BACKGROUND

In patients with chronic hypercholesterolemia, the CD40-CD40L dyad is upregulated, contributing to the initiation and progression of atherosclerosis. Our aim was to describe the role of postprandial lipemia and inflammatory stimulation on platelet and monocyte activation and CD40-ligand (CD40L) levels.

RESULTS

Before and 2 h after consumption of a defined fatty meal, whole blood samples of 31 healthy subjects were incubated with endotoxin (LPS). CD40-ligand and CD62P expression on platelets, tissue-factor expression on monocytes and platelet-monocyte aggregates were measured with flow cytometry. Soluble CD40-ligand plasma levels were measured with an ELISA. After the meal, serum triglyceride levels increased from 137.6+/-60.5 mg/dl to 201.5+/-75.0 mg/dl. Expression of CD40L and CD62P on platelets and plasma levels of soluble CD40L were significantly decreased. No significant changes after the meal were observed concerning tissue factor expression on monocytes and platelet-monocyte aggregates. Addition of LPS showed no significant effect concerning CD40L or CD62P expression on platelets, whereas the amount of platelet-monocyte aggregates significantly increased under LPS stimulation after the fatty meal.

CONCLUSIONS

Acute alimenatry lipemia leads to a decreased expression of CD40L on platelets and a reduced plasma level of sCD40L, suggesting an increased turnover in the CD40L system.

CONCLUSIONS

Before and after a fatty meal, blood samples of 31 healthy subjects were incubated with LPS. After the meal, expression of CD40L and CD62P on platelets and plasma levels of soluble CD40L were significantly decreased. Addition of LPS showed no effect concerning CD40L or CD62P expression, whereas the amount of platelet-monocyte aggregates significantly increased under LPS stimulation after the fatty meal.

OBJECTIVE

The INTERCEPT Blood System for Platelets utilizes amotosalen-HCl (S-59) in combination with ultraviolet A (UVA) light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet concentrates (PCs). To facilitate implementation of this technique into routine blood bank manufacturing procedures, this study evaluated the impact of different time settings of photochemical treatment on in vitro platelet function.

METHODS

Platelets derived from apheresis (6.5-7.0 x 10(11) platelets) were resuspended in 240 ml of autologous plasma and 360 ml of platelet additive solution (PAS III) and split into two equal-sized PC units. Whereas one unit was not treated, the other was treated with 150 microm amotosalen and 3 J/cm2 UVA light followed by a compound adsorption device (CAD) step for reduction of residual amotosalen and photoproducts. In a first series of experiments (arm A, n = 7), PC units were photochemically treated after an overnight storage period of 16-23 h followed by a CAD step of 4 h. In a second series (arm B, n = 8), photochemical treatment occurred after a short storage time of 4 h with a subsequent CAD step of 16 h. Platelet function was evaluated by assaying blood gas analysis, glucose and lactate concentration, lactate dehydrogenase (LDH), hypotonic shock response (HSR) and the expression of CD62p, over a period of 7 days.

RESULTS

Neither of the photochemical treatment procedures showed differences for pH, pCO2, pO2, HCO3, glucose consumption or platelet activation until the end of day 7. Increased lactate values detected for the treated units of arm A at the end of the storage period were independent from the PCT time setting.

CONCLUSIONS

Photochemical pathogen inactivation with different initial resting periods between 4 and 23 h, and different CAD steps of 4 and 16 h, had no influence on the platelet in vitro function during 7 days of storage.

Patients with thalassemia, an inherited hemolytic anemia, have increased risk of hypercoagulable complications. A whole blood flow cytometric (FCM) method has been used for studies of platelet activation and platelet-leukocyte aggregation in these patients. However, this FCM method presents technical difficulties because of the high proportion of immature red blood cells (RBCs) in these patients. A protocol for the simultaneous measurement of platelet activation and their aggregation with leukocyte populations in whole blood using four-color FCM which excluded immature RBC was devised, and evaluated for the evaluation of platelet function in patients with β-thalassemia/hemoglobin E (HbE). Whole blood from these patients and from healthy volunteers was stained for platelet activation and platelet-leukocyte aggregates using anti-CD42a, anti-CD62P, anti-CD45 and glycophorin A (GPA) conjugated with different fluorochromes. Our FCM method is simple, effective and based on the assumption that GPA is present on all immature RBCs, but is not expressed on CD45⁺ leukocytes. Results from the studies showed that blood samples from these patients contained a high frequency of circulating activated platelets (CD42a⁺/CD62P⁺) when compared to samples from healthy individuals. The percentage of platelet-neutrophil, platelet-monocyte-but not platelet-lymphocyte-aggregates were also elevated in both thalassemia genotypes with marked increase in patients who had undergone splenectomy. These findings suggest that platelets adhere to neutrophils and monocytes are activated which support the clinical observation that splenectomized thalassemia patients have an increased risk of arterial or venous thrombotic manifestations.

