OBJECTIVE

To investigate the effects of a 1,000-mg intravenous pulse of methylprednisolone succinate (MP) on cell adhesion molecule expression on the synovial vascular endothelium in patients with rheumatoid arthritis (RA).

METHODS

Sequential arthroscopic biopsy samples were taken before and 24 hours after MP administration (10 patients) and at the time of RA flare (2 patients) and after retreatment with MP (1 patient). Immunoperoxidase staining for E-selectin (CD62E), P-selectin (CD62P), intercellular adhesion molecule 1 (ICAM-1; CD54) and platelet-endothelial cell adhesion molecule (PECAM; CD31) was performed, and the staining was quantified by color video image analysis.

RESULTS

MP caused a rapid (within 24 hours) and substantial decrease in the expression of E-selectin on the synovial vascular endothelium, with a smaller reduction in ICAM-1 expression on synovial vascular endothelium and the synovial lining. There were no similar effects on synovial membrane P-selectin or PECAM expression.

CONCLUSIONS

A potential mechanism by which MP impairs neutrophil trafficking into inflamed RA joints might be by reducing E-selectin, and possibly, ICAM-1, expression in the synovial membrane.

The identification of stromal cell-derived factor (SDF)-1alpha as a chemoattractant for human progenitor cells suggests that this chemokine and its receptor might represent critical determinants for the homing, retention, and exit of precursor cells from hematopoietic organs. In this study, we investigated the expression profile of CXCR4 receptor and the biological activity of SDF-1alpha during megakaryocytopoiesis. CD34(+) cells from bone marrow and cord blood were purified and induced to differentiate toward the megakaryocyte lineage by a combination of stem-cell factor (SCF) and recombinant human pegylated megakaryocyte growth and development factor (PEG-rhuMGDF). After 6 days of culture, a time where mature and immature megakaryocytes were present, CD41(+) cells were immunopurified and CXCR4mRNA expression was studied. High transcript levels were detected by a RNase protection assay in cultured megakaryocytes derived from cord blood CD34(+) cells as well as in peripheral blood platelets. The transcript levels were about equivalent to that found in activated T cells. By flow cytometry, a large fraction (ranging from 30% to 100%) of CD41(+) cells showed high levels of CXCR4 antigen on their surface, its expression increasing in parallel with the CD41 antigen during megakaryocytic differentiation. CXCR4 protein was also detected on peripheral blood platelets. SDF-1alpha acts on megakaryocytes by inducing intracellular calcium mobilization and actin polymerization. In addition, in in vitro transmigration experiments, a significant proportion of megakaryocytes was observed to respond to this chemokine. This cell migration was inhibited by pertussis toxin, indicating coupling of this signal to heterotrimeric guanine nucleotide binding proteins. Although a close correlation between CD41a and CXCR4 expession was observed, cell surface markers as well as morphological criteria indicate a preferential attraction of immature megakaryocytes (low level of CD41a and CD42a), suggesting that SDF-1alpha is a potent attractant for immature megakaryocytic cells but is less active on fully mature megakaryocytes. This hypothesis was further supported by the observation that SDF-1alpha induced the migration of colony forming unit-megakaryocyte progenitors (CFU-MK) and the expression of activation-dependent P-selectin (CD62P) surface antigen on early megakaryocytes, although no effect was observed on mature megakaryocytes and platelets. These results indicate that CXCR4 is expressed by human megakaryocytes and platelets. Furthermore, based on the lower responses of mature megakaryocytes and platelets to SDF-1alpha as compared with early precursors, these data suggest a role for this chemokine in the maintenance and homing during early stages of megakaryocyte development. Moreover, because megakaryocytes are also reported to express CD4, it becomes important to reevaluate the role of direct infection of these cells by the human immunodeficiency virus (HIV)-1 in HIV-1-related thrombocytopenia.

BACKGROUND

Platelet P-selectin is a thrombo-inflammatory molecule involved in platelet activation and aggregation. This may occur via the adhesive function of P-selectin and its potential capacity to trigger intracellular signaling. However, its impact on platelet function remains elusive. This study was therefore designed to investigate the relationship between the signaling potential of platelet P-selectin and its function in platelet physiology.

