OBJECTIVE

Diabetes mellitus increases the risk of cardiovascular disease, which is accompanied by functional and structural changes in the vascular system. Microparticles (MPs) have been described as biological vectors of endothelial dysfunction in other pathologies. However, the molecular mechanisms underlying their formation and signalling are unclear. We investigated the role of MPs derived from streptozotocin (STZ)-induced diabetic rats in endothelial function.

METHODS

Male Wistar rats were injected with STZ to induce diabetes, and MPs isolated from control or STZ-induced diabetic rats were characterized by dot blotting (assessed by CD62P detections), flow cytometry (assessed by annexin V detections) and ELISA. Carotid arteries from rats were incubated with MPs, and expressions of enzymes and endothelium-dependent relaxation were analysed.

RESULTS

The circulating levels of MPs, particularly the levels of platelet-derived microparticles, from diabetic rats were higher than those present in controls. Endothelium-dependent relaxation induced by acetylcholine (ACh) was attenuated in carotid arteries from STZ-induced diabetic rats. Following the incubation of control carotid arteries with MPs isolated from STZ rats, ACh-induced endothelium-dependent relaxation was impaired, but MPs isolated from control rats had no such effect. Furthermore, the effect of MPs was mediated by a decrease in expression of endothelial nitric oxide synthase (eNOS) and the overexpression of caveolin-1.

CONCLUSIONS

Circulating MPs isolated from STZ-induced diabetic rats induce endothelial dysfunction in carotid arteries and regulate protein expressions of eNOS and caveolin-1. These data advance our understanding of the deleterious effects of circulating MPs observed in disorders with diabetic complications.

Platelets contribute to inflammation however, the role of platelet activation during the pathophysiological response to invasive bacterial infection and sepsis is not clear. Herein, we have investigated platelet activation in a mouse model of invasive Streptococcus pyogenes infection at 5, 12, and 18 hours post infection and correlated this to parameters of infection. The platelet population in ex-vivo blood samples showed no increased integrin activation or surface presentation of CD62P, however platelet-neutrophil complex formation and plasma levels of CD62P were increased during bacterial dissemination and the progression of sepsis, indicating that platelet activation had occurred in vivo. Platelet-neutrophil complex formation was the most discriminatory marker of platelet activation. Platelet-neutrophil complexes were increased above baseline levels during early sepsis but decreased to significantly lower levels than baseline during late sepsis. The removal of these complexes from the circulation coincided with a significant increase in organ damage and the accumulation of platelets in the liver sinusoids, suggesting that platelet activation in the circulation precedes accumulation of platelets in damaged organs. The results demonstrate that monitoring platelet activation using complementary methods may provide prognostic information during the pathogenesis of invasive S. pyogenes infection.

BACKGROUND

Neutrophil and mononuclear cell functional changes represent a hallmark of inflammation during cardiopulmonary bypass and cardiovascular surgery. Knowledge of mechanisms underlying monocyte functional modulation in coronary blood may be useful to develop protective interventions that can limit ischemia/reperfusion injury.

METHODS

Samples of 13 patients were drawn from venous coronary sinus before cardioplegic arrest and after reperfusion. The following parameters were studied: surface molecules expression (CD18, CD11b, CD44, CD162, CD15s, CD80, CD86, CD16, CD49d, CD29, CD25, HLA-DR, Toll-like receptor-4 [TLR-4], CXCR1, CCR2, CCR5, CX3CR1), oxidative burst response, monocyte-platelet conjugates (using antibodies against CD45, CD14, CD41a), and platelet activation (CD62P, PAC-1). Enzyme-linked immunosorbent assays were performed to measure levels of interleukin (IL)-1beta, IL-6, IL-8, IL-10, and tumor necrosis factor-alpha (TNF-alpha).

RESULTS

Coronary reperfusion down-modulated monocyte molecules expression, especially for CD18 (P = 0.048), CD44 (P = 0.0035), CD49d (P = 0.0029), CD29 (P = 0.032), HLA-DR (P < 0.0001), TLR-4 (P = 0.0109), CCR2 (P = 0.0184), CCR5 (P = 0.0396), and CX3CR1 (P < 0.0001). A marginal increase (P = 0.062) of a normalized adhesion index between monocytes and platelets was observed at reperfusion. No variations were detected for the monocyte oxidative burst and platelet activation. Increased levels of IL-6 (P = 0.013), TNF-alpha (P = 0.0272), and IL-10 (P = 0.0008) were measured after cardioplegia.

