BACKGROUND

The prevalence of food allergies and other allergic reactions is increasing worldwide, particularly in highly-urbanized populations. Cell adhesion molecules are expressed in response to various pro-inflammatory cytokines. The expression of intercellular adhesion molecule 1 - ICAM-1 (CD54), ICAM-1 (CD106), P-selectin (CD62P), and E-selectin (CD62E) on vascular endothelial cells is induced by such pro-inflammatory cytokines as tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1).

OBJECTIVE

To analyze concentrations of E-selectin and platelet endothelial cell adhesion molecule-1 (PECAM-1) in patients with an allergic type of food sensitivity co-existing with gastritis and to compare them to the values determined in individuals with dyspeptic symptoms not associated with allergic disorders.

METHODS

The study included 80 patients, among them 50 individuals with food sensitivity confirmed based on compulsory standards, and 30 subjects with dyspeptic symptoms not accompanied by allergic conditions. Venous blood samples were taken from each patient and concentrations of E-selectin and PECAM-1 were determined by means of ELISA.

RESULTS

Mean concentrations of sE-selectin and sPECAM-1 in patients with food allergy amounted to 54.0 ±21.6 ng/ml and 132.8 ±31.4 ng/ml, respectively. In individuals without food allergy, mean concentrations of sE-selectin and sPECAM-1 were 57.7 ±17.9 ng/ml and 139.6 ±31.1 ng/ml, respectively. Patients with food allergy and individuals with dyspeptic symptoms not associated with food allergy did not differ significantly in terms of sE-selectin concentrations (Mann-Whitney U-test, p = 0.453028). Similarly, no significant intergroup differences were observed with regard to sPECAM-1 concentrations (Mann-Whitney U-test, p = 0.231054).

CONCLUSIONS

Adhesion molecules play an important role in the development of inflammation. This study did not find significant differences in the concentrations of such molecules as sE-selectin and sPECAM-1 between patients with food allergy and gastritis, and subjects in whom gastritis was not accompanied by atopic disorders. A positive correlation between the concentrations of sPECAM-1 and E-selectin was observed in food allergy patients. Consequently, it can be concluded that these molecules participate in the pathogenesis of the inflammatory process independently of the etiopathogenesis of gastritis.

BACKGROUND

Cryobiologic variables responsible for cell injuries and freezing techniques applicable in medical cryopractice should be revised and/or reengineered for minimizing cryoinjuries and maximizing cell recovery. In this study, the efficacy of different cryopreservation protocols based on platelet (PLT) recovery was evaluated.

METHODS

PLTs (n = 33) were prepared from whole-blood units. Cell count and viability, PLT morphologic score (PMS), and hypotonic shock response were determined. PLT surface antigens were measured by flow cytometry. Controlled-rate (with compensated fusion heat) and uncontrolled-rate freezing methods combined with 6 percent dimethyl sulfoxide were used.

RESULTS

PLT recovery was superior in the controlled-rate setting (91.0 +/- 5.5 vs. 86.0 +/- 6.5; p < 0.05). PMS was significantly better in controlled-rate freezing (p < 0.01). GPIb/CD42b expression was reduced in both freezing groups versus control. GP140/CD62p expression was significantly (p < 0.05) lower in the controlled-rate group and in both frozen groups was significantly higher than in the control groups.

CONCLUSIONS

The use of strictly equalized (1 degrees C/min) controlled-rate freezing, combined with an intensified cooling rate (2 degrees C/min) during the liquid-to-solid-phase transition period, allows advanced quantitative and qualitative PLT recovery, even though the minor intergroup differences for some variables were observed.

This paper reports the results of an in vitro investigation into the blood response of medical grade poly (vinyl chloride) (PVC), and two types of plasticized PVC in tubing or sheet form, with di-(2-ethylhexyl)phthalate (DEHP) and di(isononyl) cyclohexane-1,2-dicarboxylate (HEXAMOLL(®) DINCH) as plasticizer, were selected for assessment of complement activation, coagulation system and platelet activation. The results of the study show that not only the plasticizers at PVC surface have an influence on complement activation, but also the incubation condition such as incubation time and the diameter of PVC tubing. Under static status, C3a, C5a and SC5b-9 concentration in the blood were higher after contacting with PVC plasticized with DEHP (PVC1) than after contacting with PVC plasticized with DINCH (PVC2). However, under dynamic circulation, the results were totally converse, which may be due to smaller diameter and higher shear rate of PVC2. In addition, there was a significant increase of activated partial thrombin time (APTT) and decrease of FIX concentration after plasma contacting with the PVC tubing, which indicated that the intrinsic pathway may be impacted when blood contacted with PVC tubing. However, there was no significant difference of APTT, FIX concentration and CD62p expression rate between the two materials. Moreover, the migration in the DINCH system was considerably lower than for DEHP, which indicates that DINCH could be a promising alterative plasticizer of DEHP.

