BACKGROUND

Platelet-leukocyte aggregates (PLAs) may be important in thrombotic and inflammatory disease states, but accurate assessment of PLA formation in vivo is hampered by the propensity for in vitro artefacts caused by sample manipulation. A whole blood flow cytometric assay for circulating PLAs, based on minimal sample manipulation, was thus developed.

METHODS

Citrated whole blood was labeled with a RPE-CD45 MAb (leukocyte marker) and an FITC-CD42a (GPIX) MAb (platelet marker). The latter was used to avoid possible influences of platelet glycoprotein proteolysis by neutrophil-derived proteases. The samples were mildly fixed with 0.5% formaldehyde saline. The cytometer was triggered by RPE-CD45 fluorescence. Leukocyte subpopulations were separated according to their typical light scattering and CD45 expression.

RESULTS

Minimal sample manipulation and mild sample fixation resulted in minor in vitro artefacts and good sample stability. Fluorescence triggering increased the efficiency of the flow cytometric analysis approximately 5-fold compared with triggering with light scatter, and allowed discrimination of leukocyte subpopulations. The majority of PLAs involved monocytes and neutrophils, rather than lymphocytes, both without and with in vitro stimulation by ADP or thrombin. A cocktail of blocking MAbs to CD62P, CD15, GPIIb/IIIa and the CD11b/CD18 complex had no effect on unstimulated samples, whilst totally inhibiting aggregation induced by 10(-5) M ADP, suggesting that the PLAs in unstimulated blood were preformed in vivo.

CONCLUSIONS

This whole blood flow cytometric assay for PLAs is simple and efficient, and appears to reflect closely platelet-leukocyte aggregates in circulating blood in vivo.

Evidence is increasing that platelets can initiate and propagate inflammatory processes by interacting with leucocytes and the vascular endothelium. Platelets have been shown to bind to neutrophils, existing as platelet/neutrophil complexes (PNC) within the circulation. We describe a simple flow cytometric method for assessing and investigating platelet interactions with neutrophils in small volumes of whole blood. Twenty-five percent (sd 6%) of circulating neutrophils from healthy adults were associated with platelets. Formation of these platelet-neutrophil complexes was CD62P (P-selectin) and divalent cation dependent. Platelet activation (with ADP or thrombin) caused a rapid and sustained rise in %PNC which differed from the pattern of free platelet activation as assessed by CD62P expression. F-met-leu-phe induced neutrophil activation but did not increase the percentage PNC. Platelet activation also caused increased neutrophil CD11b/CD18 expression which was most marked on neutrophils complexed with platelets. This straightforward technique is simple, reproducible, and allows assessment of platelet-neutrophil interactions and activation of neutrophils. It may also provide a method for estimating platelet activation in whole blood.

BACKGROUND

Blood platelets (PLTs) are critical for hemostasis, and they contain biologically active constituents with the potential to modulate inflammatory responses. This study examined the effects of photochemical pathogen inactivation treatment (PCT) on the release of cytokines and/or chemokines from PLT components.

METHODS

Double-dose apheresis PLT components were suspended in plasma-PLT additive solution mixtures and divided into paired therapeutic units. One unit served as an untreated control and the other unit was treated with PCT. PLT concentrations, pH, and levels of cytokines and/or chemokines (CD62p, platelet-derived growth factor-AB, interleukin [IL]-8, soluble CD40 ligand [sCD40L], IL-1beta, and tumor necrosis factor alpha) were measured during 7 days of storage in PLT component supernatants and PLT lysates.

RESULTS

PLT content, pH, and cytokine and/or chemokine content and release from PLT component prepared with PCT were not different (p>> 0.05) from paired control components during storage. Levels of sCD40L, however, increased significantly during storage while decreasing in parallel within PLT lysates, although no differences were detected between paired PCT and control PLT component.

CONCLUSIONS

PCT did not increase the release or secretion of PLT chemokines and/or cytokines over a 7-day period compared to conventional PLT component.

BACKGROUND

The different production methods for platelet concentrates (PCs) result in products with variable in vitro quality and in vivo viability. The aim of this study was to compare in vitro variables of PCs produced by apheresis (AP-PC) or the buffy coat (BC-PC) method by applying a number of new and established assays.