OBJECTIVE

White blood cell (WBC) fragments in platelet concentrates (PCs) may induce allo-immunization in the recipient.

METHODS

As the level of WBC fragments can differ between PCs produced using different methods, we compared PCs prepared by using the buffy-coat method (BC-PCs) in plasma or platelet additive solution (Composol) and PCs prepared using the platelet-rich plasma method (PRP-PCs).

RESULTS

Post-filtration results revealed identical levels of WBC, but significantly higher CD62p expression and a significantly lower amount of total DNA, cell-free DNA and number of WBC fragments (0.6 vs. 4.2 WBC equivalents/microl) in PRP-PCs than in BC-PCs in either plasma or Composol.

CONCLUSIONS

The number of WBC fragments is significantly higher in BC-PCs than in PRP-PCs.

Strenuous exercise may be partially responsible for cardio-vascular events. The aim was to investigate the platelet activity, reactivity and different platelet-leukocyte-conjugate formation following maximal short-term exercises. Fifteen healthy non-smokers underwent three isokinetic maximal tests on a SRM cycle ergometry system with durations of 15, 45 and 90 s. Blood samples were taken after a 30-min rest, immediately before and after exercise, and 15 min and 1 h after completion of exercise. Platelets were detected flow-cytometrically by CD41, and activated platelets by CD62P. In addition, stimulation of the platelets in vitro with 7.5 microM TRAP-6 was initiated. For testing platelet-leukocyte-conjugates, antibodies against CD45, CD14 and CD41 were used. After the exercise tests the percent of non-stimulated CD62P-positive platelets (%PC) was unchanged. In contrast, an increase in %PC (CD62P) TRAP-6 stimulated (15-s test: 37.2+/-10.3 to 46.2+/-12.3%, P < 0.05; 90-s test: 40.6+/-9.5 to 51.7+/-10.2%, P < 0.01) and in platelet-granulocyte, platelet-lymphocyte, and platelet-monocyte conjugate formation 15 min after exercise (45- and 90-s test; P < 0.05) were observed in comparison with the changes on the control day. The changes nearly reversed 1 h after exercise. Maximal short-term exercise only leads to a moderate increase of platelet reactivity and to an increase in the different platelet-leukocyte conjugates. The implications of the changes in platelet-leukocyte conjugate formation should be investigated in future studies.

The CD62P receptor and its soluble form sP-selectin is a marker of platelet (PLT) activation, and constitutes a ligand for CD24 antigen of neoplastic cells and tumor stroma components. The aim of the study was to evaluate the relationship dynamics of the percentage of CD62P+ platelets, the level of the receptor expression and the concentration of soluble form of sP-selectin in renal cancer. Examinations were performed before and after nephrectomy in patients with renal cancer (group A - 25, T2N0M0; group B - 27, T2N1-2M0) and in control group (C - 24 subjects). The two groups A and B showed an increased subpopulation of CD62P+ platelets (p<0.01) and elevated sP-selectin concentration (p<0.001) before and after nephrectomy. Although following nephrectomy sP-selectin concentration decreased markedly, it was still higher 3 months after the procedure compared to control group (p<0.05). Following nephrectomy, however, no statistically significant differences were found in the % of CD62P+ platelets and the receptor expression. Greater dynamics of changes before and after nephrectomy in the percentage of CD62P+ platelets (B1:B2 p<0.05) and the receptor expression (B1:B3 p<0.001) was observed in patients with local lymph node involvement (group B) while sP-selectin concentration was similar in both groups. Nephrectomy did not normalize intravascular activation of PLT and TNM had no significant effect on the expression of CD62P and concentration of sP-selectin.

