ser-78 is correlated with the acceleration of platelet aggregation induced by ADP in type 2 DM patients.

β1‐Adrenoceptor antibody‐induced increase in soluble CD40 ligand release in chronic periodontitis patients: role of prostaglandin E2

In this paper, we demonstrate that circulating antibodies from chronic periodontitis patients reacting with atrial β(1)-adrenoceptors (β(1)-ARs) act as an inducer of soluble CD40 ligand (sCD40L) release and prostaglandin E(2) (PGE(2)) generation. By enzyme-linked immunosorbent assay using β(1) synthetic peptide (with an amino acid sequence identical to the second loop of human myocardial β(1)-ARs) as a coating antigen, we demonstrated reactivity against the second extracellular loop on human myocardial β(1)-ARs. This autoantibody present in the serum of chronic periodontitis patients was significantly correlated with the release of sCD40L and PGE(2). The release of sCD40L was blunted by atenolol, SP600125 and β(1) synthetic peptide, and PGE(2) generation was inhibited by DuP 697 and slightly by FR122049. The effects of the antibody incubated with isolated rat atria upregulated sCD40L release with an increase of PGE(2) production and c-Jun N-terminal kinase phosphorylation. These results indicate that in chronic periodontitis patients, there is a positive association between sCD40L release and PGE(2) generation via the action of β(1)-AR antibodies.

Platelet soluble CD40L and matrix metalloproteinase 9 activity are proinflammatory mediators in Behçet disease patients

Platelets are the major source of plasma-soluble CD40L (sCD40L), an important inflammatory mediator. This study explored the impact of platelet-derived sCD40L on Behçet disease (BD), an autoinflammatory vasculitis. We also searched for influences by platelet matrix metalloproteinases (MMP) -2 and MMP-9, implicated in several inflammatory diseases, on CD40L shedding from platelet membrane. Platelet activation were studied by flow cytometry and aggregometry, surface expression of CD40L and platelet-leukocyte aggregates by flow cytometry, sCD40L by ELISA, cellular CD40L and CD40 levels by Western blot and MMPs activity by gelatin zymography. The effect of sCD40L on MMP9 expression was studied in cultured MEG-01 cells. Plasma and platelet-released sCD40L levels were higher in BD patients. No differences in platelet activation and in platelet-leukocyte aggregates formation were observed between BD patients and controls. Plasma and platelet MMP-9 levels were increased in BD patients, whereas there was no difference in platelet MMP-2 activity. Since a correlation between plasma sCD40L and platelet MMP-9 activity was observed, we studied the influence of sCD40L on MMP-9 levels in the megakaryoblastic cell line MEG-01. Treatment of MEG-01 cells with recombinant sCD40L increased MMP-9 but did not change MMP-2 levels. In conclusion, sCD40L release from platelets was mediated by MMP-9, and MMP-9 expression was in turn upregulated by sCD40L in the MEG-01 cell line. We conclude that platelets and megakaryocytes might participate in a positive feedback loop occurring between sCD40L and MMP-9 which would contribute to the proinflammatory state observed in BD.

Platelet transfusion alters CD40L blood level and release capacity in patients suffering from thrombocytopenia

Platelet (PLT)-derived cytokines, such as soluble CD40 ligand (sCD40L), play an important role in the development of adverse transfusion reactions associated with the administration of PLT products. In this study, we determined sCD40L concentration and release capacity in patients with thrombocytopenia before and after receiving a PLT transfusion. The study included 12 patients suffering from chemotherapy-induced thrombocytopenia. sCD40L levels and release capacity were measured in plasma samples of the patients before and after PLT administration as well as in the respective plateletpheresis concentrates by enzyme-linked immunosorbent assay. Sixteen healthy blood donors served as a control group. In PLT concentrates, elevated sCD40L levels (2567±134 pg/mL) were observed in comparison to plasma sCD40L levels in controls (238.4±35.3 pg/mL). sCD40L plasma concentration of patients with thrombocytopenia was significantly reduced (86.3±16.7 pg/mL) before transfusion and increased to nearly normal levels (204.4±24.8 pg/mL) after PLT administration. In parallel, the sCD40L release capacity per PLT showed no significant difference between controls and patients with thrombocytopenia before transfusion (33.3±2.6 and 29.3±4.6 ag/PLT, respectively) but was significantly reduced after PLT transfusion (22.4±2.7 compared to 29.3±4.6 ag/PLT). In patients with thrombocytopenia, sCD40L levels were clearly influenced by PLT transfusions: PLT administration led to a normalization of sCD40L plasma concentration. Nevertheless, adverse transfusion reactions did not occur in these patients. The sCD40L release capacity was enhanced by PLT administration dependent on the increase in the amount of PLT count.

Streptococcus sanguinis-induced cytokine release from platelets.