METHODS

Standard TRIMA Accel (Gambro BCT) AP-PCs (n = 20) and BC-PCs (n = 20) were stored in 100 percent plasma and changes in mitochondrial membrane potential (DeltaPsi(m)) were assessed using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethlybenzimidazolcarbocyanine iodide (JC-1) dye on Days 1, 3, 5, and 7. The capacity of platelets (PLTs) for oxidative phosphorylation was also monitored by measuring oxygen consumption using a Clark-type electrode. PLT viability was measured using a new assay that utilizes the vital stains calcein-AM and FM4-64. Expression of phosphatidylserine (PS), CD42b, CD47, CD61, and CD62P was also assessed.

RESULTS

Although the JC-1 ratio (FL2/FL1) decreased significantly in both preparations, the percentage of PLTs with depolarized DeltaPsi(m) increased significantly in BC-PCs but not in AP-PCs. However, no significant change was detected in the PLTs' ability to consume oxygen in both preparations. PLTs in BC-PCs also showed significantly lower GPIb, CD47, and CD61 expression than AP-PCs on Day 1. PLTs in both preparations, however, showed a similar increase in CD62P and PS expression during storage, without significant loss of viability.

CONCLUSIONS

PLTs in AP-PCs and BC-PCs undergo different degrees of deterioration in mitochondrial integrity and thus may undergo different degrees of apoptosis. Interventions that maintain mitochondrial integrity may improve PLT viability in vitro.

BACKGROUND

The PlA2 polymorphism of platelet glycoprotein IIIa has been identified as a prothrombotic risk factor in a number of cardiovascular settings. The aim of this study was to determine whether the PlA2 polymorphism of platelet glycoprotein IIIa and degree of platelet activation were associated with more severe myocardial injury as indicated by troponin I release following cardiopulmonary bypass.

METHODS

The PlA2 genotype was determined in 66 patients undergoing elective coronary artery bypass grafting requiring cardiopulmonary bypass. Troponin I concentrations and the percentage of circulating, activated (CD62P+) platelets were measured at predetermined intervals perioperatively.

RESULTS

Forty-six patients were Pl(A1,A1), and 20 were Pl(A1,A2) or Pl(A2,A2). Patients with at least one PlA2 allele had significantly greater postoperative troponin I concentrations than PlA1 homozygotes (P = 0.006, analysis of variance). Peak troponin I concentrations also correlated significantly with the increase in circulating, activated platelets (P = 0.02, Spearman rank correlation).

CONCLUSIONS

The PlA2 allele of platelet glycoprotein IIIa is associated with higher troponin I concentrations following cardiopulmonary bypass surgery, suggesting that this platelet polymorphism contributes to perioperative myocardial injury.

Rat CINC induced a dose- and time-dependent accumulation of neurophils into murine air-pouches, a response which was inhibited by two selective H1-antagonists, mepyramine and triprolidine (approximately 60%). As pretreatment with fucoidin abolished CINC effect, the relative time-related contribution of selectins on this process was then investigated by using specific monoclonal antibodies (mAb). Anti-CD62L mAb gave a similar degree of inhibition of CINC-induced cell accumulation both at the 2h and 4h time-point (approximately 75%). Anti-CD62P mAb, but not the anti-CD62E mAb, inhibited PMN accumulation at 2h (65%), but only co-administration of these two mAbs inhibited the cell response to CINC at the 4h time-point (90%). Thus endogenous histamine, CD62L, CD62P, and CD62E, though to a different degree, are required for PMN extravasation observed in response to CINC administration.

BACKGROUND

Uncontrolled severe hypertension is associated with alarming rates of cardiovascular events but the mechanisms of vascular injury are not well understood. Recent investigative interest has focused on platelet activation and platelet P-selectin (CD62P) as direct mediators of vascular inflammation and injury. We investigated the association of extreme blood pressure (BP) elevation with platelet P-selectin and fibrinolytic markers in high risk patients with severe hypertension.

METHODS

Cross-sectional comparison of platelet CD62, tissue plasminogen activator antigen (tPA), and plasminogen activator inhibitor-1 activity (PAI-1) among 3 BP groups: untreated severely hypertensive patients (SHT; n=18), untreated mildly hypertensive patients (MHT; n=19), and normotensive controls (NT; n=16).