Previous in vitro studies are in keeping with the finding that isolated and enriched megakaryocytes attach to bone marrow fibroblasts and generate an increased growth of these cells. This process was assumed to depend on a close spatial relationship between both cell types which supports the paracrine effect of platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-beta1. Moreover, adhesion molecules including beta1 integrin receptors and fucosylated structures were determined to play an important role in these complex interactions. However, up to now the influence of megakaryocyte expressed glycoproteins CD41a and CD42b in these processes was not investigated. In addition, the role of megakaryocytic CD62P and also of CD62L, both adhesion molecules of the selectin group, could also be of interest. Following isolation and enrichment of bone marrow megakaryocytes and fibroblasts, both cell populations were characterized regarding their expression of these factors by applying immunocytochemical techniques. Additionally, their influence on adhesion of megakaryocytes to fibroblasts as well as fibroblast growth was evaluated by comparative megakaryocyte-fibroblast co-cultures and inhibition studies using specific monoclonal antibodies (mabs). Fibroblast monocultures served as controls. In these experiments, selectin-specific antibodies significantly reduced megakaryocyte attachment to fibroblast feeder layers and fibroblast growth in the co-cultures. The effect of CD41a and CD42b specific antibodies was limited to megakaryocyte-dependent fibroblast growth. These results elucidate the involvement of the selectins CD62P and CD62L in the basal activation of megakaryocytes inducing their attachment to bone marrow fibroblasts. In contrast, the megakaryocyte glycoproteins CD41a and CD42b exert their effect on the megakaryocyte dependent fibroblast growth. Altogether, it is tempting to speculate that the various interactions of these mediators reflect certain steps in the complex pathomechanisms causing the evolution of (reactive) myelofibrosis in hematopoietic neoplasias accompanied by megakaryocytic proliferation.

Bioincompatibility is the total of side effects during hemodialysis (HD) including, amongst others, changes in platelet (PLT) level. Deviations in PLT count, immature PLT count, PLT morphology, CD62p expression, Platelet Factor 4 (PF4), β-Thromboglobulin (β-TG), serotonin, Thrombin-Antithrombin III (TAT) and Prothrombin Fragment 1+2 (F1+2) are monitored before and during treatment with HD in order to elucidate the interaction between modifications in PLT morphology, PLT activation and markers concerning activation of coagulation. Different patterns with time indicate that there is no correlation between an increased amount of depleted PLTs and increased amounts of PLT activation markers such as CD62p, PF4, β-TG and serotonin. A statistically significant correlation between increased PLT activation markers and markers for increased activation of coagulation such as TAT and F1+2 has not been established. Only a weak correlation is demonstrated between the increase of markers for activation of coagulation and the decrease in PLT counts, immature PLT counts and depleted PLTs during HD treatment. The change in the extracorporeal circuit during HD is probably a more critical factor in the mechanism leading to activation of the coagulation pathway than the modifications in PLT morphology.

OBJECTIVE

Microvesicles (MV) in the blood stream are associated with distant metastasis in cancer. Platelet or endothelial cell-related MV actively participate in thrombogenesis, which is an important step in cancer metastasis. This study investigated the correlations between MV levels of platelet-poor plasma and distant metastasis in lung cancer.

METHODS

Platelet-poor plasma from 44 treatment-naive lung cancer (23 with distant metastasis) and 19 normal subjects was used to determine the levels of glycoprotein Iβ (CD42) + platelet MV (PMV), P-selectin (CD62P) + PMV, VE-cadherin (CD144) + endothelial MV (EMV), tissue factor (CD142) + MV, thrombin-antithrombin complex and vascular endothelial growth factor (VEGF).

RESULTS

The level of CD142 + MV was significant (odds ratio 5.86, 95 % confidence interval 1.31-38.3) in predicting distant metastasis in lung cancer, and a cutoff value of 2.668 (after logarithm transformation) in the ROC curve had a specificity of 90 % and a sensitivity of 59 %. The presence of distant metastasis showed a significant correlation between CD144 + EMV and VEGF, but not between CD144 + EMV and CD42 + PMV or CD62P + PMV in lung cancer subjects.

CONCLUSIONS

The finding of CD142 + MV in platelet-poor plasma may be useful for suggesting distant metastasis in lung cancer. In addition to thrombogenesis, interaction between VE-cadherin and VEGF may be needed for successful metastasis in lung cancer.

