We investigated whether remote ischemic preconditioning (RIPC) inhibits agonist-induced conformational activation of platelet α_IIb β₃ in patients with coronary artery disease already receiving conventional antiplatelet therapy.

Consecutive patients with angiographically confirmed coronary artery disease were randomized to RIPC or sham treatment. Venous blood was collected before and immediately after RIPC/sham. Platelet aggregometry (adenosine diphosphate (ADP), arachidonic acid) and whole blood platelet flow cytometry was performed for CD62P, CD63, active α_IIb β₃ (PAC-1 binding) before and after stimulation with ADP, thrombin +/- collagen, or PAR-1 thrombin receptor agonist.

Patients (25 RIPC, 23 sham) were well matched, 83% male, age (mean ± SD) 63.3 ± 13.2 y, 95% aspirin, 81% P2Y₁₂ inhibitor. RIPC did not affect platelet aggregation, nor agonist-induced expression of CD62P, but selectively and significantly decreased α_IIb β₃ activation after stimulation with either PAR-1 agonist peptide (SFLLRN) or the combination of thrombin + collagen, but not after ADP nor thrombin alone. The effect of RIPC on platelet α_IIb β₃ activation was evident in patients receiving both aspirin and P2Y₁₂ inhibitor, and was not associated with an increase in vasodilator stimulated phosphoprotein (VASP) phosphorylation.

RIPC inhibits conformational activation of platelet α_IIb β₃ in response to exposure to thrombin and collagen in patients with coronary artery disease receiving dual antiplatelet therapy. These findings indicate agonist-specific inhibition of platelet activation by RIPC in coronary artery disease which is not obviated by the prior use of P2Y₁₂ inhibitors.

We report 4 cases of pseudothrombocytopenia due to platelet cold agglutinins. Case 1 was a 57 y.o. female whose platelet count was 97 x 10(3)/microl. Case 2 was a 37 y.o. male with a platelet count of 96 x 10(3)/microl. Case 3 was a 74 y.o. male with a platelet count of 28 x 10(3)/microl. Case 4 was a 62 y.o. female whose platelet count was 34 x 10(3)/microl. The platelet counts in these 4 cases were decreased and blood smears showed platelet clumping in blood drawn in a tube without anticoagulant just after withdrawal, as well as in blood drawn in a tube with anticoagulant. The platelets from these patients agglutinated at a temperature below 10 degrees C (case 1 and 4) and 24 degrees C (case 2). The immunoglobulin class of the platelet cold agglutinins in cases 1, 2 and 4 was IgM. Agglutinated platelets showed no activation marker, such as CD62P, CD63 or CD40L, on the surface of the platelets. The target antigen of cold agglutinins was GPIIb-IIIa in cases 1 and 2. We considered that the detection of platelet agglutination in blood without anticoagulant is important to diagnose pseudothrombocytopenia due to platelet cold agglutinins. Although this disease is considered to be very rare, we suspect that this disease may be misdiagnosed as pseudothrombocytopenia due to the presence of an anticoagulant, and overlooked.

Activated platelets form platelet-leukocyte aggregates in the circulation in inflammatory diseases. We investigated whether activated platelets in inflamed skin tissues are phagocytized and removed by neutrophils. To investigate the kinetics of platelets and neutrophils, we immunohistochemically examined the spatiotemporal distribution of them in a murine model of 2,4,6-trinitro-1-chlorobenzene (TNCB)-induced dermatitis by using confocal and structured illumination microscopy. Four hours after elicitation, aggregates of CD41-positive platelets were adhered to CD31-positive endothelial cells within the vessels, and CD62P and PF4, markers of activated platelets, were expressed on platelet aggregates. At 8 hour post-elicitation, fragmented CD41-positive platelets were located both inside and outside vessels. Twenty-four hours after elicitation, the number of Ly-6G-positive neutrophils ingesting fragmented CD41-positive platelets outside vessels was increased, and CD62P and PF4 expression on the phagocytosed platelets was no longer observed. Disc-shaped CD41-positive platelets were not found outside vessels at any time during the experiment. Our data revealed that aggregates of activated platelets inside vessels were ingested and removed by neutrophils in the early stage of TNCB-induced dermatitis, suggesting that the process of removal of activated platelets by neutrophils may play an important role not only in the early phase of skin inflammation but also in other types of acute inflammation.