RESULTS

Platelet CD62 was greatest in SHT (p=0.00008) and showed a strong correlation with both systolic (p=0.0002, r=0.52) and diastolic (p=0.0003, r=0.52) BP. tPA was greater in SHT than MHT or NT (ANOVA; p=0.02) and correlated with diastolic BP but not SBP. PAI-1 did not correlate with either SBP or DBP but was related to body mass index, diabetes, and dyslipidemia.

CONCLUSIONS

Platelet CD62 demonstrated a strong and graded association with both systolic and diastolic BP that persisted in the presence of multiple concomitant risk factors. The association of BP with CD62P was stronger than with either PAI-1 or tPA-Ag. Platelet activation and platelet CD62 increase in a BP-dependent manner and this relationship persists at extreme levels of BP. Platelet activation and platelet CD62 may participate in the accelerated target organ injury observed in high risk patients with severe hypertension.

An association between traumatic stress and cardiovascular disease (CVD) is supported by various epidemiological studies. Platelet activation and binding of activated platelets to leukocytes contributes to the pathophysiology of CVD. Evidence of hyperactive sympathetic nervous system, altered expression of platelet α(2)-adrenoreceptors (α(2)AR), and altered platelet adenylate cyclase activity in patients with posttraumatic stress disorder (PTSD) suggest that platelet reactivity in PTSD may be altered as well. We tested whether platelet reactivity to increasing doses of adenosine-diphosphate (ADP), epinephrine (EPI), or their combination differs between war veterans with PTSD (n=15) and healthy controls (n=12). For this purpose, citrated whole blood was incubated with increasing concentrations of ADP (0.1, 1, 10 μM), EPI alone (10 nM, 100 nM, 1000 nM), or EPI (10 nM, 100 nM, 1000 nM) in combination with 0.1 μM ADP. A subset of samples was also incubated with 10 μM yohimbine (YOH), α(2)AR antagonist, to distinguish receptor-specific effects. Platelet CD62P expression and formation of platelet-leukocyte aggregates (PLA) [platelet-monocyte (P-Mo), -lymphocyte (P-Ly), and -neutrophil (P-Ne) aggregates] were measured using three-color flow cytometry. Platelet reactivity was higher in war veterans with PTSD when compared to controls, as determined by greater CD62P expression and formation of PLA in response to ADP alone or in combination with EPI. Platelet reactivity also correlated with the severity of PTSD symptoms. Preliminary experiments with YOH indicate that stress-associated EPI elevations may contribute to platelet activation through a α(2)AR-dependent mechanism. The enhanced platelet reactivity observed in our study may be the underlying mechanism contributing to the development of CVD in PTSD patients.

OBJECTIVE

Our study was undertaken to determine whether increased platelet activation occurs in patients with obstructive sleep apnea syndrome (OSAS) and whether a therapy with nasal-continuous positive airway pressure (N-CPAP) alters this activation.

METHODS

We measured the positive rate of activated platelets using activation-dependent monoclonal antibodies (MoAb) and flow cytometry in whole blood from 94 patients with OSAS, and from 31 age-matched controls. Thrombotic vascular diseases were surveyed as a background of alternative of platelet activation.

RESULTS

The positive rate for activated platelets was significantly higher in patients with OSAS ( PAC1 52.6 +/- 22.9 %, CD62P 6.8 +/- 7.1%, mean +/- SD), as compared with healthy control subjects ( PAC1 16.7 +/- 8.6 %, CD62P 0.7 +/- 0.5 %, p < 0.001). The activation indexes were significantly reduced after 1 month with N-CPAP treatment as a whole (PAC1; from 52.6 +/- 22.9 to 44.2 +/- 22.4, p < 0.05, CD62P; from 6.8 +/- 7.1 to 5.3 +/- 5.5, p < 0.05). In nearly 60 % of patients, platelets activation remained high despite significant improvement of sleep apnea-episodes after N-CPAP. These patients had significantly higher incidence of previous myocardial infarction and/or cerebral infarction and abnormalities of head MRI and carotid sonograpy; indicating that the platelet activation appears to be induced by existing atheroma plaque and not by sympathetic activity in OSAS.

CONCLUSIONS

In conclusion, patients with OSAS have increased percentages of activated platelets as assessed by flow cytometrical analysis of activation dependent surface markers, and were divided into two groups, one group with response to N-CPAP treatment in the reduction of platelet activation and the other without. One possible reason of no response to N-CPAP treatment in the reduction of platelet activation was suggested to be thrombotic diseases.