RESULTS

Human and mouse platelets were freshly isolated from whole blood. Platelet activation was assessed using flow cytometry and western blot analysis, while platelet physiological responses were evaluated through aggregation, microaggregate formation and in a thrombosis model in wild-type and P-selectin-deficient (CD62P(-/-)) mice. Interaction of P-selectin with its high-affinity ligand, a recombinant soluble form of P-Selectin Glycoprotein Ligand-1 (rPSGL-1), enhances platelet activation, adhesion and microaggregate formation. This augmented platelet microaggregates requires an intact cytoskeleton, but occurs independently of platelet α(IIb)β(3). Thrombus formation and microaggregate were both enhanced by rPSGL-1 in wild-type, but not in CD62P(-/-) mice. In addition, CD62P(-/-) mice exhibited thrombosis abnormalities without an α(IIb)β(3) activation defect.

CONCLUSIONS

This study demonstrates that the role of platelet P-selectin is not solely adhesive; its binding to PSGL-1 induces platelet activation that enhances platelet aggregation and thrombus formation. Therefore, targeting platelet P-selectin or its ligand PSGL-1 could provide a potential therapeutic approach in the management of thrombotic disorders.

Recognition of microbial patterns by host receptors is the first step in a multistep sequence leading to neutrophil-dependent host resistance. Although the role of membrane-bound sensors in bacterial recognition has been examined in detail, the importance of cytosolic sensors in the lungs is largely unexplored. In this context, there is a major lack of understanding related to the downstream signaling mediators, such as cells and/or molecules, during acute extracellular Gram-negative bacterial pneumonia. In order to determine the role of NOD-like receptors (NLRs), we used an experimental Escherichia coli infection model using mice deficient in the gene coding for the NLR adaptor, receptor-interacting protein 2 (RIP2). RIP2(-/-) mice with E. coli infection displayed higher bacterial burden and reduced neutrophil recruitment and tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), macrophage inflammatory protein 2 (MIP-2), and CXCL5/LIX expression, along with attenuated histopathological changes in the lungs. Decreased IL-17A levels were observed, along with lower numbers of IL-17A-producing T cells, in RIP2(-/-) mice after infection. RIP2(-/-) mice also show reduced IL-6 and IL-23 levels in the lungs, along with decreased activation of STAT3 after infection. Furthermore, activation of NF-κB and mitogen-activated protein kinases (MAPKs) and expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in the lungs of infected RIP2(-/-) mice were attenuated following infection. Although neutrophil mobilization to the blood was impaired in RIP2(-/-) mice following infection, the expression of CD62P, CD11a/18, CD11b, and CXCR2 on blood and lung neutrophils was not altered between infected wild-type (WT) and RIP2(-/-) mice. Thus, RIP2 contributes to neutrophil-dependent host defense against an extracellular Gram-negative pathogen via (i) IL-17A regulation and (ii) neutrophil mobilization to the blood.

Binding of fluorescein-labeled coagulation factors IXa, VIII, X, and allophycocyanin-labeled annexin V to thrombin-activated platelets was studied using flow cytometry. Upon activation, two platelet subpopulations were detected, which differed by 1-2 orders of magnitude in the binding of the coagulation factors and by 2-3 orders of magnitude in the binding of annexin V. The percentage of the high-binding platelets increased dose dependently of thrombin concentration. At 100 nm of thrombin, platelets with elevated binding capability constituted approximately 4% of total platelets and were responsible for the binding of approximately 50% of the total bound factor. Binding of factors to the high-binding subpopulation was calcium-dependent and specific as evidenced by experiments in the presence of excess unlabeled factor. The percentage of the high-binding platelets was not affected by echistatin, a potent aggregation inhibitor, confirming that the high-binding platelets were not platelet aggregates. Despite the difference in the coagulation factors binding, the subpopulations were indistinguishable by the expression of general platelet marker CD42b and activation markers PAC1 (an epitope of glycoprotein IIb/IIIa) and CD62P (P-selectin). Dual-labeling binding studies involving coagulation factors (IXa, VIII, or X) and annexin V demonstrated that the high-binding platelet subpopulation was identical for all coagulation factors and for annexin V. The high-binding subpopulation had lower mean forward and side scatters compared with the low-binding subpopulation ( approximately 80% and approximately 60%, respectively). In its turn, the high-binding subpopulation was not homogeneous and included two subpopulations with different scatter values. We conclude that activation by thrombin induces the formation of two distinct subpopulations of platelets different in their binding of the components of the intrinsic fX-activating complex, which may have certain physiological or pathological significance.