To gather further information about the effects on blood platelet activation of in vivo exposure to nitric oxide (NO), platelet reactivity was studied in blood from healthy, non-smoking male volunteers before and after 30 min inhalation of 40 ppm NO. Whole blood was stimulated in vitro with adenosine diphosphate or thrombin receptor activation peptide (TRAP-6). In an ex vivo perfusion model, non-anticoagulated blood was exposed to immobilised collagen at arterial blood flow conditions (2600 s(-1)). Blood samples from both the in vitro and ex vivo experiments were stained with fluorochrome-labelled Annexin-V and antibodies against CD42a, CD45, CD49b, CD61, CD62P and fibrinogen, and analysed with a three-colour flow cytometry technique. NO inhalation reduced the platelet activation response to adenosine diphosphate (ADP) stimulation by decreasing platelet-platelet aggregation, alpha-granule release and platelet-leukocyte conjugate formation. TRAP-stimulated platelet activation, collagen-induced platelet activation and thrombus growth was unaffected by NO inhalation. We therefore suggest an ADP receptor inhibitor mode of action of inhaled NO, selective on the newly suggested G protein- and phospholipase C-coupled P2Y1 receptor. Our results demonstrate that blood platelet activation in healthy subjects is modulated by inhalation of NO in therapeutically relevant doses, although the clinical impact of our findings remains unclear.

Thrombosis is the prognostic factor with the greatest effect on survival in patients with paroxysmal nocturnal hemoglobinuria (PNH), who lack dozens of membrane surface proteins. We recently described a primary homozygous Cys89Tyr congenital nonfunctioning CD59 in humans with clinical manifestation in infancy, associated with chronic hemolysis, recurrent strokes, and relapsing peripheral demyelinating neuropathy. Here we investigated hypercoagulability mechanisms characterizing the syndrome.

Membrane attack complex (MAC) deposition (anti-SC5b-9) and free hemoglobin (colorimetric assay) were assessed. Platelet activation was identified (anti-CD61, anti-CD62P), and microparticles (MPs) of 0.5-0.9 μm, were characterized (Annexin V, anti-human GlyA, anti-CD15, anti-CD14, anti-CD61). Platelet-monocyte aggregation was assessed with FlowSight.

2/7 patients (29%) with homozygosity for Cys89Tyr and 6/12 (50%) with any of four described CD59 mutations had recurrent strokes. In plasma samples from four patients carrying identical mutations, MAC deposition was increased on RBCs (p < 0.0003), neutrophils (p < 0.009), and platelets (p < 0.0003). Free-plasma hemoglobin levels were abnormally high, up to 100 mg/dl. Patients with CD59 mutation had RBC-derived MP levels 9-fold higher than those in healthy controls (p < 0.01), and 2-2.5 fold higher than PNH patients (p < 0.09). Leukocyte-activated platelet aggregation was increased (p < 0.0062). Loss of CD59 was shown in the endothelium of these patients.

Nonfunctioning CD59 is a major risk factor for stroke and hypercoagulability. Uncontrolled hemolysis causes massive MP release and endothelial heme damage. MAC attack on unprotected endothelium and platelet activation and aggregation with leukocytes mediate additional mechanisms leading to vascular occlusion. It is suggested that CD59 loss represents a major arterial prothrombotic factor in PNH and additional diseases.

Cystic fibrosis (CF) is an inflammatory lung disease. Platelets have an emerging role in inflammation, however previous studies of platelet activation in CF have generated conflicting results. In this study, we determined platelet function in CF patients and correlated platelet activation to establish clinical and laboratory parameters. Twenty-two patients, aged 20.7 to 54.4 (mean 34.0, SD 9.45) years and with a mean FEV1%pred (forced expiratory volume in one second, % of predicted) of 72 (SD 21.4, range 32-110) were recruited. A combination of platelet assays was used: platelet aggregation, platelet activation and platelet-leukocyte complex formation. Platelets from CF patients exhibited significantly increased aggregation when stimulated ex-vivo, a tendency towards increased platelet upregulation of CD62P, but no increase of GPIIb/IIIa activation (PAC-1). Platelet-monocyte complex (PMC) formation was significantly increased in CF patients compared to controls, while platelet-neutrophil complex formation was not. In the CF group, platelet aggregation correlates with levels of anti-neutrophil cytoplasmic antibodies (ANCA) with specificity for bactericidal/permeability-increasing protein (BPI), BPI-ANCA (r=0.56). The formation of PMCs correlates with lung function decline (1-FEV1%), CRP and BPI-ANCA (r=0.61, 0.55, 0.5). We therefore confirm the presence of increased platelet activation in CF patients, and determine that further evaluation of platelet activation in relation to prognostic factors in CF is warranted.