BACKGROUND

The Intercept Blood SystemTM (Cerus) is used to inactivate pathogens in platelet concentrates (PC). The aim of this study was to elucidate the extent to which the Intercept treatment modifies the functional properties of platelets.

METHODS

A two-arm study was conducted initially to compare buffy coat-derived pathogen-inactivated PC to untreated PC (n=5) throughout storage. A four-arm study was then designed to evaluate the contribution of the compound adsorbing device (CAD) and ultraviolet (UV) illumination to the changes observed upon Intercept treatment. Intercept-treated PC, CAD-incubated PC, and UV-illuminated PC were compared to untreated PC (n=5). Functional characteristics were assessed using flow cytometry, hypotonic shock response (HSR), aggregation, adhesion assays and flow cytometry for the detection of CD62P, CD42b, GPIIb-IIIa, phosphatidylserine exposure and JC-1 aggregates.

RESULTS

Compared to fresh platelets, end-of-storage platelets exhibited greater passive activation, disruption of the mitochondrial transmembrane potential (Δψm), and phosphatidylserine exposure accompanied by a decreased capacity to respond to agonist-induced aggregation, lower HSR, and CD42b expression. The Intercept treatment resulted in significantly lower HSR and CD42b expression compared to controls on day 7, with no significant changes in CD62P, Δψm, or phosphatidylserine exposure. GPIIbIIIa expression was significantly increased in Intercept-treated platelets throughout the storage period. The agonist-induced aggregation response was highly dependent on the type and concentration of agonist used, indicating a minor effect of the Intercept treatment. The CAD and UV steps alone had a negligible effect on platelet aggregation.

CONCLUSIONS

The Intercept treatment moderately affects platelet function in vitro. CAD and UV illumination alone make negligible contributions to the changes in aggregation observed in Intercept-treated PC.

To characterize recombinant human macrophage-colony stimulating factor (rhM-CSF)-associated thrombocytopenia (TCP), in vivo studies were performed in dogs, including the biodistributions and recoveries of radiolabelled autologous and allogeneic platelets. rhM-CSF induced a reversible, dose-dependent decrease in platelet counts. The number of megakaryocytes in spleen and marrow of rhM-CSF-treated dogs was increased two to threefold. Recoveries of allogeneic platelets transfused from rhM-CSF-treated donors into tolerized recipients (n = 3) were not significantly different from allogeneic baseline studies (93 +/- 10% of baseline values at 24 h and 90 +/- 1% at 40 h), whereas autologous platelets infused back into rhM-CSF-treated donors had decreased recoveries (45 +/- 2% of baseline values at 24 h, P = 0.03 and 20 +/- 4% at 40 h, P = 0.001). Platelet biodistribution studies showed increased accumulation of radiolabelled platelets over the spleens and livers of rhM-CSF-treated dogs. Histochemistry showed increased levels of platelet-specific antigen (CD41; glycoprotein IIb) associated with Kupffer cells. The sensitivity of platelets from rhM-CSF-treated dogs to activation from thrombin, as measured by expression of P-selectin (CD62P), was not significantly different when compared with baseline studies (P = 0.18; n = 4). These results support the concept that rhM-CSF induces an activation of the monocyte-macrophage system (MMS), which causes a reversible TCP in a dog model.

BACKGROUND

Platelet inactivation technologies (PITs) have been shown to increase platelet storage lesions (PSLs). This study investigates amotosalen/ultraviolet (UV)A- and riboflavin/UVB-induced platelet (PLT) lesions in vitro. Particular attention is given to the effect of UVB alone on PLTs.

METHODS

Buffy coat-derived PLT concentrates (PCs) were treated with amotosalen/UVA, riboflavin/UVB, or UVB alone and compared to untreated PCs throughout storage. In vitro PLT function was assessed by blood gas and metabolite analyses, flow cytometry-based assays (CD62P, JC-1, annexin V, PAC-1), hypotonic shock response, and static adhesion to fibrinogen-coated wells.

RESULTS

In our experimental conditions, riboflavin/UVB-treated PCs showed the most pronounced differences compared to untreated and amotosalen/UVA-treated PCs. The riboflavin/UVB treatment led to a significant increase of anaerobic glycolysis rate despite functional mitochondria, a significant increase of CD62P on Day 2, and a decrease of JC-1 aggregates and increase of annexin V on Day 7. The expression of active GPIIbIIIa (PAC-1) and the adhesion to fibrinogen was significantly increased from Day 2 of storage in riboflavin/UVB-treated PCs. Importantly, we showed that these lesions were caused by the UVB radiation alone, independently of the presence of riboflavin.