CONCLUSIONS

The lack of CD11b and CD25 variations and of the oxidative burst exclude monocyte activation at reperfusion. The high after-cardioplegia level of IL-10, the decreased expression of HLA-DR and TLR-4, and the absence of IL-1beta and IL-8 suggest an IL-10-mediated functional depression of monocyte, including their adhesive and migratory capacities. The lack of an after-cardioplegia orientation toward IL-10 producing a "macrophage-like" CD14+/CD16+ phenotype might mean that myocardial infiltrating lymphocytes are the main source of IL-10. Moreover, the increased after-cardioplegia levels of IL-6 and TNF-alpha might be due to myocardial and endothelial activations. The increased adhesion index between monocyte and platelets, without receptor variations, suggests a monocyte membrane modification induced by extracorporeal circulation.

BACKGROUND

The preparation of platelet (PLT) concentrates (PCs) from PLT-rich plasma (PRP) requires that whole blood (WB) be processed within 8 hours of collection. Increasing WB storage time to 24 hours would be logistically attractive. This study compares the in vitro quality of blood components prepared from WB stored for 8 and 24 hours at room temperature before processing with the PRP method.

METHODS

WB units were collected from ABO-matched blood donors. To reduce individual variations, paired donations were drawn in parallel, pooled, and split back in the collection bag. One unit was held for 6 to 8 hours and the other for 22 to 24 hours at 20 to 24 degrees C. Prestorage leukoreduced components were prepared with the PRP as intermediate product and analyzed during storage.

RESULTS

RBC units prepared after an 8- or 24-hour hold were comparable in terms of hemolysis, sodium, pH, and ATP levels. RBC 2,3- diphosphoglycerate (2,3-DPG) was significantly lower in RBCs prepared from 24-hour hold donations immediately after processing but not after 20 days of storage. Residual white blood cells were approximately fivefold higher (p < 0.05) in 24-hour RBC units. For PCs, measurements for glucose, ATP, lactate, pH, extent of shape change, hypotonic shock response, and CD62p activation were similar. No differences were observed in the von Willebrand factor, factor (F)V, FVIII, and fibrinogen content of fresh-frozen plasma.

CONCLUSIONS

The decrease in FVIII and RBC 2,3-DPG can be acceptable as a compromise to improve blood component logistics, but leukoreduction efficiency must be improved before considering the adoption of an overnight storage of WB before PRP processing.

We investigated whether platelet activation could be quantitatively detected in patients with coronary artery disease using flow cytometric analysis of the expression of CD62P and CD63, which are activation-specific antigens on the platelet surface. Platelet samples were obtained from 16 healthy control subjects and 65 patients, of whom 25 had angiographically normal coronary arteries and 40 had at least one major coronary artery stenosis >> or = 50% narrowing). In both patient groups, CD62P expression was significantly higher than in the control group, but the difference between the two patient groups was not significant. In contrast, CD63 expression did not differ among the three groups. We also compared expression of these antigens after stratifying the patients according to the number of significant coronary artery stenoses. Patients with three-vessel disease had significantly increased CD62P and CD63 expression compared with the other subgroups. Our findings indicate that platelet activation occurs in patients with severe coronary artery stenosis.

OBJECTIVE

To study patients with coronary artery disease (CAD) scheduled for coronary angioplasty and to examine platelet activation in response to mental stress as a potential mechanism involved in the association between psychosocial factors and cardiac outcomes. Psychosocial factors have been identified as risk factors for CAD and adverse cardiac outcomes, although the underlying mechanisms are poorly understood.

METHODS

Markers of platelet activation and platelet reactivity in response to experimentally induced mental stress (mental arithmetic and anger recall) were examined, using flow cytometry analysis and beta-thromboglobulin (BTG) assays among 249 CAD patients (age = 60.3 +/- 9.0 years, 15% women) who were scheduled to undergo elective percutaneous coronary intervention.

RESULTS

Mental stress-induced increases in platelet activation (CD41 (GP IIb/IIIa), p = .002; percent of mononuclear cells positive for CD41, p = .01; CD62P (P-selectin) expression, p = .005; and percent platelets positive for CD62P, p < .001). The degree of platelet reactivity was not related to demographic, clinical, or psychological variables, or cardiovascular hemodynamic changes.

CONCLUSIONS

Experimentally induced mental stress induced platelet activation in patients with CAD. This mechanism may partially explain the link between psychosocial variables and the development of adverse cardiac outcomes in patients with CAD.