Platelet activation is involved in the pathogenesis of cerebrovascular ischemia, but the major agonist involved has yet to be identified. To investigate the role of thrombin in platelet activation in patients with acute ischemic stroke, and while thrombin is the most likely candidate for activation of the thrombin receptor PAR-1 in vivo, we assessed its cleavage and internalization using the antibodies SPAN12, binding to uncleaved PAR-1, and WEDE15, recognizing cleaved and uncleaved, but not internalized PAR-1. In contrast to healthy age-matched controls, platelets from stroke patients exhibited significant cleavage and internalization of PAR-1 (P<0.001) and failed to respond to thrombin in vitro. Enhanced surface expression of CD62P, CD63, TSP-1 and less mepacrine uptake showed platelet degranulation during stroke. Platelets from patients with acute cerebral ischemia are exhausted and desensitized to thrombin through cleavage of PAR-1, indicating that high concentrations of thrombin occur with acute cerebrovascular ischemic events in vivo.

Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses.

The objective of the study was to investigate platelet activation markers in whole blood in idiopathic nephrotic syndrome (INS) in children. The study group consisted of 34 children with 45 relapses of INS, 35 children in long-term remission of INS, and 26 healthy controls. Using flow cytometry we measured the percentage of platelet microparticles, platelet-platelet aggregates, and surface expression of CD62P (P-selectin) and CD42b (a component of von Willebrand factor receptor). We found an increased percentage of microparticles and platelet-platelet aggregates, decreased expression of CD42b in the first 2 weeks of INS relapse. CD62P expression was elevated only at the onset of INS relapse when compared with the long-term remission group and healthy subjects. Children in long-term INS remission did not differ from healthy controls. There was no significant correlation between platelet activation markers and selected biochemical factors of blood in children with INS. Activation of the coagulation cascade was confirmed by an elevated serum concentration of F1+2 prothrombin fragment during follow-up. These findings suggest that platelets may contribute independently to the prothrombotic state in the early stages of INS, but their role in triggering relapses remains to be investigated.

Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physical health. Recent work demonstrated that exercise triggers the release of extracellular vesicles (EVs) into the circulation, possibly contributing to exercise-associated adaptive systemic signalling. Circulating EVs comprise a heterogeneous collection of different EV-subclasses released from various cell types. So far, a comprehensive picture of the parental and target cell types, EV-subpopulation diversity and functional properties of EVs released during exercise (ExerVs) is lacking. Here, we performed a detailed EV-phenotyping analysis to explore the cellular origin and potential subtypes of ExerVs. Healthy male athletes were subjected to an incremental cycling test until exhaustion and blood was drawn before, during, and immediately after the test. Analysis of total blood plasma by EV Array suggested endothelial and leukocyte characteristics of ExerVs. We further purified ExerVs from plasma by size exclusion chromatography as well as CD9-, CD63- or CD81-immunobead isolation to examine ExerV-subclass dynamics. EV-marker analysis demonstrated increasing EV-levels during cycling exercise, with highest levels at peak exercise in all EV-subclasses analysed. Phenotyping of ExerVs using a multiplexed flow-cytometry platform revealed a pattern of cell surface markers associated with ExerVs and identified lymphocytes (CD4, CD8), monocytes (CD14), platelets (CD41, CD42, CD62P), endothelial cells (CD105, CD146) and antigen presenting cells (MHC-II) as ExerV-parental cells. We conclude that multiple cell types associated with the circulatory system contribute to a pool of heterogeneous ExerVs, which may be involved in exercise-related signalling mechanisms and tissue crosstalk.

BACKGROUND

Blood flow induced shear stress plays an important role in platelet and endothelial cell functions. The goal of this study was to investigate the effect of physiologically relevant dynamic shear stress on platelet and endothelial cells.

METHODS

Pulsatile shear stress waveforms mimicking the flow in a normal left coronary artery (0.1-1 Pa), at a 60% stenosis (0.2 - 6 Pa) and in the recirculation zone (0.01 - 0.5 Pa) behind a stenosis were used to stimulate platelets and endothelial cells in a cone and plate shearing device. Platelet activation was measured by CD62P expression and thrombogenicity. Meanwhile, endothelial cell activation and damage was measured by cell surface ICAM-1 and tissue factor expression using fluorescence microscopy. Endothelial tissue factor activity was measured using a commercial kit.

RESULTS

Results showed that for platelets, a short exposure to elevated shear stress at the stenosis throat did not induce significant increase in platelet activation or thrombogenicity. While the low pulsatile shear stress had a potential for enhanced thrombosis. Both low and high pulsatile shear stress led to a significant increase in ICAM-1 expression on endothelial cell surface, but only low shear stress caused tissue factor over expression and enhanced tissue factor activity.