CONCLUSIONS

The amotosalen/UVA-treated PCs confirmed previously published results with a slight increase of PSLs compared to untreated PCs. Riboflavin/UVB-treated PCs present significant in vitro PSLs compared to untreated PCs. These lesions are caused by the UVB radiation alone and probably involve the generation of reactive oxygen species. The impact of these observations on clinical use must be investigated.

Adaptor proteins play a critical role in the assembly of signalling complexes after engagement of platelet receptors by agonists such as collagen, ADP and thrombin. Recently, using proteomics, the Dok (downstream of tyrosine kinase) adapter proteins were identified in human and mouse platelets. In vitro studies suggest that Dok-1 binds to platelet integrin β3, but the underlying effects of Dok-1 on αIIbβ3 signalling, platelet activation and thrombosis remain to be elucidated. In the present study, using Dok-1-deficient (Dok-1-/-) mice, we determined the phenotypic role of Dok-1 in αIIbβ3 signalling. We found that platelets from Dok-1-/- mice displayed normal aggregation, activation of αIIbβ3 (assessed by binding of JON/A), P-selectin surface expression (assessed by anti-CD62P), and soluble fibrinogen binding. These findings indicate that Dok-1 does not affect "inside-out" platelet signalling. Compared with platelets from wild-type (WT) mice, platelets from Dok-1-/- mice exhibited increased clot retraction (p < 0.05 vs WT), increased PLCγ2 phosphorylation, and enhanced spreading on fibrinogen after thrombin stimulation (p < 0.01 vs WT), demonstrating that Dok-1 negatively regulates αIIbβ3 "outside-in" signalling. Finally, we found that Dok-1-/- mice exhibited significantly shortened bleeding times and accelerated carotid artery thrombosis in response to photochemical injury (p < 0.05 vs WT mice). We conclude that Dok-1 modulates thrombosis and haemostasis by negatively regulating αIIbβ3 outside-in signalling.

Blood platelets bind rapidly to foreign surfaces and interact with adsorbed proteins and neutrophil granulocytes. We demonstrate by use of luminol-amplified chemiluminescence under stirred and non-stirred conditions that platelets at IgG-coated surfaces amplify the neutrophil extracellular release of reactive oxygen species (ROS). The neutrophil response involved tyrosine phosphorylation, but was only in part induced by neutrophil F(c gamma)-receptor stimulation. The platelet mediated effects were contact-dependent since the respiratory burst was inhibited when the IgG-stimulated platelets were removed by filtration, but not when they were fixed in paraformaldehyde. Bodipyphallacidin-staining of filamentous actin (F-actin) revealed that an actin-dependent platelet adhesion supported the subsequent adhesion and spreading of neutrophils. The neutrophil ROS-response was lowered when the interaction between platelet P-selectin (CD62P) and neutrophil P-selectin glycoprotein ligand-l (PSGL-1 or CD162) was inhibited. The blocking of L-selectin (CD62L) or blocking of the interaction between platelet glycoprotein (Gp) IIb/IIIa and neutrophil complement receptor 3 (CR3) showed no effect. We conclude that platelet activation on immobilized IgG trigger a contact-dependent "frustrated" phagocytosis by neutrophils, associated with a release of toxic ROS.

Soluble P-selectin (CD62P) may arise from platelets, the endothelium, or both, and raised levels are found in those with thrombotic disease and atherosclerosis. To determine whether these increased levels in atherosclerosis are related to hypercholesterolaemia, blood samples were obtained from 86 patients (43 with symptomatic vascular disease) attending a hypercholesterolaemia clinic, and 86 age- and sex-matched controls. Parallel measurement of endothelial cell product von Willebrand factor helped define the origin of sP-selectin. Using ELISAs, soluble P-selectin was higher (median 290 ng/ml, range 80-735, P < 0.05) in patients with vascular disease than in both patients with uncomplicated hypercholesterolaemia (median 210 ng/ml, range 55-550), and controls (median 190 ng/ml, range 48-500). Von Willebrand factor was raised in both patients with uncomplicated hypercholesterolaemia (115 +/- 26 IU/dl, P < 0.05) and patients with hypercholesterolaemia and vascular disease (129 +/- 32 IU/dl, P < 0.02) compared with controls (102 +/- 30 IU/dl). Levels of soluble P-selectin did not correlate with von Willebrand factor, total low-density-lipoprotein (LDL) or high-density-lipoprotein (HDL) cholesterol or triglycerides levels, blood pressure or smoking, but von Willebrand factor correlated with LDL cholesterol (r = 0.42, P < 0.05). We conclude that plasma lipoproteins are not a major influence on levels of soluble P-selectin.