OBJECTIVE

Neurocognitive decline occurs frequently after cardiac surgery and persists in a significant number of patients. Magnesium is thought to provide neuroprotection by preservation of cellular energy metabolism, blockade of the N-methyl-D-aspartate receptor, diminution of the inflammatory response, and inhibition of platelet activation. We therefore hypothesized that intraoperative magnesium administration would decrease postoperative cognitive impairment.

METHODS

After approval by the Duke University Health System Institutional Review Board, 389 patients undergoing cardiac surgery were enrolled in this prospective, randomized, double-blind, placebo-controlled clinical trial. Subjects were randomized to receive magnesium as a 50 mg/kg bolus followed by another 50 mg/kg infusion for 3 hours or placebo bolus and infusion. Cognitive function was assessed preoperatively and again at 6 weeks postoperatively using a standardized test battery. Mean CD11b fluorescence and percentage of platelets expressing CD62P, which are markers of leukocyte and platelet activation, respectively, were assessed by flow cytometry as a secondary outcome. The effect of magnesium on postoperative cognition was tested using multivariable regression modeling, adjusting for age, years of education, baseline cognition, sex, race, and weight.

RESULTS

Among the 389 allocated subjects (magnesium: n=198; placebo: n=191), the incidence of cognitive deficit in the magnesium group was 44.4% compared with 44.9% in the placebo group (P=0.93). The cognitive change score and platelet and leukocyte activation were also not different between the groups. Multivariable analysis revealed a marginal interaction between treatment group and weight such that heavier subjects receiving magnesium were less likely to have cognitive deficit (P=0.06).

CONCLUSIONS

Magnesium administered intravenously during cardiac surgery does not reduce postoperative cognitive dysfunction.

BACKGROUND

http://www.clinicaltrials.gov. Unique identifier: NCT00041392.

Although hematological disorders with salient features of thrombocytopenia have been well documented in dengue patients, the role of CD61-expressing platelets and the megakaryocytic cell lineage in the pathogenesis of dengue virus (DENV) infection remains largely unexplored. A prospective observational study was performed using blood samples and PBMCs from dengue-confirmed patients, as well as from rhesus monkeys (RM) experimentally infected with DENV. Immunohistochemical staining and FACS techniques were applied to evaluate the frequencies of CD61(+) cells that contained DENV antigen. Highly enriched population of CD61(+) cells was also isolated from acute DENV-infected RM and assayed for DENV RNA by quantitative RT-PCR. Results revealed that DENV antigen was found in small vesicles of varying size, and more frequently in anucleated cells associated with platelets in dengue patients. The DENV antigen-containing cells were CD61(+) and appeared to share characteristics of megakaryocytes. Kinetic profiles of CD61(+) cells from DENV-infected RM revealed a transient increase in CD61(+)CD62P(+) cells early after DENV infection. DENV RNA in a highly enriched population of CD61(+) cells from the infected RM was observed during acute stage. Our results indicate that virus containing CD61(+) cells may be directly linked to the platelet dysfunction and low platelet count characteristics of dengue patients.

BACKGROUND

One blood system for PLTs (INTERCEPT, Baxter Transfusion Therapies) is based on photochemical treatment (PCT) with small molecules that target cross-link nucleic acids (Helinx technology, Cerus Corp.) with amotosalen-HCl (S-59) and UVA light (320-400 nm) to inactivate pathogens and WBCs.

METHODS

A two-arm in vitro study was conducted to compare pooled buffy-coat-derived PLT concentrates (PCs) treated with the INTERCEPT blood system, resuspended in PLT additive solution (PAS) III (InterSol, Baxter Transfusion Therapies), and stored for up to 7 days (test units, n = 20) with unpaired, nontreated PCs, resuspended in PAS II (T-Sol, Baxter Transfusion Therapies), and prepared at the same center in the same manner (control units, n = 18).

RESULTS

PLT dose (x 1011/unit +/- SD) on Day 1 immediately following PCT was 3.0 +/- 0.4 for test units and 3.2 +/- 0.4 for control units. After 7 days of storage, the pH of all test units was maintained above 6.8. No marked trend was observed in the hypotonic shock response (HSR). Values among study groups were similar at the end of observation period: 68 +/- 11 percent for control unites versus 67 +/- 8 percent for test units (p>> 0.05). Aggregation response to ristocetin was slightly lower in test units: at Day 7, 65 +/- 10 percent versus 76 +/- 6 percent (p < 0.05). Significantly higher (p < 0.001) glucose consumption, lactate production, and CD62P expression were observed in test units.