Production and storage of platelet concentrates (PC) induce protein changes in platelets leading to impaired platelet function. This study aimed to identify signaling pathways involved in the development of early platelet storage lesions in apheresis-PCs stored in plasma or additive solution (PAS). Apheresis-PCs from four donors were stored in plasma or in PAS at 22°C (n=4 each). Platelets were analyzed at day 0 (production day) and after 1, 6 and 9 days of storage. Platelet response to agonists (TRAP, collagen, ADP) and to hypotonic shock decreased, CD62P expression increased in both storage media over time. Using DIGE 1550 protein spots were monitored and compared to baseline values at day 0. Platelets in plasma displayed changes in 352 spots (166/day 1, 263/day 6 and 201/day 9); in PAS 325 spots changed (202/day 1, 221/day 6, 200/day 9). LC-ESI-MS/MS analysis of 405 platelet proteins revealed 32 proteins changed during storage in plasma (9/day 1, 15/day 6 and 26/day 9) and 28 in PAS (5/day 1, 20/day 6, 26/day 9). Ingenuity pathway analysis found integrin-αII(b)β(3) and focal adhesion signaling pathways involved in early alterations, being confirmed by Western blotting. Corresponding mRNAs in platelets were identified by next generation sequencing for 84 changed proteins. Integrin-αII(b)β(3) and focal adhesion signaling cause irreversible early storage lesions in apheresis platelets. This article is part of a Special Issue entitled: Integrated omics.

This study evaluates transcoronary changes in neutrophil and platelet activation and conjugate formation in patients with angina pectoris secondary to coronary artery disease. We examined parameters of neutrophil and platelet activation as well as the neutrophil-platelet conjugate formation in patients who underwent diagnostic coronary angiography. Thirty-nine patients with chest pain referred for cardiac catheterization were studied (23 patients with unstable angina pectoris [UAP] and 16 with stable angina pectoris [SAP]). Before coronary angiography, blood samples were obtained simultaneously from the aortic root and coronary sinus to assess leukocyte (CD11b) and platelet (CD62P) activation and leukocyte-platelet conjugates. There was a 94% increase in CD62-expressing platelets from the aorta to the coronary sinus in patients with UAP compared with a 49% increase in patients with SAP. The percentage of neutrophil-platelet conjugates increased by 22% in patients with UAP compared with a 16% decrease in those with SAP (p <0.01). In contrast, monocyte-platelet binding across the coronary bed increased to a similar degree in both groups. This study demonstrates an increase in neutrophil-platelet conjugates across the coronary circulation in UAP, compatible with a higher activation state in both cell types.

Two ADP receptors have been identified on human platelets: P2Y(1) and P2Y(12). The P2Y(12) receptor blocker clopidogrel is widely used to reduce the risks in acute coronary syndromes, but, currently, there is no P2Y(1) blocker in clinical use. Evidence for variable responses to clopidogrel has been described in several reports. The mechanistic explanation for this phenomenon is not fully understood. The aim of this study was to examine mechanisms responsible for variability of 2MeS-ADP, a stable ADP analogue, induced platelet reactivity in clopidogrel-treated patients. Platelet reactivity was assessed by flow cytometry measurements of P-selectin (CD62P) and activated GpIIb/IIIa complex (PAC-1). Residual 2MeS-ADP activation via the P2Y(12) and P2Y(1) receptors was determined by co-incubation with the selective antagonists AR-C69931 and MRS2179 in vitro. P2Y(1) and P2Y(12) receptor expression on both RNA and protein level were determined, as well as the P2Y(12) H1 or H2 haplotypes. Our data suggest that the residual platelet activation of 2MeS-ADP after clopidogrel treatment is partly due to an inadequate antagonistic effect of clopidogrel on the P2Y(12) receptor and partly due to activation of the P2Y(1) receptor, which is unaffected by clopidogrel. Moreover, a correlation between increased P2Y(12) protein expression on platelets and decreased response to clopidogrel was noticed, r(2)=0.43 (P<0.05). No correlation was found between P2Y(12) mRNA levels and clopidogrel resistance, indicating post-transcriptional mechanisms. To achieve additional ADP inhibition in platelets, antagonists directed at the P2Y(1) receptor could be more promising than the development of more potent P2Y(12) receptor antagonists.

Platelet-derived microparticles, activated platelets, and monocyte-derived microparticles were measured in 73 patients with diabetes mellitus. A comparative study of these parameters was performed before and after administration of ticlopidine. The number of platelet-derived microparticles and activated platelets was increased significantly in diabetic patients. Monocyte-derived microparticles were also increased significantly. After administration of ticlopidine, platelet-derived microparticles and activated platelets corrected positively, not only CD62P- and CD63-positive platelets, but also platelet-derived microparticles and monocyte-derived microparticles showed a significant decrease. These data suggest that in patients with diabetes, platelet-derived microparticles and activated platelets stimulate the activation of monocytes and promote the production of monocyte-derived microparticles, and that ticlopidine is useful for hypercoagulabillity in diabetic patients.