We evaluated the plasma concentrations of soluble adhesion molecules, platelet activation markers, and platelet-derived microparticles (PMPs) in patients with connective tissue diseases who had secondary hyperlipidemia caused by long-term steroid administration (n = 22) before and after treatment with bezafibrate. There were differences in levels of platelet activation markers both before and after treatment (platelet CD62p: 15.11+/-2.03 vs 10.38+/-8.53%, P < 0.05; platelet CD63: 12.12+/-9.17 vs 9.90+/-7.20%, P < 0.05). There were also differences in the levels of PMPs and soluble adhesion molecules both before and after treatment (PMP: 514+/-273 vs 401+/-201 /10(4) platelet. P < 0.05; soluble vascular cell adhesion molecule-1: 724+/-191 vs 666+/-157 ng/mL, P < 0.01). After 6 months of treatment, serum lipid concentrations were reduced by 9% for total cholesterol (TC) and 32% for triglyceride (TG). The level of PMPs, activated platelets, and soluble adhesion molecules were all significantly decreased after treatment with bezafibrate. These findings suggest that bezafibrate may be useful for inhibiting both PMP-dependent and -independent vascular damage in patients with connective tissue diseases complaining of secondary hyperlipidemia.

Acute inflammatory diseases, such as colic, septicemia and endotoxemia are common in equines and have been shown to be correlated to vascular injury and thrombosis. In humans with similar thrombotic conditions, P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1)-mediated platelet-leukocyte adhesion contributes to the pathogenesis of these disorders through the generation of inflammatory mediators and tissue factor. As such, we hypothesized that a P-selectin-PSGL-1 (platelet-leukocyte) interaction, similar to that in humans, may also exist in the horse. The objective of this study was to investigate phenotypic and morphological properties of equine platelet activation with a focus on CD62P (P-selectin) expression and CD62P mediated platelet-leukocyte interactions. To study high levels of platelet activation, we used 1 U/ml thrombin to induce secondary, irreversible aggregation in both human and equine platelets. Addition of glycyl-L-prolyl-L-arginyl-L-proline amide (GPRP) prior to thrombin activation blocked fibrin polymerization, allowing the use of flow cytometry to study alpha-granule expression as a measure of platelet activation. Thrombin activation resulted in high levels of activation, measured as P-selectin expression, in both humans and equines. Interestingly, our research illustrates that in healthy horses, P-selectin is also constitutively expressed on 20-25% of resting platelets. This finding is in direct contrast to humans, in which P-selectin expression is negligible (<5%) in the absence of agonist activation. The high baseline level of P-selectin expression among equine platelets may suggest that they are primed for leukocyte adhesion, possibly resulting in prothrombotic conditions. This phenomenon could be of significant clinical relevance, as it may be related to the rapid clinical decline often seen in equine patients with colic and endotoxemia, where vascular injury and thrombotic complications compromise patient survival. Based on these findings, further investigation into the mechanisms of platelet P-selectin-mediated inflammation and platelet-leukocyte mediated vascular injury in the horse appears warranted.

We measured and compared levels of platelet-derived microparticles (PMPs), monocyte-derived microparticles (MMPs), CD62P on activated platelets, soluble E-selectin (sE-selectin), and anti-oxidized low density lipoprotein (LDL) antibody in hyperlipidemia patients and control subjects. Binding of anti-GPIIb/IIIa and anti-GPIb monoclonal antibodies to platelets was not significantly different between hyperlipidemia patients and controls. However, expression of CD62P on platelets and levels of PMPs were higher for hyperlipidemia patients than in controls, although the difference between groups in CD62P expression was not significant (PMPs: 534 +/- 63 vs. 388 +/- 47, p < 0.05; CD62P: 9.1% +/- 1.45 vs. 7.3% +/- 1.15, N.S.). Although there were no differences in expression of CD36 and CD40 by monocytes between the two groups, levels of MMPs were higher in hyperlipidemia patients than in controls (MMPs: 147 +/- 21 vs. 59 +/- 8, respectively, p < 0.01). Levels of anti-oxidized LDL antibody and sE-selectin were also higher in hyperlipidemia patients. We studied the effects of Saiko-ka-ryukotsu-borei-to on levels of these factors in patients with elevated triglyceride levels. After Saiko-ka-ryukotsu-borei-to treatment, levels of CD62P, PMPs, sE-selectin, and anti-oxidized LDL antibody were reduced significantly. Levels of triglycerides, total cholesterol and MMPs also decreased, but the changes were not significant. These findings suggest that Saiko-ka-ryukotsu-borei-to prevents the development of vascular complications in hyperlipidemia patients.

BACKGROUND

In our previously described primate renal allograft model, T cell ablation leads to long-term graft survival. The role of endothelial cell alteration in chronic rejection was examined in our model.