Adhesion of inflammatory cells to vascular endothelium is mediated by specific cell adhesion receptors on both leukocytes and endothelial cells. One of the adhesion molecules on the endothelium is P-selectin. Decreased vascular P-selectin expression has been associated with tumor progression in melanoma patients. We now report on the expression of endothelial P-selectin in colorectal cancer (CRC). We studied a colorectal tissue specimen series ranging from normal colorectal tissue via unmetastasized primary tumors to tumors with the same depth of invasion at the primary site but with liver metastases. Moreover, P-selectin expression levels in liver metastases were determined. The number of P-selectin positive vessels as a fraction of the total number of vessels, both intra- and peritumorally, was determined by staining for CD62P and CD34, respectively. Furthermore, by immunostaining for leukocytes (CD45) and macrophages (CD68), it was evaluated whether levels of P-selectin expression influenced infiltrate density and composition. The results showed that levels of peritumoral P-selectin expression were reciprocal to the degree of progression in CRC. This relation was even more pronounced intratumorally: in metastasized primary tumors and in the metastatic lesions, P-selectin expression was virtually absent. This distribution pattern was reflected in the numbers of leukocytes that accumulated in the various tissues, since in the primary tumors with metastases, and in the metastatic lesions, hardly any infiltrating cells were observed. In these lesions, leukocytes were present in the peritumoral zone, but seemed unable to enter the tumor tissue. In primary tumors without metastasis, the intratumoral leukocyte infiltration density was significantly higher. Recruitment levels of macrophages remained constant throughout the different tissues. We suggest that downregulation of endothelial P-selectin expression is a mechanism by which CRC lesions evade inflammatory regression and, thereby, progress to a more advanced stage of malignancy.

BACKGROUND

Recent studies have shown that platelets play an important role in the pathogenesis of reperfusion injury. Using an inferior epigastric artery skin flap as a flap ischemia/reperfusion (I/R) injury model, we investigated whether the administration of a nitric oxide (NO) donor, nitrosoglutathione (GSNO), could decrease platelet activation and modulate the NO synthase (NOS) activity of platelets and promote flap survival.

METHODS

Thirty minutes before flap reperfusion, normal saline (1 mL), nitrosoglutathione (GSNO 0.2, 0.6, 3 mg/kg), or N(G)-nitro-L-arginine-methyl ester (450 mg/kg) was injected intravenously in 10 rats, respectively. The p-selectin (CD62P) expression of platelet activation was detected by a flow cytometry. Immunohistochemical staining was performed to investigate the CD62P deposition on the microvasculature of the flap vessels. NOS isoform expression in the platelets was evaluated by Western blot. Tissue perfusion was monitored by using laser-Doppler flowmetry. Survival areas were assessed at 7 days postoperatively

RESULTS

An optimal dose of GSNO (0.6 mg/kg), significantly decreased in CD62P expression on platelets (P < 0.001) and its deposition on the flap vessels, selectively suppressed iNOS induction of platelet, and significantly improved blood perfusion and the flap survival rate (59.8 +/- 4.9% versus 22.1 +/- 6.1%, P < 0.001). In contrast, the NO synthase inhibitor, N(G)-nitro-l-arginine methyl ester, although inhibiting iNOS expression of platelets, compromised platelet activation, tissue perfusion, and flap survival.

CONCLUSIONS

This study suggests that GSNO can appropriately donate NO to suppress platelet activation and platelet iNOS induction, resulting in less platelet activation, better blood perfusion, and flap survival after I/R injury.

OBJECTIVE

This study was undertaken to define the pattern of cell adhesion receptor expression by the CD34+ progenitor cells from mobilized peripheral blood and bone marrow from normal and autologous donors, and to correlate the adhesion receptor profile with time to blood cell recovery for patients undergoing autologous transplant.

METHODS

Blood cell recovery was determined by absolute neutrophil count >> 500/microL), time to last red cell transfusion, and platelet count >> 50,000/microL and>> 100,000/microL). The analysis for expression of adhesion receptors alphaL (CD11a), alpha2 (CD49b), alpha3 (CD49c), alpha4 (CD49d), alpha5 (CD49e), alpha6 (CD49f), beta1 (CD29), L-selectin (CD62L), ICAM-1 (CD54), PECAM-1 (CD31), HCAM (CD44), and P-selectin (CD62P) was performed by two-color flow cytometry. The Kruskal-Wallis test and Spearman's rank correlation were used for statistical analysis.

RESULTS

Statistical analysis of adhesion expression by the CD34+ population for patients undergoing autologous transplant demonstrated that the higher percent expression of PECAM-1 correlated with longer time to platelet recovery >> 100,000/microL, p = 0.049). In contrast, the higher the percent expression of alpha6 (p = 0.013) and the increased density of expression of alpha2 (p = 0.035), alpha3 (p = 0.023), alpha4 (p = 0.044), beta1 (p = 0.027), and ICAM-1 (p = 0.010) correlated with shorter time to platelet recovery. Neutrophil recovery time decreased with increased density of expression of alphaL (p = 0.014) and P-selectin (p = 0.007) receptors. Increased density of expression of CD44 (HCAM) was associated with longer time to red blood cell recovery (p = 0.05).