P-selectin (CD62P), expressed on stimulated endothelial cells and activated platelets, reacts with P-selectin glycoprotein ligand-1 (PSGL-1, CD162) for leukocyte rolling. It also binds to heparin and heparan sulfate proteoglycans (HSPGs), which attenuates P-selectin mediated adhesions of leukocytes and cancer cells. Here we report that P-selectin mediated adhesion, but not rolling, of the HSPGs bearing human malignant melanoma A375 cells under shear stress. To understand its underlying molecular mechanism, we measured the biophysical properties of this interaction. Heparin inhibited the adhesion of A375 cells to immobilized P-selectin under flow (IC(50) = 3 microM heparin) and neutralized the binding of P-selectin to A375 cells (IC50 = 4 microM heparin). Using surface plasmon resonance technique, we found that P-selectin bound to heparin with a dissociation constant (K(d)) of 115 +/- 6 nM. The measured off rate (k(off)) was 3.15 +/- 0.34 x 10(-3) s(-1) and the calculated on rate (k(on)) was 2.75 x 10(4) M(-1) s(-1). Taken together, our data suggest that the very slow k(off) and the reduced k(on), but apparently not the K(d), are responsible for adhesion, but not rolling of A375 cells, to P-selectin under flow.

BACKGROUND

Proinflammatory cytokines and platelets play a key role in the systemic inflammatory response associated with cardiopulmonary bypass (CPB). The aim of this study was to evaluate the effects of both hypothermic and normothermic CPB on platelet activation, cytokine production, as well as their possible correlations.

METHODS

Twenty patients who underwent CABG were randomly assigned into two groups receiving hypothermic and normothermic CPB. Blood samples were obtained through a venous catheter at 6 time points. The following parameters were measured: in vitro platelet aggregation, in vivo platelet activation, complete and differential blood cell counts, plasma soluble P-selectin levels, plasma IL-6, IL-1beta and TNFalpha levels.

RESULTS

The results demonstrated that platelet abnormalities could be observed to a greater extent during hypothermic rather than normothermic CPB. The occurrence of in vivo platelet activation was suggested by the presence of a significantly increased percentage of platelets expressing CD62P on their surface, as well as by a decreased in vitro platelet aggregation induced by different agonists. Complete and differential blood cell counts showed no substantial decrease in platelet number without differences between groups. The results obtained also showed the presence of a significant release of sP-selectin during CPB, as well as a more pronounced increase of plasma sP-selectin levels in patients undergoing hypothermic compared to normothermic CPB. A comparison of cytokine levels demonstrated a significant elevation of plasma IL-6 levels during either hypothermic or normothenmic CPB, paralleling the neutrophil rise, while no differences were observed for TNF-alpha levels. Conversely, plasma IL-1beta levels were significantly elevated during hypothermic, but not during normothermic CPB.

CONCLUSIONS

Hypothermic CPB is responsible for a greater platelet activation and endothelial dysfunction than normothermic CPB, leading to more profound changes in the hemostatic and inflammatory systems, which, in turn, might be responsible for the higher incidence of postoperative complications reported during hypothermic CPB.

The nanoparticle (NP) response of platelets is shown to be critically dependent on extent of preactivation of platelets by an agonist like ADP. A transition from de-aggregatory to aggregatory state is triggered in the presence of gold NPs (AuNP) only in such critical conditions. Adhered and suspended platelets respond differentially to NPs. Preactivation in the adhered state induced by shear force explains such observation. The NP effect is associated with enhanced release reaction, tyrosine phosphorylation and CD62P expression level. Unlike cancer cells, whose response is maximal when NP size is optimal (within the range 50 - 70 nm), the platelet response monotonically increases with reduction of the AuNP size. The uptake study, using quenching of quinacrine hydrochloride fluorescence by AuNP, indicates that accumulation 18 nm AuNP is several-fold higher than the 68 nm AuNP. It is further shown that AuNP response can provide a simple measure for thrombotic risk associated with nano-drugs.

UNASSIGNED

Platelet aggregation can be triggered in the presence of gold nanoparticles (AuNP). Platelet response monotonically increases with reduction of the AuNP size. AuNP response can provide a simple measure for thrombotic risk associated with nano-drugs.

BACKGROUND

Cardiovascular disease (CVD) is the leading cause of death in patients with end-stage renal disease (ESRD). Platelet (PLT) dysfunction, which is a well-known phenomenon in advanced chronic renal failure, corresponds positively with CVD in these patients. The accumulation of retained uraemic toxins might play an important role in this respect. During haemodialysis (HD), both an increase in the expression of the platelet (PLT) cell surface molecule P-selectin (CD62p) and the release of intra-granular substances, such as platelet factor 4 (PF4) and ss-thromboglobulin (BTG), have been described. As the removal of uraemic toxins is superior during haemodiafiltration (HDF), this form of treatment may have quite another impact on PLTs than HD.