CONCLUSIONS

Compared to nontreated PLTs, the PCT process was associated with a variety of differences of in vitro analyses. Although significant, these changes were relatively small in most cases. Characteristics correlated with survival in vivo such as HSR and swirling were comparable between both study groups, indicating that the viability of the majority of cells appears to have persisted throughout 7 days of storage. The impact of this finding, however, remains to be investigated in clinical trials performed with 7-day stored PLTs.

Hormones such as prolactin and leptin have recently been recognized as potent platelet aggregation co-activators, and have therefore been postulated as an additional risk factor for both arterial and venous thrombosis. Clinical situations exist that are known to be associated with higher leptin and/or prolactin levels (obesity, pregnancy, prolactinomas and anti-psychotic therapy respectively) and increased venous thrombosis or atherosclerosis risk. Therefore, we compared the impact of both hormones on platelet activation in vitro and in vivo. First, we investigated platelet aggregation and P-selectin expression after stimulation with 1,000 mU/l prolactin or 100 ng/ml leptin in five healthy volunteers in vitro. Prolactin revealed significant higher levels of P-selectin expression and platelet aggregation than leptin in all subjects. We also compared the correlation of prolactin and leptin values with the P-selection expression on platelets. Previously, we detected a significant correlation between prolactin values and ADP-stimulated P-selectin expression on platelets in pregnant women, patients with pituitary tumours, and patients on anti-psychotic therapy. In contrast, leptin did not correlate with P-selectin expression in all subject groups investigated. However, leptin correlated with body mass index in the subjects investigated. Our data indicate that prolactin has a stronger effect on platelet activation as leptin in vitro and in vivo. Moreover, our data suggest that the stronger effect of prolactin on ADP-stimulated platelet aggregation, compared to leptin, depends on higher stimulation of CD62p expression by prolactin.

Heparin induced thrombocytopenia (HIT) is a well-known complication of heparin administration but usually resolves upon discontinuation without sequelae. However, a small proportion of HIT patients develop thrombosis associated with HIT, designated as HITT, which is often life-threatening and may lead to gangrene and amputations. Existing laboratory methods of confirming HIT/HITT do not distinguish between HIT and HITT. We report a flow cytometric assay of platelet activation marker CD62P to distinguish the effects of addition of HIT vs. HITT plasma to normal blood. Briefly, normal whole blood was incubated with platelet-poor plasma from 12 patients with HITT, 30 with HIT, and 65 controls, in presence and absence of heparin, and expression of CD62P was assayed by flow cytometry. When the ratios of fluorescent intensity of CD62P with heparin divided by that without heparin were compared, HITT plasma induced significantly higher ratios than HIT plasma (HITT ratios approximately 2.5 vs. HIT ratios approximately 1.2; p <0.001). Eleven of 12 HITT patients were positive by this test but only 5 of 30 HIT patients were positive (p <0.0005). In a case of HIT with silent thrombosis, this assay gave a positive results prior to clinically evident thrombosis. In conclusion, this method distinguishes HITT from HIT and may be clinically useful in the detection of HITT, allowing early intervention for preventing catastrophic thrombosis.

BACKGROUND

This study was performed to analyze the influence of either aspirin, ticlopidine, or their combination on platelet activation and aggregation parameters after stent implantation.