CONCLUSIONS

These results suggest that low pulsatile shear stress may be more atherogenic, compared to elevated shear stress induced by stenosis.

Clostridium perfringens gas gangrene is a fulminant necrotizing infection in which inflammatory cells are notably absent from infected tissues but are often massed within adjacent vessels. It has been shown that C. perfringens phospholipase C (PLC) stimulates formation of large intravascular platelet/leukocyte complexes and that PLC-induced activation of platelet gpIIbIIIa plays a major role. In vivo, such aggregates contribute to microvascular thrombosis and ischaemic necrosis of tissue. However, the effects of adherent platelets on neutrophil diapedesis have not been established. The present work investigated (1) the contribution of platelet P-selectin (CD62P) to PLC-induced cellular complex formation and (2) the effects of platelet adhesion on neutrophil diapedesis. The effects of anti-gpIIbIIIa and anti-CD62P strategies on PLC-induced complex formation were measured by flow cytometry and followed by light microscopy. Both platelet gpIIbIIIa and CD62P contributed to the formation of platelet/leukocyte complexes. Specifically, gpIIbIIIa mediated the formation of large platelet/platelet aggregates that were tethered to the leukocyte principally via CD62P. Neutrophil diapedesis, quantified by a transendothelial cell migration assay and visualized by electron microscopy, was significantly reduced (>60%) by the adherence of large platelet aggregates. It was concluded that the absence of a tissue inflammatory response in C. perfringens gas gangrene is due, in part, to impaired neutrophil mobility caused by large aggregates of adherent platelets induced by PLC. Further, an adjunctive immunotherapeutic strategy targeting both gpIIbIIIa and CD62P may improve the tissue inflammatory response, prevent vascular occlusion, maintain tissue viability, and reduce the need for radical amputation in patients with clostridial gas gangrene.

BACKGROUND

Congestive heart failure (CHF) is associated with increased risk of venous thromboembolism, stroke and sudden death. This may be related to abnormalities of thrombogenesis and platelet activation. A comprehensive assessment of platelet (dys)function in acute decompensated heart failure (AHF) is lacking, and we hypothesised that such patients would show greater abnormalities in platelet indices, compared to stable CHF and healthy controls.

METHODS

We measured soluble P-selectin (sP-sel, by ELISA); platelet surface P-selectin (CD62P%G) and CD63%G expression by flow cytometry; and platelet structural indices [mean platelet volume (MPV), mean platelet mass (MPM) and mean platelet component (MPC)] in 22 patients with AHF (pre- and posttreatment), who were compared to 68 patients with stable congestive heart failure (CHF, all with left ventricular ejection fraction (LVEF) <50%) and 23 healthy controls.

RESULTS

There were significant differences between the 3 study groups in MPV (p<0.001), MPC (p=0.001), platelet surface P-selectin (CD62P%G, p<0.0001) and platelet surface CD63P%G (p=0.017). On post-hoc analyses, AHF patients had higher platelet surface P-selectin (CD62P%G) compared to stable CHF patients and healthy controls (Tukey's test, all p<0.05), whilst CD63%P was similarly high in both disease groups, compared to healthy controls. Platelet surface P-selectin (p=0.032), CD63 (p=0.024) and CD40L (p=0.024) were significantly reduced following treatment of AHF, though platelet morphology and sP-sel levels were not significantly changed.

CONCLUSIONS

AHF patients demonstrate some abnormalities of platelet activation compared to stable CHF patients and healthy controls. These platelet abnormalities are modified by treatment, raising the possibility that platelets may partly contribute to the pathophysiology of adverse complications associated with AHF.

BACKGROUND

Platelet aggregation mediated by inflammation played a critical role in the development of coronary heart diseases (CHD). Our previous clinical researches showed that Th17 cells and their characteristic cytokine IL-17A were associated with the plaque destabilization in patients with acute coronary syndrome (ACS). However, the potent effect of IL-17A on platelets-induced atherothrombosis remains unknown.