METHODS

Nineteen chronic HD patients who were treated with low-flux HD for at least 2 months were included in the Dutch CONvective TRAnsport STudy (CONTRAST). After randomization, 10 patients continued low-flux HD and 9 patients switched to post-dilution HDF. The present study describes various parameters of PLT activation and degranulation at baseline (during HD) and after 3 months (during HDF) in the latter group of patients. At both time points, multiple blood samples were drawn. During the first 30 min of treatment, differences over the extracorporeal circuit (ECC) were calculated by taking samples from both afferent (arterial) and efferent (venous) lines. Correlations between various parameters were calculated in the total group of patients after 3 months.

RESULTS

Immediately after the start of HD, PLT counts dropped over the ECC. During HDF, PLT counts decreased even more and reached a nadir at t30. CD62p expression increased early during HD and returned to baseline thereafter. During HDF, these changes were more pronounced and more protracted. With respect to degranulation, rather dissimilar results were obtained. During HD, both PF4 and BTG increased over time, whereas during HDF, PF4 increased but BTG did not change. Haemoconcentration and transmembrane pressure (TMP) within the dialyser were, respectively, approximately 10 and 3x higher during HDF than during HD. There was a striking correlation between the changes in haemoconcentration and the changes in both PLT counts and CD62p over the ECC.

CONCLUSIONS

PLT activation, as measured by the expression of CD62p, was more pronounced and more protracted during HDF than during HD. During HDF, PLTs were trapped abundantly within the ECC, not only after first passage, but also thereafter. The degranulation product BTG increased during HD, but did not change during HDF. These observations may well be explained by the greater haemoconcentration and/or higher TMP during HDF on the one hand, and superior convective transport at the other. Whether the potential harmful effects of enhanced PLT activation are counterbalanced by the beneficial effects of an increased convective transport of degranulation products remains to be established.

Niacin is a natural pyridine derivative, proven to favorably modulate the blood lipid profile by increasing levels of high-density lipoprotein (HDL) cholesterol, and by reducing total cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, and Lp (a) lipoprotein concentrations. Considering that platelet activity is important in predicting vascular outcomes, and that HDL heavily constitutes platelet cellular membranes, we sought to evaluate the effect of niacin on human platelet activity indices. The blood obtained from 30 aspirin-naïve volunteers was preincubated with escalating concentrations of niacin in vitro. Platelet tests included whole blood and plasma aggregometry, rapid cartridge-based analyser, expression of major surface receptors by flow cytometry, and plasma prostaglandins by ELISA. Preincubation of blood with niacin at 0.3, 1.0 and 3.0 mM resulted in significant inhibition of maximal adenosine diphosphate (ADP)- (p=0.03), and collagen-induced platelet aggregation (p=0.01), and reduced activity by VerifyNow (p=0.007) bedside analyser. Surface platelet PAR-1 (MoAb WEDE-15; p=0.04), and vitronectin (CD51/CD61; p=0.02) receptors were up-regulated. Niacin was associated with a two- to three-fold increase of thromboxane B2, prostaglandins D2, and E2. Formation of platelet-monocyte microparticles (CD14+CD151), and expression of PECAM-1 (CD31), thrombospondin (CD36), GP IIb/IIIa (CD41a) antigen, and activity with MoAb PAC-1, GPIb (CD42b), P-selectin (CD62p), LAMP-3 (CD63), LAMP-1 (CD107a), CD40-ligand (CD154), GP37 (CD165), were not affected by niacin, suggesting no effect on prostacyclin release. In conclusion, niacin in vitro affects platelet activity by mildly inhibiting aggregation, and stimulating significant prostaglandin release, with mostly intact major platelet receptor expression. The effect of niacin is unique, differs from other known antiplatelet agents, and suggests potential opportunities for therapeutic combination, particularly in patients with low levels of HDL-C. These preliminary data, while intriguing, require confirmation in subjects receiving orally dosed extended-release niacin in order to determine whether these findings are clinically relevant.

OBJECTIVE

Abciximab, a nonselective glycoprotein IIb/IIIa inhibitor, was shown to reduce peri-interventional stroke rate in carotid stenting. We evaluated the effect of adjunct abciximab therapy on monocyte-platelet cross talk and neurological deficit in unprotected carotid stenting and compared its efficacy with distal filter protection.