METHODS

Renal transplants were performed in rhesus monkeys using a T cell- depleting immunotoxin, FN18-CRM9. Sections from 10 rejected kidneys (5 acute and 7 chronic rejection) were examined after immunohistochemical staining for expression of endothelium-related proteins [von Willebrand factor (vWF), CD62P, and CD31], fibrinogen, and a macrophage marker (CD68). Glomerular staining for each antigen was graded on a semiquantitative scale.

RESULTS

Intense staining for vWF was consistently observed in glomerular endothelium, subendothelium, and mesangium in all kidneys removed due to chronic rejection. vWF staining was weak in kidneys showing acute rejection. The difference in glomerular staining was statistically significant. Staining for vWF in extraglomerular vessels was nearly identical in kidneys showing acute and chronic rejection. Expression of CD62P was increased in extraglomerular vessels in allografts with chronic rejection, but the glomeruli showed little or no staining. There was no significant difference in the glomerular staining for CD62P or CD31 in organs showing acute and chronic rejection. Fibrinogen staining of glomerular mesangium was seen in kidneys with chronic rejection. Macrophages (CD68+) infiltrating glomeruli were more numerous in kidneys showing chronic rejection.

CONCLUSIONS

Increased glomerular deposition of vWF in renal allografts showing chronic rejection, without increased staining for CD62P or CD31, suggests increased constitutive secretion of vWF from endothelial cells as a component of the mechanism of chronic rejection in our model.

Dopamine (DA) is a co-agonist for platelet activation; yet, donor DA treatment is associated with improved transplantation outcome in renal and heart recipients. Recently, N-octanoyl-dopamine (NOD) was developed which displays superior effects compared to DA in terms of graft protecting properties. Whereas DA is a known platelet co-agonist, the effect of NOD on platelet function is unknown. This is a hypothesis generating study with the aim to assess the effects and molecular mechanisms of NOD and NOD-like compounds on platelet function. The influence of DA, NOD, and NOD-like compounds on platelet responses to classical agonists (adenosine 5'-diphosphate (ADP), U46619) was investigated in six healthy donors by applying whole blood aggregometry (Multiplate®) and flow cytometry for Pac-1, CD62P, and CD63 expression. Changes in platelet cAMP concentrations were assessed by ELISA. While DA showed synergy in platelet activation by ADP and U46619, NOD caused significant inhibition of platelet function both in whole blood aggregometry and flow cytometry. The inhibitory effect of NOD was not mediated via cAMP levels. The nonredox-active NOD-analog N-octanoyl-tyramine had no effects on platelet function. Acetylated NOD conferred to NOD by intracellular esterases showed similar inhibitory effects as NOD. In contrast to DA, NOD is a potent inhibitor of platelet function most likely through intracellular redox-active processes. This adds to the overall protective effect of NOD on pre-transplantation injury and makes NOD an attractive candidate compound for donor or organ conditioning prior to transplantation.

Platelets in stirred whole blood can be induced to form aggregates and also to form heterotypic platelet-monocyte (P/M) and platelet-neutrophil (P/N) conjugates. Here we have investigated the effects of three GPIIb-IIIa antagonists (GR144053F, MK-852 and Reopro, a CD62P-blocking antibody, GA6, and EDTA on the conjugate formation that occurs on stirring whole blood and in response to adding ADP and PAF. We have confirmed the identities of the conjugates by light microscopy after cell sorting. Platelet aggregation was measured by platelet counting. Monocytes, neutrophils, P/M and P/N were detected and quantitated using immunofluorescence and flow cytometry. Stirring whole blood resulted in both platelet aggregation and formation of P/M but not P/N. Adding ADP or PAF to whole blood caused rapid platelet aggregation and generation of both P/M and P/N. All of the GPIIb-IIIa antagonists studied had similar effects: inhibition of stirring-induced platelet aggregation and P/M formation, and inhibition of ADP-induced platelet aggregation and P/N formation. In contrast, they accelerated ADP induced-P/M conjugate formation and PAF-induced formation of both P/M and P/N. Both EDTA and GA6 completely inhibited P/M and P/N, which is commensurate with CD62P being involved in platelet-leucocyte conjugate formation. The results of these investigations suggest that GPIIb-IIIa has a dual role in determining the interaction between platelets and leukocytes.