RESULTS

Sixty-one patients with successful implantation of a single Palmaz-Schatz stent in a native coronary artery were randomly assigned to either group A (aspirin 300 mg/d+ticlopidine 2X250 mg/d), group B (ticlopidine 2X250 mg/d), or group C (aspirin 300 mg/d). Platelet activation was evaluated on days 1, 7, and 14 by flow cytometry measurement of expression of CD62p (p-selectin) and the binding of fibrinogen to the platelet surface glycoprotein IIb/IIIa receptor. Platelet aggregation was induced by addition of ADP or collagen. Differences between treatment groups were compared by ANOVA. Between days 1 and 14, we observed a significant decrease in collagen-induced platelet aggregation in group A (62.2+/-2.5% versus 36.9+/-3.1%), whereas an increase was seen in group B (58.3+/-2.5% versus 67.7+/-3.2%) and no change was seen in group C (P<.0001). The ADP-induced aggregation declined significantly in group A (74.7+/-1.4% versus 55.3+/-2.6%), whereas a delayed reduction was seen in group B (72.0+/-3.0% versus 52.6+/-4.2%) and no change was seen in group C (P=.0017). The CD62p expression declined significantly in groups A (68.2+/-2.7% versus 41.3+/-2.7%) and B (64.8+/-2.9% versus 39.3+/-3.5%) but not in group C (P<.0001). Moreover, the fibrinogen binding decreased significantly in group A (61.0+/-4.3% versus 36.3+/-4.2%) and with delay in group B (58.3+/-2.2% versus 39.4+/-3.0%), whereas no alterations were seen in group C (P=.012).

CONCLUSIONS

Our results demonstrate synergistic and accelerated platelet inhibitory effects of ticlopidine plus aspirin in patients after stent implantation compared with a monotherapy with either ticlopidine or aspirin alone.

BACKGROUND

The noble gas helium is devoid of anesthetic effects, and it elicits cardiac preconditioning. We hypothesized that inhalation of helium provides protection against postocclusive endothelial dysfunction after ischemia-reperfusion of the forearm in humans.

METHODS

Eight healthy male subjects were enrolled in this study with a crossover design. Each volunteer was randomly exposed to 15 min of forearm ischemia in the presence or absence of helium inhalation. Helium was inhaled at an end-tidal concentration of 50 vol% from 15 min before ischemia until 5 min after the onset of reperfusion ("helium conditioning"). Hyperemic reaction, a marker of nitric oxide bioavailability and endothelial function, was determined at 15 and 30 min of reperfusion on the forearm using venous occlusion plethysmography. Expression of the proinflammatory markers CD11b, ICAM-1, PSGL-1, and L-selectin (CD62L) on leukocytes and P-selectin (CD62P), PSGL-1, and CD42b on platelets were measured by flow cytometry during reperfusion.

RESULTS

Ischemia-reperfusion consistently reduced the postocclusive endothelium-dependent hyperemic reaction at 15 and 30 min of reperfusion. Periischemic inhalation of helium at 50 vol% did not improve postocclusive hyperemic reaction. Helium decreased expression of the proinflammatory marker CD11b and ICAM-1 on leukocytes and attenuated the expression of the procoagulant markers CD42b and PSGL-1 on platelets.

CONCLUSIONS

Although inhalation of helium diminished the postischemic inflammatory reaction, our data indicate that human endothelium, which is a component of all vital organs, is not amenable to protection by helium at 50 vol% in vivo. This is in contrast to sevoflurane, which protects human endothelium at low subanesthetic concentrations.

BACKGROUND

The characterisation and quantification of cell-derived microparticles (MPs) using flow cytometry are often complicated by a low staining intensity and a non-discrete signal pattern of many cell surface antigens. Fluorescence-labelled isotype controls (ICs) are commonly used to set limits for the discrimination of antigen positive vs. negative events.

OBJECTIVE

The influence of different ICs on the characterisation and quantification of MPs was studied. Antigen negative MPs stained with an antibody of interest were evaluated as an alternative control.

METHODS

MPs were prepared from platelets, endothelial cell lines and leucemic cell lines and stained with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labelled antibodies or isotype controls. Results are given as the mean fluorescence intensity (MFI) or percentage of "false-positive" events above a fluorescence intensity>> 1.

RESULTS

Using identical instrument settings, seven different ICs (FITC-conjugates N = 3, PE-conjugates N = 4) resulted in a wide range of MFI and percentage of positive events with a mean coefficient of variation (CV) of 0.77. Instead, NMPs showed less variability with a mean CV of 0.50 and allowed a reliable and reproducible quantification of MPs when set as controls with < 2% false-positive events above an FI>> 1. As a result, the expression of certain antigens (e.g. CD62P) was lower compared to previous reports in the literature.

CONCLUSIONS

Diversity in the staining intensity of isotype controls is a potential source of error in the characterisation and quantification of MPs by flow cytometry. The use of antigen negative MPs to adjust instrument settings is suggested.