CONCLUSIONS

These data suggest that upregulation of specific adhesion receptors or selection of certain cell populations could result in earlier blood cell recovery after transplant.

Dasatinib, a tyrosine kinase inhibitor, has a reduced plasma half-life and a more extensive inhibition profile, including targeting of Src family kinases. We monitored the peripheral blood count and the serum concentration of dasatinib over time. Interestingly, we found a transient fluctuation of blood cells, which correlated with the dasatinib level. The peripheral blood count before intake of dasatinib was compared with counts measured 2 h later in blood samples from 23 patients. Total white blood cells (WBCs) increased by 2,186 ± 1,960/μL from baseline (P = 0.00002), whereas platelets decreased from a baseline of 185 ± 47 × 10(3)/μL to 164 ± 52 × 10(3)/μL (P = 0.0007). Similar phenomena were not observed in patients treated with imatinib or nilotinib. In addition, in contrast to imatinib, dasatinib strongly attenuated the expression of CD18, CD62P and CD63 by blood cells both in vivo and in vitro. These results suggest that this drug may influence the distribution of blood cells in vivo by regulating its specific adhesion molecule expression on blood cells.

The sodium-hydrogen exchanger isoform 1 (NHE-1) contributes to platelet activation at elevated pH. Effects of NHE-1 inhibitors on platelet degranulation and formation of proinflammatory and procoagulatory platelet-leukocyte aggregates (PLA) and possible interactions with P2Y(12) inhibitors--which also affect platelet degranulation--have not been investigated. Whole blood from healthy human subjects was incubated with the NHE-1 inhibitor cariporide and the P2Y(12) inhibitor AR-C 69331 MX at clinically reasonable concentrations, in the presence of normal pH or in a propionate model to activate the NHE-1 (approximately pH 7.0). The degranulation marker CD62p, the expression of the activated GPIIb/IIIa receptor (PAC-1), and formation of platelet-leukocyte (monocyte) aggregates (PLA) was assessed by flow cytometry. Cariporide at concentrations up to 20 microg/ml had no effects on ADP- (5 microM) or TRAP- (2 microM) induced CD62p expression or PLA formation at normal pH. At pH 7.0 and stimulation with ADP, PLA decreased from 64+/-24% (control) to 47+/-23% under cariporide at 2 microg/ml (p<0.05), and the MFI of PLA (i.e. the platelet mass attached at monocytes) decreased from 547+/-203 to 360+/-96 units (p<0.05). PAC-1 MFI decreased from 66+/-23 to 34+/-18 units (p<0.05) after ADP and from 74+/-29 to 42+/-17 units (p<0.05) after TRAP, respectively. AR-C 69331 MX (10 nM) had inhibitory effects on all parameters irrespectively of the pH, and the combination of both agents at pH 7.0 shows additive effects. In conclusion, our investigation points to-perhaps clinically relevant-effects of NHE-1 inhibition on the degranulation of platelets and formation of platelet-leukocyte aggregates.

It has been hypothesized that, in addition to freezing injury, some damage to platelets may result from the cell packing that occurs during removal of the cryoprotectant. This study examined DMSO removal by fluid exchange across hollow-fiber (HF) filters as an alternative to centrifugation. The DMSO solution with or without cell suspension was passed once through the filter. The optimum exchange during unloading of DMSO was determined by varying the flow rates in the external and internal compartments of the HF filter. Initially, buffered solutions of a 5% DMSO solution in the absence of platelets were pumped into the fibers and exchanged against PBS. The residual DMSO was determined by osmometry. The exchange of DMSO across the membrane was flow dependent and also influenced by the chemical nature of the HF fibers. No protocol using a reasonable rate flow through the fibers removed more than 95% of the DMSO in a single pass. The optimum protocol was achieved with polysynthane fibers with an internal flow rate of approximately 20 mi/min and an external flow rate of 100 ml/min. Subsequently, frozen/thawed platelet concentrates in DMSO were washed using centrifugation and compared to the HF filtration method. Platelet quality was assayed by flow cytometry, cell count, morphology and osmotic stress test. Both filtration and centrifugal washing techniques resulted in comparable morphological scores and numbers of discoid cells. When agents reducing platelet activation were added, platelet quality was improved after washing by either technique. The lower platelet osmotic response with HF filtration than with centrifugation while using activation inhibitors was attributed to the remaining amount of the inhibitors. All other parameters tested were similar. The expression of CD62P was equivalent with both techniques, and centrifugation did not activate platelets more than filtration contrary to what was originally anticipated. In conclusion, platelet quality was comparable after washing by either technique but hollow fiber filtration does remove cryoprotectant more rapidly than does centrifugation.