METHODS

Fifty patients were randomized to either standard antithrombotic therapy (n=30) consisting of aspirin, clopidogrel, and heparin or adjunct bolus (0.25 mg/kg) and 12-hour infusion (0.125 microg x kg(-1) x min(-1)) of abciximab (n=20). A third cohort of patients was stented with filter protection (n=30). Monocyte-platelet aggregate formation and monocyte tissue factor expression were determined by whole blood flow cytometry, and F1.2 generation and soluble CD40 ligand (sCD40L) were determined by immunoassay.

RESULTS

The incidence of peri-interventional ischemic episodes (23% versus 10%; P=0.2) and the number of de novo ischemic lesions detected by diffusion-weighted MRI (47% versus 30%; P=0.17) were not significantly different between standard antithrombotic therapy and adjunct abciximab but were reduced with filter protection (P=0.023). However, the number of transient ischemic attacks was lower (P=0.05) and the National Institutes of Health Stroke Score rapidly decreased in patients with adjunct abciximab. This clinical improvement was paralleled by a reduction in the postinterventional percentage of activated monocyte-platelet aggregates (CD62P+/CD14+; P=0.018) and the number of tissue factor-positive monocytes (TF+/CD14+; P=0.005). Both abciximab and filter protection suppressed F1.2 generation and significantly reduced sCD40L.

CONCLUSIONS

Abciximab limits thrombus propagation and thrombus stabilization after carotid stenting by reducing monocyte-platelet cross talk and sCD40L. Although abciximab seems inferior to filter devices in peri-interventional cerebral protection, it may be considered in patients who do not allow placement of protection devices.

The platelet-rich fibrin-like matrix (PRFM) is usually prepared onsite and immediately used for regenerative therapy. Nonetheless, to meet the clinical necessity of preserving the PRFM without quality deterioration, we developed a method for preparation of PRFMs from short-term-stored whole blood (WB) samples. In this study, to evaluate the practical expiration date of storage, we extended the storage time of WB samples from 2 to 7 days and assessed the quality of the resulting PRFMs. WB samples collected with acid-citrate-dextrose were stored with gentle agitation at ambient temperature. To prepare PRFMs, the stored WB samples were mixed with CaCl₂ in glass tubes and centrifuged. Fibrin fiber networks, CD41 and CD62P expression, and Platelet Derived Growth Factor-BB (PDGF-BB) levels were examined by scanning electron microscopy (SEM), flow cytometry, and an Enzyme-Linked ImmunoSorbent Assay (ELISA), respectively. Long-term storage had no significant effect on either blood cell counts or platelet functions tested. The resulting PRFMs were visually identical to freshly prepared ones. PDGF-BB levels did not markedly decrease in a time-dependent manner. However, fibrin fibers gradually became thinner after storage. Although the coagulation activity may diminish, we propose that PRFMs can be prepared-without evident loss of quality-from WB samples stored for up to 7 days by our previously developed method.

Hydrophilic and hydrophobic titanium and glass were exposed to capillary whole blood between 5s and 24h. The time-sequence for adsorption of thrombin, kallikrein and complement C5b-9, and their relationship with adherent platelets and polymorphonuclear granulocyte (PMN) activation were investigated. Adsorbed thrombin and kallikrein were measured by cleavage of specific chromogenic substances, S-2238 and S-2303, respectively. Complement C5b-9 and expression of CD11b, CD66b, CD62P and Pan-platelets were measured by immunofluorescence. Thrombin and kallikrein were present on the surfaces during the whole investigated periods. Platelet adhesion and PMN cell adhesion and activation on all surfaces and activation of platelets on hydrophobic surfaces showed a similar pattern to thrombin adsorption. Kallikrein adsorption had a different pattern on each surface. C5b-9 was detected between 32min and 24h of blood exposure and a varying pattern of C5b-9 coverage was observed on each surface. In conclusion, our results indicate that the interaction between material and blood coagulation and kinin-activating proteins regulate the adhesion and activation of blood cells, whereas after longer time the coagulation and kallikrein-kinin system play minor roles and the complement system is decisive for mediating and elongating the inflammatory process.