This study was to determine the impact of adjunctive Buchang Naoxintong Jiaonang (BNJ) to clopidogrel on volunteers with the CYP2C19*2 gene mutation accompanied with qi deficiency and blood stasis (QDBS) constitution. Eighteen males with QDBS constitution were selected, who were 6 CYP2C19*1/*1, 6 CYP2C19*1/*2, and 6 CYP2C19*2/*2, and signed informed consent. Results showed that the maximal platelet aggregation (Agg(max)) and 5 min aggregation (Agg(late)) with 5-μmol/L ADP in three different CYP2C19*2 genotypes were significantly decreased after any drug therapy compared with corresponding baseline measurements (all values P < .05). But percent inhibitions of Agg(max) and Agg(late) (IPAs) in CYP2C19*2/*2 genotype at 4 hours, 24 hours, 3 days, and 7 days after clopidogrel administration were all the lowest among three CYP2C19*2 genotypes (P < .01), and IPAs in CYP2C19*1/*2 genotype were between CYP2C19*1/*1 and CYP2C19*2/*2. IPAs had no significant difference among three different CYP2C19*2 genotypes after BNJ or adjunctive BNJ. In addition, changes of CD62P, PAC1, and sCD40L were similar to changes of ADP-induced platelet aggregation in three different CYP2C19*2 genotypes. Conclusion was that adjunctive BNJ to clopidogrel can enhance the antiplatelet effect in volunteers with the CYP2C19*2 gene mutation.

Platelets functions in hemostasis have been widely studied. Currently, growing evidence shows that platelets have also a role in the immune innate response. Recently, protein expression of Toll-like receptors (TLR's) 2, 4, 7, 8, and 9, and the presence of TLRs 1 and 6 mRNA in human platelets was described. Up to now the functionality of TLR-2, 4 and 9 in human platelets has been demonstrated. Due to the relevance of TLRs functions to PAMPS (pathogen-associated molecular patterns) recognizing, we evaluated the presence of TLR3 in human platelets founding low percentages of platelets expressing surface or intracellular TLR3 protein. The activation with thrombin induced an increase in the percentage of platelets expressing surface TLR3 and higher levels of TLR3 expression in the whole population. Human platelets responded to poly I:C by increasing [Ca(2+)]i, the percentages of cells expressing TLR4 and CD62P, and by releasing CXCL4 and IL-1β in comparison to unstimulated platelets. These results demonstrate that human platelets express TLR3 and are capable of responding to poly I:C, suggesting that these cells might influence the immune innate response when detecting viral dsRNA.

The immuno-inflammatory responses to titanium miniplates used in the treatment of mandibular fractures were studied immunohistochemically at light and electron microscope levels. Titanium miniplates were stably situated on the cortical bone surface. In the soft tissue adjacent to the surface of titanium miniplates, double layered connective tissue was observed, which consisted of dense fibrous connective tissue, and relatively loos connective tissue contained proliferated blood vessels with hypertrophied endothelial cells. These vascular endothelial cells expressed HLA-DR, CD54 and CD62P antigens. In some cases they were CD62Epositive. CD68+ and CD11c+ round or spindle-shaped macrophages had infiltrated around the small vessels. Fine titanium particles were observed in the cytoplasm of these macrophages. Both CD4+ and CD8+ T lymphocytes had also infiltrated around venules in some cases. They were CD4+ T lymphocyte-dominant. Immunoelectron microscopically, CD68+ and CD11c+ macrophages contained titanium particles in the lysosomes. Most of the macrophages showed varying degrees of degenerative change. The presence of titanium was confirmed by energy-dispersive X-ray analysis.

BACKGROUND

Deterioration of platelet (PLT) quality during storage is accompanied by an increase in lactate production, indicating a decrease in mitochondrial function. In this study, the optimal conditions under which the fluorescent dye JC-1 can be used to detect changes in mitochondrial function in PLTs were established.

METHODS

PLTs were incubated at 37 degrees C in synthetic medium under various conditions of JC-1 loading. In the presence of a high membrane potential, this dye accumulates in the mitochondria with a concomitant increase in red fluorescence. After JC-1 loading, the ratio of red (FL2) to green (FL1) fluorescence was determined by flow cytometry.