The significance of three platelet membrane glycoproteins, (CD62p, CD63, and CD36) was studied in platelet interaction with native and copper-oxidized plasma low-density lipoproteins (LDL). Native LDL acquired platelet-activating ability only after oxidation. Flow-cytometric studies showed that CD62P and CD63 were rapidly expressed on platelets at a low concentration of oxidized LDL, indicating that they are more sensitive markers than platelet aggregation for detection of platelet activation by oxidized LDL. CD36 was found to be contributing to the binding and activation of platelets with oxidized LDL, but not to the binding of platelets with native LDL. Although the presence of oxidized LDL in plasma is known, its effect on platelet function remains to be studied.

OBJECTIVE

To evaluate the effects of ticagrelor and high-dose clopidogrel on the platelet functions in patients with inadequate response to clopidogrel.

METHODS

In this prospective, randomized and controlled study, patients who had been diagnosed as acute coronary syndrome (ACS) with inadequate response to clopidogrel in the Second Hospital of Hebei Medical University from July 2015 to June 2016 were enrolled. Inadequate response to clopidogrel was defined as absolute reduction of platelet aggregation rate (PAR) <30% or PAR >70%. Eligible patients were randomly assigned to two groups, the high-dose group and the ticagrelor group. Clinical information and intervention protocols were compared. The PAR of the two groups were measured at the time of baseline, the 24th hour, 72nd hour, and the 7th day after treatments, the other platelet-related parameters were measured including platelet count (PLT), mean platelet volume (MPV), platelet distribution width (PDW) at the same time points. Besides, the markers of platelet activation human P-selectin (CD62P) and thromboxane A2 (TXA2) were also recorded to compare. The incidence of major adverse cardiac events (MACE) and the side effects between two groups were followed up for three months.

RESULTS

A total of 74 patients were finally enrolled, 38 of whom were assigned to the ticagrelor group and the rest of them to the high-dose clopidogrel group. The baseline clinical and procedural characterists were similar. There were no significant differences in baseline levels of PAR between the two groups [(79.38±11.20)% vs. (73.97±12.74)%, P>0.05]. For both groups, the levels of PAR significantly decreased at each time point (P<0.001). Besides, the levels of PAR in ticagrelor group were lower than those in high-dose clopidogrel group at the 24th hour, 72nd hour and 7th day after treatments: [(25.92±10.31)% vs. (37.95±11.63)%, P<0.001], [(28.02±14.61)% vs. (30.64±10.73)%, P<0.001], [(37.17±11.11)% vs. (36.80±7.26)%, P<0.001]. The baseline levels of platelet related parameters were similar between the two groups (P>0.05), and there were no significant differences in the levels of PLT, PDW, and MPV at the 24th hour, 72nd hour and 7th day. It was lower in ticagrelor group than that in clopidogrel group at the 24th hour [(5.47±1.03) ng/ml vs. (8.02±1.45) ng/ml, P<0.001] while the CD62P concentrations in the two groups significantly decreased comparing to the baseline levels (P<0.001). During 3-month follow-up, the incidences of MACE and side effects were not significantly different between the two group.

CONCLUSIONS

ticagrelor could further decrease levels of platelet aggression rate and CD62p compared with high-dose clopidogrel, without serious side effects.

Platelet flow cytometry is restricted by spontaneous platelet activation in unfixed samples or by significant alterations of platelet performance caused by the use of fixatives. Aim of this study was to evaluate the new platelet stabilizer ThromboFix for clinical diagnostics.

METHODS

Whole blood samples with or without addition of a weak (ADP) or strong (TRAP-6) agonist were fixed with either ThromboFix or paraformaldehyde (PFA) and stored for different periods of time for up to 7 days. Samples were then incubated with either CD41 and CD62P or CD42b and CD45 monoclonal antibodies and analyzed by flow cytometry.

RESULTS

The numbers of platelets, microparticles and aggregates remained stable for 7 days after treatment with ThromboFix but not with PFA due to an increasing aggregate formation after 3 days. Platelet activation was restricted to less than 1% of CD62P positive events in resting samples without a significant difference compared to an unfixed reference sample. Fixation, however, significantly reduced CD62P expression after stimulation (P < 0.05). Stabilized by ThromboFix, the level of platelet activation remained unchanged in resting and ADP stimulated samples for 7 days but decreased moderately with time after a strong stimulation with TRAP-6 (P < 0.01). After PFA fixation, intact CD62P antigen disappeared from the platelet surface within hours (P < 0.01). ThromboFix reduced the formation of platelet-leukocyte conjugates significantly (P < 0.05) and, in contrast to PFA, failed to stabilize the already formed conjugates.