RESULTS

In this study, we detected the plasma IL-17A levels and platelet aggregation in patients with stable angina (SA), unstable angina (UA), acute myocardial infarction (AMI) and chest pain syndrome (CPS). In addition, the markers of platelet activation (CD62P/PAC-1) and the mitogen-activated protein kinases (MAPKs) pathway were detected in platelets from ACS patients. We found that plasma IL-17A levels and platelet aggregation in patients with ACS (UA and AMI) were significantly higher than patients with SA and CPS, and the plasma IL-17A levels were positively correlated with the platelet aggregation (R = 0.47, P<0.01). In addition, in patients with ACS, the platelet aggregation, CD62P/PAC-1 and the phosphorylation of ERK2 signaling pathway were obviously elevated in platelets pre-stimulated with IL-17A in vitro. Furthermore, the specific inhibitor of ERK2 could attenuate platelet aggregation and activation triggered by IL-17A.

CONCLUSIONS

Our experiment firstly proved that IL-17A could promote platelet function in patients with ACS via activating platelets ERK2 signaling pathway and may provide a novel target for antiplatelet therapies in CHD.

Tissue factor (TF) is the most important initiator of intravascular coagulation. Platelets contribute to TF exposure on monocytes, but the mechanism is not completely understood. Here we examined the possibility that platelets may release TF that can be transferred to monocytes by platelet-derived microvesicles. When human citrated platelet-rich plasma was incubated with collagen there was an increase in the plasma levels of TF and CD62P. Incubation of plasma obtained from collagen-stimulated PRP with a sediment of red and white blood cells resulted in an increase in the number of monocytes that express TF, CD62P and the platelet-specific antigen CD42a on their surface. This transfer of platelet-derived antigens to monocytes was reduced when CD62P was blocked by a specific antibody or when platelet-derived microvesicles were removed from the plasma either by high speed centrifugation (17,500 x g for 30 min) or by filtration (pore size 0.2 microm). The data indicate that platelet-derived microvesicles that are released from collagen-stimulated platelets may carry TF, CD62P and CD42a and may transfer these antigens to the surface of monocytes. The interaction of platelet-derived microvesicles with monocytes and the transfer of TF to monocytes strongly depend on CD62P.

BACKGROUND

Short-term refrigeration of platelets (PLTs) in the absence of plasma results in their rapid clearance after transfusion. Blocking beta-N-acetylglucosamine (beta-GlcNAc) residues of glycoprotein Ibalpha (GPIbalpha) with galactose prevents binding of refrigerated human and mouse PLTs to macrophages and prolongs the circulation times of refrigerated mouse PLTs. PLT-associated galactosyltransferase efficiently galactosylates chilled PLTs in the presence of its substrate UDP-galactose is added to PLT-rich plasma.

METHODS

To characterize the hemostatic function of refrigerated and galactosylated human PLTs processed in the blood bank, PLT aggregation was studied in vitro under static and flow conditions and expression of integrin beta3 (CD61), CD62P (P-selectin), GPIbalpha (CD42b), annexin V binding, and integrin alphaIIbeta3 activation with flow cytometry. Affinity of macrophages for galactosylated refrigerated PLTs was evaluated with THP-1 cells, which recognize and phagocytize refrigerated PLTs.

RESULTS

PLTs refrigerated and galactosylated for 14 days 1) maintained their ability to aggregate when exposed to agonists in a standard aggregometry assay, 2) showed less pronounced changes in surface expression of GPIbalpha compared with room temperature (RT)-stored PLTs, 3) increased P-selectin expression, and 4) were poorly phagocytized by differentiated THP-1 cells in vitro. In addition, it is shown that refrigeration of PLTs does not affect their adhesive properties under in vitro flow conditions.

CONCLUSIONS

It is shown that refrigerated human PLTs retain in vitro function better than RT PLTs during storage and demonstrate that galactosylation prevents recognition of stored refrigerated PLTs by macrophages in vitro.

The pathophysiology of platelet dysfunction in the Wiskott-Aldrich immune deficiency syndrome (WAS) remains unclear. Using flow cytometry, we have characterized the functional properties of platelets from 10 children with WAS. Patients with WAS had thrombocytopenia, small platelets, increased platelet-associated IgG and reduced platelet-dense granule content. Levels of reticulated 'young' platelets were normal in the WAS patients. Although the mean numbers of platelet glycoprotein (GP) Ib, GPIIbIIIa and GPIV molecules per platelet appeared lower in WAS patients than in healthy controls, analysis of similar-sized platelets revealed the mean number of GPIb molecules per platelet to be comparable in patients and normal controls. Surface GPIIbIIIa and GPIV expression was, however, significantly lower on the WAS platelets than on normal platelets. Compared with normal platelets, WAS platelets showed a reduced ability to modulate GPIIbIIIa expression following thrombin stimulation. In addition, thrombin- and ADP-induced expression of CD62P and CD63 was defective in WAS platelets. Phallacidin staining of the WAS platelets revealed less F-actin content than in normal platelets. Together, these data suggest that the reduced platelet number and function in WAS reflects, at least in part, a defect in bone marrow production as well as an intrinsic platelet abnormality.