There is increasing evidence that both chronic and acute infections play a role in the development and progression of atherothrombotic disorders. One potential mechanism is the direct activation of platelets by bacteria. A wide range of bacterial species activate platelets through heterogeneous mechanisms. The oral micro-organism S. sanguinis stimulates platelet aggregation in vitro in a strain-dependent manner, although there are no reports of associated cytokine production. The aim of the present study was to determine whether platelet activation by S. sanguinis involved the release of pro-inflammatory and immune modulating factors, and whether activation was enhanced by epinephrine. Four strains of S. sanguinis and one of S. gordonii stimulated the release of RANTES, PF4, sCD40L and PDGF-AB, whereas only one S. sanguinis strain caused the release of sCD62p. Epinephrine enhanced S. sanguinis-induced platelet aggregation and phosphorylation of phospholipase Cγ2 and Erk, but inhibited RANTES, PF4, sCD40L and PDGF-AB release. Wortmannin inhibited S. sanguinis-induced aggregation and release; however, only aggregation was partially reversed by epinephrine. The present study demonstrates that platelets respond to S. sanguinis with both prothrombotic and pro-inflammatory/immune-modulating responses. Epinephrine, potentially released in response to infection and/or stress, can significantly enhance the prothrombotic response, thereby providing a putative link between bacteraemia and acute coronary events during stress. In contrast, epinephrine inhibited the pro-inflammatory/immune-modulating response by an undetermined mechanism.

Determination of thromboxane formation, soluble CD40L release and thrombopoietin clearance in apheresis platelet concentrates

All deleterious changes in platelet morphology, structure and function that occur in platelet concentrates (PC) during storage are titled as the ‘platelet storage lesion’. No single in vitro test currently available is sufficient in assessing these changes of platelet quality. The release of soluble CD40 Ligand (sCD40L), the formation of thromboxane (TXB2) and the thrombopoietin (TPO) clearance reflect different aspects of platelet metabolism and activitiy, and were used to examine platelet quality in apheresis platelet products. At days 1, 3 and 5, in single-donor apheresis platelet products (n = 10) under routine storage conditions, sCD40L (measured by ELISA) and TXB2 (measured by RIA) were determined after platelet stimulation (recalcification and clot formation). TPO (measured by ELISA) was determined after an incubation time of 5 h at 37°C with platelet-rich plasma (adjusted initial TPO concentration of about 500 pg/mL). Results were related to a therapeutic unit (TU = 2 × 1011 platelets). Immediately after platelet preparation, sCD40L release was 41 ± 7.6 ng/TU, TXB2 formation 1688 ± 374 ng/TU and TPO clearance 1.22 ± 0.32 ng/h/TU. At days 1, 3 and 5, sCD40L was reduced to 89 ± 7%, 71 ± 12% and 57 ± 9%, TXB2 release to 91 ± 6%, 74 ± 12% and 58 ± 9% and TPO clearance to 90 ± 15%, 84 ± 5% and 79 ± 10% of the respective control values. In conclusion, in single-donor apheresis PC, sCD40L release and TXB2 formation as well as TPO clearance by the platelets were dependent on storage duration and reduced to about 60% to 80% of the respective control values after a storage period for 5 days. These findings are in line with literature data, indicating that a loss of platelet functionality of about 30% will occur after 5 days of storage.

Inhibitory effects of lipoteichoic acid from Staphylococcus aureus on platelet function and platelet–monocyte aggregation

Lipoteichoic acid (LTA) from Staphylococcus aureus has been demonstrated to inhibit agonist-stimulated platelet aggregation. However, its effects on platelet inflammatory mediator release and platelet-monocyte aggregation are still unclear. In the present study, LTA is examined for its anti-inflammatory properties and effects on platelet-monocyte aggregation. Blood samples were obtained from 5 healthy volunteers who had taken no medicine in the previous 2weeks. Washed platelets were prepared and incubated with LTA (0.5-2.0μg/mL), then platelet aggregation, P-selectin expression, and soluble CD40L (sCD40L) release were measured by light transmission aggregometry, flow cytometry and enzyme-linked immunoassays, respectively. Platelet-monocyte aggregate formation in whole blood was measured by flow cytometry. Thrombin was used as a stimulant. LTA dose-dependently decreased platelet aggregation from 89.32±10.24% to 36.28±9.01% (P<0.05), sCD40L release from 3.28±0.76 to 1.13±0.45ng/mL (P<0.05) and surface P-selectin expression from 82.01±11.20 to 22.78±6.42% (P<0.05). In human whole blood, 1.0μg/mL LTA inhibited platelet-monocyte aggregation from 78.19±10.94 to 38.24+8.74% (P<0.05). These results indicate that LTA from S. aureus can inhibit platelet-dependent inflammatory mediator release and platelet-monocyte aggregation. These findings suggest that LTA-mediated functional alteration of platelets may contribute to immune evasion of S. aureus.