In order to identify the factors that control the binding of blood leucocytes to cerebral blood vessels we have modified and applied the frozen section assay of Stamper and Woodruff to the study of human brain. Cryostat sections of brain tissue obtained at post mortem were overlaid with blood lymphocytes and experimental conditions were defined which permitted optimum binding of the cells to transected blood vessel walls. The maximal binding of lymphocytes to cerebral vessels occurred when 6 x 10(6) lymphocytes were overlaid onto brain sections for 30 min at 7 degrees C with gentle agitation. Only a small proportion (0.01%) of the added lymphocytes bound to exposed cerebral vessels. However, lymphocytes were far more adherent than monocytes and polymorphonuclear cells (7-fold and 11-fold respectively: p < 0.001) and activation of lymphocytes with IL-2 enhanced their binding to blood vessel walls (mean 130% increase; p < 0.03). Further analysis revealed that CD4-positive T lymphocytes were the predominant cell population binding to the blood vessels. Antibody blocking studies showed that lymphocyte binding to cerebral blood vessels was inhibited by pretreating the lymphocytes with anti-CD11a, anti-CD18 or anti-CD49d (p < or = 0.02) and immunohistochemical studies revealed the presence of the counter-receptors ICAM-1 (CD54) and VCAM-1 (CD106) for these adhesion molecules in addition to the presence of E-selectin (CD62E) and P-selectin (CD62P) on the cerebral blood vessels. The establishment of a technique in situ which measures selective binding of CD4-positive peripheral lymphocytes to sections of cerebral blood vessels will assist in the molecular characterization of factors that control the interaction of leucocytes with the blood-brain barrier in health and disease.

This study evaluated the expression by seeded endothelial cells (S-EC) of P-selectin (CD62P/GMP-140/PADGEM), an adhesion molecule implicated in endothelial-leukocyte interactions. Endothelial cells were seeded onto knitted polyethylene terephthalate (PET, Dacron(R)) prostheses and compared with control endothelial cells (C-EC) cultured in flasks to the same stage. Using flow cytometry techniques, we observed that CD62P expression by PET S-EC was significantly increased (p<0.05) compared to C-EC. Moreover, RT PCR techniques showed that the CD62P RNA level was higher on S-ECs compared to C-ECs. Following adhesion assays, increased polymorphonuclear neutrophil (PMN) attachment to the PET-seeded prostheses as compared to control cultures (p<0.001) was observed. PMN adherence was enhanced by TNFalpha activation. PMN adhesion was decreased significantly (p<0.001) after the incubation of resting EC or TNFalpha-activated EC-seeded prostheses with a blocking monoclonal antibody (LYP20) directed against the P-selectin. Such results suggest that: (1) PET prosthetic material may induce the expression of P-selectin by S-EC; (2) seeding conditions provoke an increase in PMN adhesion; (3) increased PMN interactions with seeded PET material is partially dependent upon P-selectin expression by the S-EC.

Interaction of endothelial P-selectin with sialyl Lewis(x)-glycoprotein or P-selectin glycoprotein ligand (PSGL)-1 on leukocytes represents an early step in leukocyte recruitment. Redistribution of P-selectin to the endothelial cell surface occurs rapidly after challenge with several proinflammatory agents, for example, histamine, leucopterins, or lipopolysaccharide. We present evidence that prostaglandin E2 (PGE2) is an efficient inductor of surface P-selectin on cultured human umbilical vein endothelial cells (HUVEC). The increase in P-selectin-immunoreactivity coincided with redistribution of cytoplasmic P-selectin-reactive granulae to the endothelial cell surface, as visualized by confocal laser microscopic examination. CD4-T-cell adhesion to PGE2-stimulated HUVEC was also enhanced by a factor of 4, and blocking mAb directed against the binding site of P-selectin almost completely abrogated this increase in CD4-T-cell adhesion. In summary, our findings show that liberation of PGE2 is an important inductor of P-selectin surface expression on endothelial cells, resulting in enhanced recruitment of inflammatory cells.

OBJECTIVE

Type 1 diabetes is associated with increased platelet reactivity. We investigated whether α-lipoic acid (ALA) has any effect on platelet reactivity in these patients.

METHODS

We randomly assigned 51 type 1 diabetic patients to ALA (600 mg once daily) or placebo for 5 weeks. Platelet reactivity was evaluated by the PFA-100 method and by measuring CD41 and CD62 platelet expression. C-reactive protein (CRP) and 8-iso-prostaglandin F2α serum levels also were measured.

RESULTS

Baseline variables were similar in the two groups. After treatment, closure time was longer (P = 0.006) and CD62P platelet expression was lower, both before (P = 0.002) and after (P = 0.009) ADP stimulation in the ALA group compared with the placebo group. CRP and 8-iso-prostaglandin F2α levels showed no differences between the two groups.

CONCLUSIONS

Our data show that ALA reduces measures of platelet reactivity ex vivo in type 1 diabetic patients, independently of antioxidant or anti-inflammatory effects.