CD39/ATP diphosphohydrolase is expressed on B lymphocytes, cytotoxic T lymphocytes, monocytes, platelets, and endothelial cells, and it has a critical role in the inhibition of platelet responsiveness. To determine whether strenuous exercise could acutely change expression of CD39 in platelets and lymphocytes, eight healthy sedentary men, 34 yr old (SD 7), and eight physically active men, 34 yr old (SD 6), performed graded upright cycle ergometry to volitional exhaustion. Blood samples collected both at baseline and after exercise test were employed to measure CD39 expression in platelets and lymphocytes. The percentage of circulating platelet-platelet aggregates, the "in vitro" ADP and collagen-induced platelet aggregation, and the expression of both platelet glycoprotein IIb-IIIa (PAC-1) and P-selectin (CD62) were also considered markers of platelet activation. After strenuous exercise, all subjects demonstrated significant platelet activation as judged by the increased percentage of platelet-platelet aggregates. The in vitro ADP-induced platelet aggregation and the expression of CD62P on ADP-stimulated platelets significantly increased in sedentary but not in active subjects. After exercise, all of the subjects showed a significant reduction of CD39 expression in platelet [sedentary: from 2.2 (SD 0.8) to 1.1% (SD 0.8), P = 0.008; active: from 0.6 (SD 0.2) to 0.35% (SD 0.1), P = 0.009] and an increase of CD39 expression in B lymphocytes [sedentary: from 47 (SD 13) to 60% (SD 11), P = 0.0039; active: from 46 (SD 11) to 59% (SD 11), P = 0.0038]. Taken together, these findings confirm the critical role of this ADPase in inhibition of platelet responsiveness, also suggesting a possible role of B lymphocytes in thromboregulation mechanism.

Nicotine is a primary constituent of tobacco and smoke, and its roles in causing addiction and causing disease are commonly conflated. In the present work, we investigated whether nicotine protects smokers' platelets against smoke-induced activation in vivo, raising a possible dilemma in harm-reduction strategies. In vivo platelet activation state (PAS) was measured by fixing blood at drawing and measuring a standard marker, platelet P-selectin (CD62P). We conducted two studies: (1) 32 smokers smoked three medium-nicotine (0.6 mg nicotine) cigarettes for 1 h. Following this initial conditioning phase, 16 subjects continued with five of the same cigarettes from 1-2.5 h, resulting in a 33% increase in PAS. The other 16 subjects smoked five low/zero-nicotine cigarettes (0.05 mg nicotine), causing a 94% increase in PAS. The increase in PAS caused by nicotine withdrawal in the second group is very significant (p<.02). Any compensation in smoke-intake due to nicotine withdrawal in the second group was not measured in this study. (2) To determine whether nicotine modulates platelet activation by secondhand smoke (SHS), 16 nonsmokers were exposed to medium-nicotine smoke and 16 to low/zero-nicotine smoke for 1.5 h on two consecutive days. Exposure to SHS increased PAS by 60% (p<.01), but no difference in the medium and zero nicotine groups was observed (p>.09). We conclude that in smokers, nicotine modulates platelet activation, and it may significantly moderate the risk of cardiovascular disease caused by non-nicotine smoke components. Conversely, reduced-nicotine cigarettes may increase harm.

BACKGROUND

Sevoflurane can be used as sedative-analgesic drug with endothelial protective properties. We tested whether low-dose sevoflurane inhalation provides sustained inhibition of detrimental granulocyte-platelet aggregation in humans.

METHODS

Ten healthy male volunteers were enrolled in this crossover study. Each subject inhaled sevoflurane for 1 h at 0.5-1 vol % end-tidal concentration in oxygen (50 vol %). Inhaling oxygen (50 vol %) alone served as control. Venous blood samples were collected at baseline before inhalation, immediately after inhalation, and 24 h thereafter, and were used for flow cytometry to determine platelet surface marker (CD41, CD42b, CD62P/P-selectin, and PAC-1) on platelets and granulocytes and for kaolin-induced clot formation, as assessed by thromboelastography. In flow cytometry experiments, platelets were stimulated with arachidonic acid (AA, 30 microM), adenosine diphosphate (ADP, 1 microM), and thrombin receptor agonist peptide-6 (TRAP-6, 6 microM).