P-selectin (CD62P), a Ca(2+)-dependent lectin expressed on activated platelets and endothelial cells, functions as a receptor for myeloid and monocytoid cells. Previous reports have described a homodimeric sialoglycoprotein from human leukocytes and HL-60 cells specifically recognized by P-selectin. We describe here a panel of monoclonal antibodies prepared against high molecular weight fractions of HL-60 cell membranes. These antibodies are of IgM isotype, bind to a approximately 240-kDa protein from human leukocyte membranes which is also reactive with P-selectin. They recognize a Ca(2+)-dependent, sialidase-sensitive determinant on myeloid and monocytoid cell lines. Each antibody specifically inhibits adhesion of neutrophils or HL-60 cells to: 1) purified P-selectin, 2) thrombin-stimulated platelets, and 3) phorbol 12-myristate 13-acetate-activated endothelial cells. These results suggest that the sialoglycoprotein recognized by this panel of monoclonal antibodies may function as a cell surface ligand for P-selectin.

Silicon membranes with highly uniform nanopore sizes fabricated using microelectromechanical systems (MEMS) technology allow for the development of miniaturized implants such as those needed for renal replacement therapies. However, the blood compatibility of silicon has thus far been an unresolved issue in the use of these substrates in implantable biomedical devices. We report the results of hemocompatibility studies using bare silicon, polysilicon, and modified silicon substrates. The surface modifications tested have been shown to reduce protein and/or platelet adhesion, thus potentially improving biocompatibility of silicon. Hemocompatibility was evaluated under four categories-coagulation (thrombin-antithrombin complex, TAT generation), complement activation (complement protein, C3a production), platelet activation (P-selectin, CD62P expression), and platelet adhesion. Our tests revealed that all silicon substrates display low coagulation and complement activation, comparable to that of Teflon and stainless steel, two materials commonly used in medical implants, and significantly lower than that of diethylaminoethyl (DEAE) cellulose, a polymer used in dialysis membranes. Unmodified silicon and polysilicon showed significant platelet attachment; however, the surface modifications on silicon reduced platelet adhesion and activation to levels comparable to that on Teflon. These results suggest that surface-modified silicon substrates are viable for the development of miniaturized renal replacement systems.

Neutrophils and platelets interact both physically and metabolically during inflammation and thrombosis, but the mechanisms responsible for their adhesion remain incompletely understood. Neutrophil-platelet adhesion was measured after specific stimulation of neutrophils, platelets, or both and quantified by flow cytometry. Specific stimulation of either the neutrophil or the platelet led to a marked increase in the percentage of neutrophils that bound platelets, although platelet stimulation led to a large increase and neutrophil stimulation to only a small increase in the number of platelets per neutrophil. Stimulation of both cells further increased the number of neutrophil-platelet adhesive events and led to large numbers of platelets binding to each neutrophil. Confirming previous observations, blocking antibodies to platelet P-selectin (CD62P) partially inhibited adhesion. However, blockade of the neutrophil beta2 integrin CD11b/CD18 also inhibited the percentage of neutrophils that bound platelets. Combining P-selectin and CD11b/18 blockade further inhibited the stimulated increase in the percentage of neutrophils binding platelets and the increased number of platelets per neutrophil. Both cell adhesion molecules were active even when only a single cell type was primarily activated, supporting physiologically important transcellular activation. These data suggest that: (1) neutrophil-platelet adhesion can be initiated by specific activation of either the neutrophil or the platelet and that specific activation of either cell type leads to distinct patterns of adhesion, and (2) neutrophil-platelet adhesion uses both platelet P-selectin and the neutrophil beta2 integrin CD11b/CD18 when the cells are primarily or secondarily activated.

BACKGROUND

Platelets are known to participate in vascular pathologies; however, their role in neuroinflammatory diseases, such as multiple sclerosis (MS), is unknown. Autoimmune CD4 T cells have been the main focus of studies of MS, although the factors that regulate T-cell differentiation toward pathogenic T helper-1/T helper-17 phenotypes are not completely understood.