Low-grade inflammation, endothelial dysfunction, and platelet hyper-reactivity to agonists are associated with an increased risk of cardiovascular events. In vitro and animal studies infer an inverse mechanistic relationship between platelet activation and the production of endothelium-derived nitric oxide and prostacyclin. This concept is supported by evidence of an inverse relationship between endothelial function and platelet activation in high-risk cardiac patients. The aim of this study was to investigate what relationship, if any, exists between platelet and endothelial function in healthy, middle-aged, and elderly adults. In 51 participants (18 male, 33 post menopausal female), endothelial function was assessed by flow-mediated dilation (FMD). Platelet function was assessed by flow cytometric determination of glycoprotein IIb/IIIa activation (measured by PAC-1 binding), granule exocytosis (measured by surface P-selectin expression), and monocyte-platelet aggregates (MPAs), with and without stimulation by canonical platelet agonists adenosine diphosphate (ADP), arachidonic acid (AA), and collagen. Correlation analysis indicated there was no significant (all P >> 0.05) relationship between FMD and any marker of in vivo platelet activation (MPAs R = 0.193, PAC-1 R = -0.113, anti-CD62P R = -0.078) or inducible platelet activation by ADP (MPA R = -0.128, anti-CD62P R = -0.237), AA (MPA R = -0.122, PAC-1 R = -0.045, anti-CD62P R = -0.142), or collagen (MPA R = 0.136, PAC-1 R = 0.174, anti-CD62P R = -0.077). Our findings contrast with two previous studies performed in high-risk cardiac patients, which reported inverse relationships between platelet activation and endothelial function, suggesting that some compensatory redundancy may exist in the relationship between platelet and endothelial function in preclinical populations.

Platelet function is influenced by changes in membrane fluidity that has an important role in the expression of platelet receptors and in modulating the activity of proteins like phospholipase C or proteinkinase C. In freshly prepared platelets, membrane fluidity modifies the aggregation/agglutination function. Reactive oxygen species (ROS) represent another important parameter involved in platelet receptor activation. There is a certain association of high levels of ROS and iron overload. Patients with hemochromatosis have low platelet aggregation induced by thrombin; little is known about the anemia and effects of iron overload on platelet activation in myelodysplastic syndromes (MDS) patients. Study of platelet membrane fluidity and ROS production changes in patients with MDS and possible correlations with altered platelet function as reflected in aggregation curves and platelet receptor expression. To find out possible correlations of fluidity of platelet membrane and ROS level with hematologic parameters and iron levels. The prospective study included 34 patients with myelodysplastic syndromes classified according to French-American-British cooperative group proposals and 29 healthy volunteers. Platelet membrane fluidity was quantified by fluorescence anisotropy measurements using the marker 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate. ROS production was evaluated by fluorescence measurements using 2',7'-dichlorodihydrofluorescein diacetate. Platelet function was analyzed by optical aggregometry using the agonists adenosine diphosphate, collagen, ristocetin and epinephrine. The expression of platelet receptors CD41/CD61, CD42a/CD42b and CD62P/CD63 was evaluated by flow cytometry. Platelet membrane fluidity in patients with MDS was similar to that of healthy volunteers and did not vary according to the risk category. Patients with MDS had increased platelet ROS production compared with the control group without statistical correlation with membrane fluidity. We found a negative correlation of ROS levels with the severity of anemia (R = -0.587, P = 0.017). Platelet response was reduced in patients with MDS compared with volunteers, for all reagents. The response was different according to the risk category only in case of ristocetin or collagen. Patients with anemia presented a decreased platelet aggregation induced by collagen or ristocetin (collagen: R = 0.395, P = 0.003; ristocetin: R = 0.420, P = 0.002). The membrane fluidity of platelets from MDS patients appeared unmodified, but the ROS production was increased in all risk categories of MDS. The levels of ROS were correlated with the degree of anemia, which, in turn, had a negative impact on the platelet aggregation function induced by collagen or ristocetin.