Platelet activation and adhesion to endothelial cells and extracellular matrix proteins are crucial events in the development of arterial cardiovascular diseases. Platelet activation is initiated by stimulation of intracellular signaling cascades, including the p42 mitogen-activated protein kinase (MAPK) and p38 MAPK pathways, followed by major changes in the platelet cytoskeleton and expression and activation of platelet surface receptors, such as P-selectin (CD62P) and CD40 ligand (CD40L). Activated platelets directly bind to vascular endothelial cells via CD40L/CD40 interactions and induce inflammatory reactions that initiate or aggravate atherosclerotic lesions. The aim of this study was to investigate effects of two known platelet inhibitors-the cAMP-elevating prostaglandin E(1) (PG-E(1)) and the cGMP-elevating sodium nitroprusside (SNP)-on platelet p42 MAPK and p38 MAPK activation as well as on surface expression of CD62P and CD40L. MAPK activation was analyzed by Western blot experiments using phosphorylation-specific antibodies, and surface CD40L and CD62P expression was determined by flow cytometry analysis. PG-E(1) and SNP strongly inhibited p42 and p38 MAPK phosphorylation as well as CD40L and CD62P expression in response to thrombin, a thromboxane A(2) analog, and ADP. These data indicate that adenosine and guanosine 3',5'-cyclic monophosphate-dependent protein kinases not only inhibit platelet pathways leading to activation and aggregation, but also those resulting in enhanced surface expression of protein ligands involved in inflammation. Expression of CD40L and CD62P was found to be independent of MAPK activation, since it was not inhibited by specific MAPK inhibitors. Inhibition of platelet-induced inflammatory responses including CD62P- and CD40L-mediated interaction of platelets with leukocytes and endothelial cells, respectively, is suggested to be an important component of the long-term vasoprotective effects of cyclic nucleotide-elevating prostaglandins and NO donors.

Recent work has indicated that platelets, which are anucleate blood cells, significantly contribute to inflammatory disorders. Importantly, platelets also likely contribute to various inflammatory secondary disorders that are increasingly associated with Human Immunodeficiency Virus Type-1 (HIV) infection including neurological impairments and cardiovascular complications. Indeed, HIV infection is often associated with increased levels of platelet activators. Additionally, cocaine, a drug commonly abused by HIV-infected individuals, leads to increased platelet activation in humans. Considering that orchestrated signaling mechanisms are essential for platelet activation, and that nuclear factor-kappa B (NF-κB) inhibitors can alter platelet function, the role of NF-κB signaling in platelet activation during HIV infection warrants further investigation. Here we tested the hypothesis that inhibitory kappa B kinase complex (IKK) activation would be central for platelet activation induced by HIV and cocaine. Whole blood from HIV-positive and HIV-negative individuals, with or without cocaine abuse was used to assess platelet activation via flow cytometry whereas IKK activation was analyzed by performing immunoblotting and in vitro kinase assays. We demonstrate that increased platelet activation in HIV patients, as measured by CD62P expression, is not altered with reported cocaine use. Furthermore, cocaine and HIV do not activate platelets in whole blood when treated ex vivo. Finally, HIV-induced platelet activation does not involve the NF-κB signaling intermediate, IKKβ. Platelet activation in HIV patients is not altered with cocaine abuse. These results support the notion that non-IKK targeting approaches will be better suited for the treatment of HIV-associated inflammatory disorders.

The clinical courses of polycythemia vera (PV) and essential thrombocythemia (ET) are characterized by thrombohemorrhagic diathesis. Several groups have suggested an association between JAK2V617F mutation and thrombosis. We hypothesized a relationship between JAK2V617F allele burden, cellular activation parameters, and thrombosis. We evaluated a group of PV and ET patients using flow cytometry: platelet CD62P, CD63, and dense granules, platelet-leukocyte aggregates (PLA), leukocyte CD11b and monocyte tissue factor (TF) expression. All patients had increased baseline platelet CD62P and CD63 expression (p < 0.05); 71 % of PV and 47 % of ET presented with a storage pool disease. Leukocyte CD11b, TF, and PLA were elevated in all patients. TF was higher in PV compared to ET (p < 0.05) and platelet-neutrophil [polymorphonuclear (PMN)] aggregates were increased in ET versus PV (p < 0.05). In ET, PLA were correlated with platelet numbers (p < 0.05). In all patients, JAK2V617F allele burden was directly correlated with monocyte CD11b. Patients with JAK2V617F allele burden >50 % presented higher levels of leukocyte activation. In ET, thrombosis was associated with JAK2V617F mutation (p < 0.05, χ (2) = 5.2), increased monocyte CD11b (p < 0.05) and with platelet-PMN aggregates (p < 0.05). In ET patients, hydroxyurea does not significantly reduce the activation parameters. Our data demonstrate that JAK2V617F allele burden is directly correlated with activation parameters that drive mechanisms that favor thrombosis.