OBJECTIVE

We investigated the role of platelets in the modulation of CD4 T-cell functions in patients with MS and in mice with experimental autoimmune encephalitis, an animal model for MS.

RESULTS

We found that early in MS and experimental autoimmune encephalitis, platelets degranulated and produced soluble factors serotonin (5-hydroxytryptamine), platelet factor 4, and platelet-activating factor, which specifically stimulated differentiation of T cells toward pathogenic T helper-1, T helper-17, and interferon-γ/interleukin-17-producing CD4 T cells. At the later stages of MS and experimental autoimmune encephalitis, platelets became exhausted in their ability to produce proinflammatory factors and stimulate CD4 T cells but substantially increased their ability to form aggregates with CD4 T cells. Formation of platelet-CD4 T-cell aggregates involved the interaction of CD62P on activated platelets with adhesion molecule CD166 on activated CD4 T cells, contributing to downmodulation of CD4 T-cell activation, proliferation, and production of interferon-γ. Blocking of formation of platelet-CD4 T-cell aggregates during progression of experimental autoimmune encephalitis substantially enhanced proliferation of CD4 T cells in the central nervous system and the periphery leading to exacerbation of the disease.

CONCLUSIONS

Our study indicates differential roles for platelets in the regulation of functions of pathogenic CD4 T cells during initiation and progression of central nervous system autoimmune inflammation.

BACKGROUND

Many complications associated with congestive heart failure (CHF) have a thrombosis-related aetiology. Platelets play an important role in thrombogenesis, but it is not clear whether circulating platelets actively participate in thrombosis-related complications associated with CHF.

OBJECTIVE

To determine whether soluble P-selectin, platelet surface P-selectin, and total platelet P-selectin as indices of platelet activation in CHF patients-compared to 'disease controls' and 'healthy controls'-and to assess their prognostic value in CHF.

METHODS

We measured soluble P-selectin (sP-sel, by enzyme-linked immunosorbent assay, ELISA), total platelet P-selectin (pP-sel, by a novel 'platelet lysate' assay), platelet surface P-selectin (CD62P%G) and platelet surface CD63 (CD63%G) expression by flow cytometry-in 108 patients with stable congestive heart failure (all with left ventricular ejection fraction (LVEF) <50%). Levels were compared with 50 healthy controls and 70 'disease controls' (patients with coronary artery disease with normal left ventricular systolic function).

RESULTS

CHF patients and disease controls had higher sP-sel, CD62P%G and CD63%G than healthy controls. There were no significant correlations between sP-sel, pP-sel, CD62P%G and CD63%G with ejection fraction (all P>0.05). There were no differences in these markers when ischaemic and non-ischaemic aetiologies of CHF were compared. After a median follow-up of 490 days (range 340-535), there were 7 deaths, 15 hospitalizations for worsening heart failure, 1 for cardiac resynchronization therapy, 4 for revascularizations, 4 for myocardial infarctions, and 1 stroke. None of the platelet markers were predictive of the composite end-point at follow-up.

CONCLUSIONS

Patients with stable CHF exhibit evidence of abnormal platelet activation, despite usage of antiplatelet agents. These abnormalities did not determine prognosis and were broadly similar to those seen in 'disease controls' indicating that platelet abnormalities in CHF may simply be related to associated comorbidities.

Platelet activation in preeclampsia is reflected by elevated levels of platelets exposing P-selectin. In plasma, a non-cell bound (soluble) form of P-selectin is present. Elevated levels of this soluble form have been reported in preeclampsia. Plasma P-selectin may consist of two fractions: microparticle (MP)--associated P-selectin and non-MP--associated P-selectin. In the present cross-sectional study, we investigated to which extent plasma P-selectin is MP--associated and whether such MP are elevated in preeclamptic patients. Preeclamptic patients (n=10) were matched with normotensive pregnant women (n=10) and non-pregnant controls (n=10). Plasma P-selectin was measured by ELISA. MP were isolated, double labelled with anti-CD61 (GPIIIa) and anti-CD62P (P-selectin) and subsequently analyzed with flowcytometry. Plasma P-selectin concentration was elevated in preeclamptic patients compared to non-pregnant controls (p=0.007), but not compared to normotensive pregnant women (p=0.210). Plasma P-selectin is partially MP--associated (3-5%). In pregnancy, the fraction of P-selectin exposing platelet-derived MP (PMP) (10.9%) was increased compared to non-pregnant controls (8%). This fraction further increased in preeclamptic patients (15.4%), and significantly differed from normotensive pregnant women (p=0.02). A minor fraction of plasma P-selectin is associated with PMP. The fraction of PMP exposing P-selectin is increased in preeclamptic patients and to a lesser extent in normotensive pregnancy. Because MP associated P-selectin exclusively originates from platelets, this fraction indicates platelet activation. Platelet activation is prominent in preeclampsia and this study proves that at least a part of the plasma P-selectin originates from platelets.