RESULTS

The FL2-to-FL1 ratio of PLTs (3 x 10(7)/mL, loaded with 0.5 micromol/L JC-1) amounted to about 5 in 1-day-old PLTs. At higher dye concentrations, the FL2-to-FL1 ratio was significantly lower, suggesting uncoupling by the dye itself. Plasma concentrations above 3 percent significantly affected the JC-1 signal. The FL2-to-FL1 ratio showed a dose-dependent decrease to an uncoupler of oxidative phosphorylation or to inhibition of the respiratory chain. JC-1-loaded PLTs showed a clear decrease in FL2-to-FL1 ratio after prolonged storage or upon ultraviolet (UV) illumination. Only after UV treatment did changes in JC-1 signal correlate with changes in CD62P expression.

CONCLUSIONS

The FL2-to-F1 ratio of PLTs loaded with JC-1 is a reliable and sensitive indicator of the mitochondrial membrane potential, provided that the proper experimental conditions have been applied.

OBJECTIVE

Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in patients with dyslipidemic disorders. Although oxLDL stimulates activating signaling, researchers have not clearly determined how these events drive accelerated thrombosis. Here, we describe the mechanism by which ROS regulate autophagy during ox-LDL-induced platelet activation by modulating the PI3K/AKT/mTOR signaling pathway.

METHODS

For in vitro experiments, ox-LDL, the ROS scavenger N-acetylcysteine (NAC), the mTOR inhibitor rapamycin and the autophagy inhibitor 3-MA were used alone or in combination with other compounds to treat platelets. Then, platelet aggregation was evaluated on an aggregometer and platelet adhesion was measured under shear stress. The levels of a platelet activation marker (CD62p) were measured by flow cytometry, reactive oxygen species (ROS) levels were then quantified by measuring DCFH-DA fluorescence intensity via flow cytometry. Nitric oxide (NO) and superoxide (O2·-) levels were determined by the nitric acid deoxidize enzyme method and lucigenin-enhanced chemiluminescence (CL), respectively. Transmission electron microscopy was used to observe the autophagosome formation, immunofluorescence staining was employed to detect LC3 expression and western blotting was used to measure the levels of PI3K/AKT/mTOR pathway- and autophagy-related proteins.

RESULTS

Ox-LDL-induced platelets showed a significant increase in platelet aggregation and adhesion, CD62p expression, ROS level and O2·- content, with an elevated LC3II/LC3I ratio and Beclin1 expression, but a dramatic reduction in the levels of p62 and pathway-related proteins (all P < 0.05). However, platelet activation and autophagy were aggravated by the Rapamycin treatment, and decreased following treatment with NAC, 3-MA, or NAC and 3-MA, together with increased activity of the PI3K/AKT/mTOR pathway. Additionally, decreased platelet activation and autophagy were observed in platelets treated with NAC and Rapamycin or Rapamycin and 3-MA compared with platelets treated with Rapamycin alone, suggesting that both NAC and 3-MA reversed the effects of Rapamycin.

CONCLUSIONS

Inhibition of ROS production may reduce autophagy to suppress ox-LDL-induced platelet activation by activating PI3K/AKT/mTOR pathway.

We tested the ability of platelet-derived extracellular vesicles (PEV) to promote adhesion of flowing neutrophils to endothelial cells (EC). PEV were collected from platelets stimulated with collagen-related peptide, and differential centrifugation was used to collect larger vesicles enriched for platelet membrane microvesicles (PMV) or smaller vesicles enriched for platelet exosomes (Pexo). Vesicle binding and resultant activation of neutrophils and EC were assessed by flow cytometry. Flow-based adhesion assays assessed binding of neutrophils directly to deposited vesicles or to EC, after neutrophils or EC had been treated with vesicles. PEV bound efficiently to neutrophils or EC, with resultant upregulation of activation markers. Binding was Ca++-dependent and dominantly mediated by CD62P for neutrophils or by integrins for EC. Deposited PEV supported mainly transient attachments of flowing neutrophils through CD62P and some stable adhesion through CXC-chemokines. Neutrophil adhesion to EC was promoted when either cell was pre-treated with PEV, although the effect was less prominent when EC were pre-activated with tumor necrosis factor-α. The pro-adhesive effects on neutrophils could largely be attributed to the larger PMV rather than Pexo. Thus, surface-bound PEV can capture flowing neutrophils, while PEV also activate neutrophils and EC to promote interactions. PEV may potentiate inflammatory responses after tissue injury.