ABSTRACT-We examined the mechanisms and the adhesive molecules mediating platelet-neutrophil adhesion in patients with septic shock. Neutrophils, platelets, and platelet poor plasma (NPPP) were isolated from 12 normal volunteers. Platelets and neutrophils were stimulated with platelet poor plasma (SPPP) removed from 12 patients in septic shock. Cell adhesion was assessed by filtration through 5-microm pore filters and by flow cytometry. Blocking monoclonal antibodies were used against the platelet and neutrophil surface receptors glycoprotein complex IIb/IIla, P-selectin, ICAM-2, CD11a, CD11b, and CD18. The filtration pressure (Pi) of cells suspended in SPPP was significantly greater than that of cells suspended in NPPP (24 +/- 1.0 mmHg vs. 14 +/- 1.0 mmHg; P< 0.05). The difference between the Pi of cells suspended in SPPP or NPPP (deltaPi SPPP-NPPP) in the presence of monoclonal antibodies anti-CD41, anti-CD62P, abciximab, anti-CD11a, anti-CD11b, and anti-CD18 was significantly less than the APi SPPP-NPPP of cell suspensions without the addition of these monoclonal antibodies (P < 0.01). The greatest reduction in Pi occurred when platelet receptor P-selectin was blocked simultaneously with the CD11b receptor on the neutrophil as compared to all other single blocking monoclonal antibodies or combinations of monoclonal antibodies. The mean fluorescence of activated platelet CD63-PE binding to neutrophils suspended in SPPP was significantly greater than that of cells suspended in NPPP (780 +/- 130 Ifu vs. 295 +/- 35 Ifu; P < 0.05). The greatest attenuation in mean fluorescence occurred by blocking the P-selectin receptor on the platelet simultaneously with CD11b receptor on the neutrophil. We conclude that platelet-neutrophil aggregation is increased in septic shock. This aggregation is mediated by the interaction of multiple platelet and neutrophil surface receptors. The platelet receptor P-selectin and the neutrophil receptor CD11b/CD18 appear to play the most important role in these interactions.

BACKGROUND

Glutamate is stored in platelet dense granules and large amounts (>400 μM) are released during thrombus formation. N-methyl-d-aspartate glutamate receptors (NMDARs) have been shown in platelets but their roles are unclear.

METHODS

Platelet activation indices (CD62P expression and PAC-1 binding) and platelet aggregation were tested in the presence of well-characterized agonists (glutamate, NMDA, glycine) and antagonists (MK-801, memantine, AP5) of neuronal NMDARs. Expression of NMDAR subunits in platelets was determined.

RESULTS

NMDAR agonists facilitated and NMDAR antagonists inhibited platelet activation and aggregation. Low concentrations (100 μM) of MK-801 and memantine reduced adrenaline-induced CD62P expression by 47 ± 5 and 42 ± 3%, respectively, and inhibited adrenaline-induced platelet aggregation by 17 ± 6 and 25 ± 5%, respectively (P<0.05). AP5 caused less inhibition of platelet function, requiring concentrations of at least 250 μM to inhibit aggregation. NMDAR agonists did not aggregate platelets by themselves but enhanced aggregation initiated by low concentrations of ADP. Exogenous glutamate helped reverse inhibition of platelet aggregation by riluzole (inhibitor of glutamate release). Compared with seven possible NMDAR subunits in neurons, human platelets contained four: GluN1, GluN2A, GluN2D and GluN3A, a combination rarely seen in neurons. The presence of NMDAR transcripts in platelets implied platelet ability to regulate NMDAR expression presumably 'on demand'. Flow cytometry and electron microscopy demonstrated that in non-activated platelets, NMDAR subunits were contained inside platelets but relocated onto platelet blebs, filopodia and microparticles after platelet activation.

CONCLUSIONS

Our results support an active role for NMDARs in platelets, in a process that involves activation-dependent receptor relocation towards the platelet surface.

BACKGROUND

Impaired maternal immune tolerance resulting in systemic inflammation plays a pivotal role in the pathogenesis of preeclampsia. Phenotypical changes of monocytes and neutrophil granulocytes have already been studied in preeclampsia, and some studies also included T lymphocyte activation markers; however, the results are controversial and a comprehensive analysis of activation markers is lacking. The characteristics of cellular adhesion molecules in preeclampsia are yet to be described.

METHODS

Peripheral blood samples of 18 preeclamptic patients and 20 healthy pregnant women in the third trimester were evaluated using flow cytometry to characterize the cell surface expression of T lymphocyte activation markers and selectins.

RESULTS

We found an elevated ratio of HLA-DR and CD122-, CD62E-, and CD62L-expressing cells among the CD4+ T lymphocytes in PE in comparison to healthy pregnancy. No alterations were found in the prevalence of CD69-, CD25-, and CD62P-expressing lymphocytes and CD11c-expressing monocytes.

CONCLUSIONS

Our findings support the role of activated T lymphocytes and specific cell adhesion molecules in the pathogenesis of preeclampsia.