RESULTS

AA, ADP, and TRAP-6 markedly increased the expression of CD62P on platelets, whereas CD42b (shedding) and PAC-1 (heterotypic conjugates) expression decreased. The amount of granulocyte-platelet aggregates increased upon agonist stimulation. Low-dose sevoflurane inhalation reduced ADP-induced CD62P expression on platelets 24 h after inhalation, and inhibited the formation of granulocyte-platelet aggregates under stimulation with AA and ADP after 1 and 24 h, and with TRAP-6 after 24 h compared with control. Inhibition of granulocyte-platelet aggregates was accompanied by reduced clot firmness 24 h after sevoflurane inhalation compared with control.

CONCLUSIONS

We demonstrated for the first time that inhaling low-dose sevoflurane (<1 vol % end-tidal) inhibits agonist-induced granulocyte-platelet interactions 24 h after administration and thus counteracts thromboinflammatory processes.

Mass cytometry (MC) uses mass spectrometry to simultaneously detect multiple metal-conjugated antibodies on single cells, thereby enabling the detailed study of cellular function. Here, for the first time, we applied MC to the analysis of platelets. We developed a panel of 14 platelet-specific metal-tagged antibodies (targeting cluster of differentiation [CD] 9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD107a, CD154, glycoprotein [GP] VI and activated integrin αIIbβ3) and compared this panel with two fluorescence flow cytometry (FFC) panels (CD41, CD42b, and CD61; or CD42b, CD62P, and activated integrin αIIbβ3) in the evaluation of activation-dependent changes in glycoprotein expression on healthy subject and Glanzmann thrombasthenia (GT) platelets. High-dimensional analysis of surface markers detected by MC identified previously unappreciated subpopulations of platelets in healthy donors. As expected, MC and FFC revealed that GT platelets had significantly reduced CD41, CD61, and activated integrin αIIbβ3 surface expression. MC also revealed that surface expression of CD9, CD42a and CD63 were elevated, CD31, CD154 and GPVI were reduced and CD29, CD36, CD42b, CD62P and CD107a were similar on GT platelets compared to healthy donor platelets. In summary, MC revealed distinct platelet subtypes in healthy subjects and novel alterations in surface glycoproteins on GT platelets.

To elucidate a mechanism of prothrombotic effects of carbon nanotubes (CNTs), we report here that multiwalled CNTs activate blood platelets by inducing extracellular Ca(2+) influx that could be inhibited by calcium channel blockers SKF 96365 and 2-APB. We also demonstrate platelet aggregating activity of different single-walled and multiwalled CNTs. In addition, we show that CNT-induced platelet activation is associated with a marked release of platelet membrane microparticles positive for the granular secretion markers CD62P and CD63.

BACKGROUND

Hepatic impairment, portal hypertension, and multi-systemic damage could occur during liver cirrhosis's late stage. Bleeding is a complication of hepatic cirrhosis along with several changes including blood platelet count (BPC), mean platelet volume (MPV), platelet crit (PCT) and expression of platelet CD62P. Blood platelet count (BPC), mean platelet volume (MPV), platelet distribution width, and other indices are indirect reflections of CD62P parameters.

OBJECTIVE

To investigate the changes in platelet functional parameters and CD62 P expression in liver cirrhosis as a possible guide in clinical treatments and prognoses of liver cirrhosis.

METHODS

CD62P was tested by flow cytometry in liver cirrhosis. BPC, MPV, and PCT in peripheral blood were tested using an auto blood cell analyzer. Data were analyzed using SPSS11.0.

RESULTS

The values of CD62P and MPV in patients was significantly higher than those of healthy donors (P<0.01), while the values of BPC and PCT were significantly lower than those of the control group (P<0.01).

CONCLUSIONS

CD62P, BPC, MPV, and platelet crit (PCT) show several changes in liver cirrhosis. It is useful to understand the relationship between hepatic cirrhosis severity and CD62P, BPC, MPV, PCT, timely monitoring of CD62P for treatment of hepatic cirrhosis in clinical treatment and prognosis.