Human immunodeficiency deficiency virus (HIV) infection is associated with chronic inflammation and an increased risk of thrombotic events. Activated platelets (PLTs) play an important role in both thrombosis and inflammation, and HIV has been shown to induce PLT activation by both direct and indirect mechanisms. P-selectin (CD62P) is a well-described marker of PLT activation, and PLT glycoprotein (GP) IV (CD36) has been identified as a marker of PLT aggregation. Data on PLT function in the context of HIV infection remain inconclusive. Laboratory techniques, such as flow cytometry, enable the assessment of PLTs in their physiological state and environment, with minimal artifactual in vitro activation and aggregation. In this study, we describe a novel flow cytometry PLT assay, which enabled the measurement of PLT function in HIV infection. Forty-one antiretroviral-naïve HIV-positive individuals and 41 HIV-negative controls were recruited from a clinic in the Western Cape. Platelet function was evaluated by assessing the response of platelets to adenosine diphosphate (ADP) at two concentrations (0.04 mM, 0.2 mM). The percentage expression and mean fluorescence intensity (MFI) of CD62P and CD36 was used to evaluate platelet function. These were then correlated with platelet (PLT) count; CD4 count; % CD38/8; viral load and D-dimers. The % CD62P levels were higher in HIV-positive patients (HIV % CD62P 11.33[5.96-29.36] vs. control 2.48[1.56-6.04]; p < 0.0001). In addition, the HIV group showed higher CD62P MFI levels (HIV CD62P MFI 3.25 ± 7.23 vs. control 2.35 ± 1.31, p = 0.0292). Baseline levels of %CD36 expression were significantly higher in HIV-positive patients (%CD36 12.41[6.31-21.83] vs. control 6.04[1.34-13.15]; p = 0.0091). However, the baseline CD36MFI showed no significant difference between the two groups (HIV CD36 MFI 3.09 ± 0.64 vs. control 2.44 ± 0.11, p = 0.4591). The HIV group showed higher levels of % CD36 expression post stimulation with 0.04 mM ADP 43.32 ± 27.41 vs. control 27.47 ± 12.95; p < 0.0214) and no significant difference at 0.2 mM ADP (HIV % CD36 39.06 ± 17.91 vs. control 44.61 ± 18.76; p = 0.3277). Furthermore, the HIV group showed a single phase response to ADP as compared to the control group, which showed a normal biphasic response. We concluded that PLT flow cytometry is valuable in the assessment of levels of PLT activation, and further, that the addition of an endogenous agonist, such as ADP, enabled the measurement of PLT function in HIV infection. We were able to show that, although PLTs are significantly activated in HIV compared to uninfected controls, they retain their functional capacity.

BACKGROUND

Adhesion of activated platelets to neutrophils and monocytes has an important role in the regulation of inflammatory processes. This study investigates whether halothane and isoflurane affect binding of activated platelets to leukocytes in human whole blood.

METHODS

Citrated whole blood was incubated for 60 min with either 1 or 2 minimum alveolar concentration (MAC) halothane or isoflurane. After stimulation with adenosine-5-diphosphate (ADP) or the thrombin receptor agonist protein TRAP-6, platelet-leukocyte adhesion and surface expression of CD62P on platelets were evaluated by flow cytometry.

RESULTS

Halothane led to an inhibition of agonist-induced adhesion of activated platelets to neutrophils and monocytes. One MAC halothane reduced the formation of TRAP-6-induced platelet-monocyte conjugates. After exposure to 2 MAC halothane, agonist-induced platelet-monocyte and platelet-neutrophil adhesion were inhibited. Surface expression of CD62P on ADP- and TRAP-6-stimulated platelets were significantly reduced after 1 and 2 MAC halothane. After 2 MAC isoflurane, the authors observed an increase of the percentage of lymphocytes with bound platelets after activation with ADP. The percentage of neutrophils with bound platelets after activation with ADP or TRAP-6 was also increased in this group. Two MAC isoflurane led to an increase of the percentage of platelets expressing CD62P in the unstimulated and TRAP-6 stimulated samples, and of the amount of CD62P epitopes on the surface of platelets in the ADP-stimulated samples.

CONCLUSIONS

This study indicates that halothane inhibits, whereas isoflurane enhances, adhesion of agonist-activated platelets to leukocytes. Interaction of both anesthetics with the expression of CD62P on platelets contribute to theses effects.

BACKGROUND

Evidence has shown that platelets play an important role in the pathogenesis of flap failure. Employing a rat inferior epigastric artery skin flap as a flap reperfusion injury model, we investigated whether platelet activation was involved in the skin flap failure and whether administration of abciximab (ReoPro, chimeric 7E3 Fab) could decrease platelet activation/aggregation and promote flap survival.

METHODS

Normal saline and abciximab (0.06 mg/kg; 0.2 mg/kg; 1 mg/kg) were injected intravenously into skin flaps 30 min before reperfusion and 1 h after reperfusion (each subgroup n = 6). Platelet activation as demonstrated by P-selectin (CD62P) was analyzed by flow cytometry. P-selectin expression on flap vessels was detected by immunohistochemical staining. Platelet aggregation was induced with adenosine diphosphate (ADP). Laser Doppler flowmetry monitored tissue perfusion. The surviving area was evaluated 7 days postoperatively.

RESULTS

CD62P progressively increased after reperfusion. The peak CD62P occurred after reperfusion for 12 h. Immunohistochemical staining showed CD62P significantly deposited on the endothelium after reperfusion. Administration of abciximab (1 mg/kg) effectively improved flap survival rate (P = 0.003), significantly decreased ADP-induced platelet aggregation (P < 0.001), and suppressed CD62P expression on blood platelets (P = 0.002) and its deposition on the flap vessels.

CONCLUSIONS

Abciximab promotion of skin flap survival is due to blocked platelet activation/aggregation and decreased activated-platelet deposition on the vascular endothelium. Thus, administration of a platelet glycoprotein IIb/IIIa receptor antagonist such as abciximab may save the skin flap from reperfusion injury after a long period of ischemia.