CAMP regulates ADP-induced HSP27 phosphorylation in human platelets

Elevation of cAMP in platelets is recognized to play a suppressive role in platelet functions. We have previously shown that adenosine diphosphate (ADP)-induced phosphorylation of heat shock protein 27 (HSP27) via p38 mitogen-activated protein (MAP) kinase is correlated with platelet-derived growth factor (PDGF)-AB secretion and soluble CD40 ligand (sCD40L) release. In the present study, we investigated the relationship between cAMP and HSP27 phosphorylation in platelet function. 8-Bromoadenosine-3',5'-cyclic monophosphate (8-bromo-cAMP), a plasma membrane-permeable cAMP analogue, or cilostazol, an inhibitor of cAMP phosphodiesterase, markedly attenuated the ADP-induced phosphorylation levels of p38 MAP kinase. In addition, the ADP-induced HSP27 phosphorylation was suppressed by 8-bromo-cAMP or cilostazol. 8-Bromo-cAMP, forskolin and cilostazol remarkably reduced the ADP-stimulated PDGF-AB secretion and sCD40L release. These results strongly suggest that cAMP regulates ADP-stimulated platelet activation due to inhibition of HSP27 phosphorylation via p38 MAP kinase.

Comparison of soluble CD40L concentrations and release capacities in apheresis and prestorage pooled platelet concentrates

Soluble CD40L (sCD40L) is expressed by platelets and is involved in the stabilization of arterial thrombi. Additionally, it was shown that sCD40L accumulation occurred in stored blood products triggering adverse transfusion reactions like TRALI. To study the influence of the preparation technique on sCD40L accumulation and platelet function we examined CD40L concentrations in prestorage pooled platelet concentrates compared to apheresis products. In addition, sCD40L release capacity was determined as a marker for platelet viability. sCD40L concentrations were determined in prestorage pooled platelet concentrates (n = 8) and in platelet apheresis concentrates (n = 8) before and after platelet stimulation (recalcification and clot formation) at day 1, 3 and 5 under routine storage conditions. sCD40L concentrations were determined by a commercially available ELISA kit. sCD40L concentrations in storage medium increased over time in prestorage pooled platelet concentrates (from 1,185 pg/mL ± 87 pg/mL at day 1 to 4,464 pg/mL ± 212 pg/mL at day 5) as well as in apheresis products (from 581 pg/mL ± 124 pg/mL at day 1 to 2,718 pg/mL ± 154 pg/mL at day 5) in a hyperbolic manner. Recalcification and clot formation caused an increase in sCD40L concentrations (for example 3,842 pg/mL ± 769 pg/mL before platelet activation to 31,219 pg/mL ± 2,063 pg/mL after platelet activation at day 3), and we observed comparable release capacities for both preparation techniques, however, decreasing over storage time up to 50% (day 5) of the respective control value (day 1). Amounts of sCD40L accumulation and release capacity during storage of platelet concentrates were dependent on storage duration, but showed no relevant differences regarding the preparation technique. After 5 days of storage, CD40L basal levels were increased, in contrast sCD40L release capacity was decreased. By recalcification and clot formation sCD40L release capacity could be easily induced and is assumed to be used as a marker for platelet viability.

Antiplatelet Activity of Valproic Acid Contributes to Decreased Soluble CD40 Ligand Production in HIV Type 1-Infected Individuals

CD40L is a type II membrane glycoprotein of the TNF family that is found on activated T cells, B cells, and platelets. We previously reported that the soluble form of this inflammatory mediator (sCD40L) is elevated in the plasma and cerebrospinal fluid of HIV-1-infected, cognitively impaired individuals. In this study, we demonstrate that the mood-stabilizing drug valproic acid (VPA) reduces sCD40L levels in plasma samples of HIV-1-infected patients (n = 23) and in washed human platelets, which are the main source of circulating sCD40L. VPA also inhibited HIV-1 transactivator of transcription-induced release of sCD40L and platelet factor 4 in C57BL/6 mice. The mechanism by which VPA was able to do so was investigated, and we demonstrate that VPA, a known glycogen synthase kinase 3β inhibitor, blocks platelet activating factor-induced activation of glycogen synthase kinase 3β in platelets in a manner that alters sCD40L release from platelets. These data reveal that VPA has antiplatelet activity, and they convey important implications for the potential of VPA as an adjunct therapy not only for cognitively impaired patients with HIV-1 infection, but also numerous inflammatory diseases for which such antiplatelet therapies are currently lacking.

Internalization of IgG-Coated Targets Results in Activation and Secretion of Soluble CD40 Ligand and RANTES by Human Platelets

Platelets are crucial elements for maintenance of hemostasis. Other functions attributable to platelets are now being appreciated, such as their role in inflammatory reactions and host defense. Platelets have been reported to bind immunological stimuli like IgG complexes, and for nearly 50 years it has been speculated that platelets may participate in immunological reactions. Platelets have been reported to bind and internalize various substances, similar to other leukocytes, such as macrophages and dendritic cells. In the present study, we tested the hypothesis that human platelets can bind and internalize IgG-coated particles, similar to leukocytes. To this end, we observed that interaction with IgG-coated beads resulted in platelet activation (as measured by CD62P expression), internalization of targets, and significant soluble CD40 ligand (sCD40L) and RANTES (regulated upon activation, normal T cell expresses and secreted) secretion. Blocking FcγRIIA with monoclonal antibody (MAb) IV.3 or inhibiting actin remodeling with cytochalasin D inhibited platelet activation, internalization, and cytokine production. These data suggest that platelets are capable of mediating internalization of IgG-coated particles, resulting in platelet activation and release of both sCD40L and RANTES.