Platelets (PLTs) act in antimicrobial host defense by releasing PLT microbicidal proteins (PMPs) or PLT kinocidins (PKs). Receptors mediating staphylocidal efficacy and PMP or PK release versus isogenic PMP-susceptible (ISP479C) and -resistant (ISP479R) Staphylococcus aureus strains were examined in vitro. Isolated PLTs were incubated with ISP479C or ISP479R (PLT/S. aureus ratio range, 1:1 to 10,000:1) in the presence or absence of a panel of PLT inhibitors, including P2X and P2Y receptor antagonists of increasingly narrow specificity, and PLT adhesion receptors (CD41, CD42b, and CD62P). PLT-to-S. aureus exposure ratios of>> or = 10:1 yielded significant reductions in the viability of both strains. Results from reversed-phase high-performance liquid chromatography indicated that staphylocidal PLT releasates contained PMPs and PKs. At ratios below 10:1, the PLT antistaphylococcal efficacy relative to the intrinsic S. aureus PMP-susceptible or -resistant phenotype diminished. Apyrase (an agent of ADP degradation), suramin (a general P2 receptor antagonist), pyridoxal 5'-phosphonucleotide derivative (a specific P2X(1) antagonist), and cangrelor (a specific P2Y(12) antagonist) mitigated the PLT staphylocidal response against both strains, correlating with reduced levels of PMP and PK release. Specific inhibition occurred in the presence and absence of homologous plasma. The antagonism of the thromboxane A(2), cyclooxygenase-1/cyclooxygenase-2, or phospholipase C pathway or the hindrance of surface adhesion receptors failed to impede PLT anti-S. aureus responses. These results suggest a multifactorial PLT anti-S. aureus response mechanism involving (i) a PLT-to-S. aureus ratio sufficient for activation; (ii) the ensuing degranulation of PMPs, PKs, ADP, and/or ATP; (iii) the activation of P2X(1)/P2Y(12) receptors on adjacent PLTs; and (iv) the recursive amplification of PMP and PK release from these PLTs.

BACKGROUND

Aspirin-exacerbated respiratory disease (AERD) is characterized by respiratory reactions on ingestion of COX-1 inhibitors and cysteinyl leukotriene overproduction. The hypersensitivity reaction is induced by low doses of aspirin that inhibit COX-1 in platelets.

OBJECTIVE

We sought to explore the role of platelets in the pathogenesis of AERD in patients under stable conditions and during an aspirin challenge test.

METHODS

Stable patients with AERD (n = 30), aspirin-tolerant asthma (ATA; n = 21), or idiopathic chronic eosinophilic pneumonia (n = 10) were enrolled. Platelet activation was estimated based on expression levels of P-selectin (CD62P), CD63, CD69, and GPIIb/IIIa (PAC-1) in peripheral platelets; percentages of circulating platelet-adherent leukocytes; and plasma levels of soluble P-selectin (sP-selectin) and soluble CD40 ligand (sCD40L).

RESULTS

In the stable condition, expression of all surface markers on platelets, the percentage of platelet-adherent eosinophils, and the plasma levels of sP-selectin and sCD40L were significantly higher in patients with AERD compared with those in patients with ATA. P-selectin and CD63 expression on platelets and plasma sP-selectin and sCD40L levels were positively correlated with the percentage of platelet-adherent eosinophils. Among these markers, P-selectin expression and plasma sP-selectin levels positively correlated with urinary concentrations of leukotriene E4. Additionally, plasma sP-selectin and sCD40L levels were negatively correlated with lung function. In contrast, platelet activation markers in patients with AERD did not change during the aspirin challenge test.

CONCLUSIONS

Peripheral platelets were activated more in patients with stable AERD compared with those in patients with stable ATA, patients with idiopathic chronic eosinophilic pneumonia, and control subjects. Platelet activation was involved in cysteinyl leukotriene overproduction and persistent airflow limitations in patients with AERD.