P-selectin (CD62p) exposure is an established marker for platelet activation. P-selectin exposure can trigger variety of thrombotic and inflammatory reactions. In patients with coronary artery disease (CAD), platelets are activated, and hence, there is increased P-selectin exposure. The role of P-selectin exposure in patients on treatment with statins and anti-platelets is conflicting. A case-control study was performed to determine P-selectin exposure in consecutively recruited 142 patients (age ≤ 55 years) with angiographically proven CAD on treatment and 92 asymptomatic controls. P-selectin exposure was determined by flow cytometry. Data on conventional risk factors were obtained along with estimation of levels of thrombotic [fibrinogen, lipoprotein (a), tissue plasminogen activator, plasminogen activator inhibitor-1, homocysteine and von Willebrand factor] and anti-thrombotic factors (antithrombin III). The P-selectin exposure was compared among patient groups who had different modes of presentation of CAD and categories of CAD disease severity. The patients were followed up for a period of 26 months. The results indicate that P-selectin exposure was significantly elevated in patients (mean ± SD 9.24 ± 11.81) compared to controls (mean ± SD 1.48 ± 2.85) with p < 0.0001. Similarly, conventional risk factors were significantly elevated in patients. P-selectin exposure showed significant negative correlation with antithrombin III levels. P-selectin exposure was higher in patients who presented with acute coronary syndromes than those who presented with effort angina. Cardiovascular event rate was 6 % on follow-up. The study establishes that thrombotic-inflammatory pathways enhancing P-selectin exposure unrelated to treatment might be activated in patients, while the event rate remained lowered, and hence, treatment strategies should be inclusive to control these factors.

OBJECTIVE

Enhanced platelet activity has previously been reported in the acute phase after ischemic stroke. We tested the hypothesis that activated platelets (expressed by CD62p) are substantially increased in the acute stage after a stroke and decrease thereafter, and that antiplatelet therapies can suppress CD62p expression.

METHODS

We serially examined platelet CD62p expression using flow cytometry after acute ischemic stroke in 87 consecutive patients. The CD62p expression was also evaluated in 20 healthy volunteers and 33 at-risk control subjects.

RESULTS

CD62p expression was significantly higher in the acute phase after ischemic stroke than in normal and at-risk control subjects (both P<0.0001). CD62p expression decreased to a significantly lower level on day 21, and to a substantially lower level on day 90. CD62p expression was not significantly suppressed by warfarin. However, CD62p expression was significantly suppressed by aspirin treatment (P=0.024) and more substantially suppressed by clopidogrel (P<0.0001) on day 90. Furthermore, only clopidogrel treatment (P=0.0016) was significantly independently associated with decreased CD62p expression on day 90.

CONCLUSIONS

Platelet activation was significantly increased in acute ischemic stroke and substantially decreased thereafter. The lesser long-term pharmacodynamic potency of aspirin relative to clopidogrel raises the prospect of the need for more effective antiplatelet agents or a synergistic combination therapy for stroke prevention in the future.

OBJECTIVE

To explore the effect of Danhong Injection (DHI) on platelet activation and inflammatory factors in patients of acute coronary syndrome (ACS) after intervention therapy.

METHODS

One hundred ACS patients were randomly assigned to the DHI group and the control group equally. Both were treated with the conventional treatment, including aspirin, clopidogrel, beta-receptor blocker, angiotensin converting enzyme inhibitor, etc. for 2 weeks after percutaneous coronary intervention (PCI), and to the patients in the DHI group, intravenous dripping of DHI was given simultaneously. Fasting venous blood of patients were collected before PCI and on the next morning of PCI to determine the platelet activation indices, expression of CD62p and receptor complex of glucose protein (GP) II b/III a by flow cytometry; plasma fibrinogen C (FIB-C) by scattering turbidimetry, and serum high-sensitivity C-reactive protein (hs-CRP) with emulsoid immuno-enhancing turbidimetric test kit. The outcomes were compared with those determined in 40 healthy persons for control.

RESULTS

Serum levels of CD62p, GP II b/III a, FIB-C and hs-CRP in ACS patients were significantly higher than those in the healthy control (all P<0.01), and those were significantly higher after PCI than before PCI (P <0.05 or P<0.01). After being treated for 2 weeks, the 4 platelet activation indices were lowered to different extent in both groups, but the lowering in the DHI group was more significant than that in the control group (P<0.05).

CONCLUSIONS

DHI can inhibit the platelet activation and inflammatory reaction in ACS patients after PCI.