CONCLUSIONS

In clinical situations without immediate access to a flow cytometer, ThromboFix is helpful in the flow cytometric analysis of the platelet activation marker CD62P. It should not be used for the investigation of platelet-leukocyte conjugate formation.

: Freeze-drying is an effective means of storing platelets. In this study, we investigated the effects of a protective agent on freeze-dried platelet-rich plasma (FD-PRP) after a 12-week preservation period. Platelet structure was measured by transmission electron microscopy (TEM), and the expression levels of procaspase activating compound (PAC)-1 and CD62P were measured by flow cytometry. The levels of transforming growth factor-beta (TGF-β), platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) were determined by ELISA. The effect of FD-PRP on cell proliferation was measured by cell counting. TEM revealed that most platelets were intact, and their internal structure was evident. The expression levels of the platelet activation marker CD62P in FD-PRP and fresh PRP were 36.83% ± 8.21 and 35.47% ± 4.11, respectively, without a significant difference (P > 0.05). The expression levels of PAC-1 in FD-PRP and fresh PRP were 3.23% ± 0.49 and 2.83% ± 0.44, respectively, without a significant difference (P > 0.05). Upon activation of FD-PRP and fresh PRP by thrombin, the levels of TGF-β, PDGF and VEGF were not significantly decreased in FD-PRP. Moreover, FD-PRP promoted cell proliferation in a manner similar to that of fresh PRP. The protective agent maintained the biological activity of FD-PRP after a 12-week preservation period.

Alzheimer's disease (AD) is a neurodegenerative disease with a complex and not fully understood pathogenesis. Besides brain-intrinsic hallmarks such as abnormal deposition of harmful proteins, i.e., amyloid beta in plaques and hyperphosphorylated Tau in neurofibrillary tangles, blood-derived elements, in particular, platelets have been discussed to be involved in AD pathogenesis. The underlying mechanisms, however, are rather unexplored. Here, we investigate a potential role of platelets in an AD transgenic animal model with severe amyloid plaque formation, the APP-PS1 transgenic mice, and analyzed the presence, spatial location and activation status of platelets within the brain. In APP-PS1 mice, a higher number of platelets were located within the brain parenchyma, i.e., outside the cerebral blood vessels compared to WT controls. Such platelets were activated according to the expression of the platelet activation marker CD62P and to morphological hallmarks such as membrane protrusions. In the brain, platelets were in close contact exclusively with astrocytes suggesting an interaction between these two cell types. In the bloodstream, although the percentage of activated platelets did not differ between transgenic and age-matched control animals, APP-PS1 blood-derived platelets showed remarkable ultrastructural peculiarities in platelet-specific organelles such as the open canalicular system (OCS). This work urges for further investigations on platelets and their yet unknown functional roles in the brain, which might go beyond AD pathogenesis and be relevant for various age-related neurodegenerative diseases.

This prospective observational study was designed to analyze platelet functions across time in 50 patients scheduled for liver transplantation (LT) secondary to decompensated cirrhosis or hepatocellular carcinoma. Platelet functions were assessed before LT (pre-LT), one week (D7) and 1 month (D28) after LT. Platelet count significantly increased from pre-LT time to day 28 as well as circulating CD34+hematopoietic stem cells. To avoid any influence of platelet count on assays, platelet function was evaluated on platelet-rich-plasma adjusted to pre-LT platelet count. Although platelet secretion potential did not differ between time-points, as evaluated by the expression of CD62P upon strong activation, platelet aggregation in response to various agonists significantly increased along time, however with no concomitant increase of circulating markers of platelet activation: platelet microvesicles, platelet-leukocyte complexes, soluble CD40L and soluble CD62P. In the multivariate analysis, hepatic function was associated with platelet count and function. A lower platelet aggregation recovery was correlated with Child C score. History of thrombosis or bleeding was associated with respective higher or lower values of platelet aggregation. This longitudinal analysis of platelet functions in LT patients showed an improvement of platelet functions along time together with platelet count increase, with no evidence of platelet hyperactivation at any time-point.

OBJECTIVE

Platelet-leukocyte aggregation is believed to contribute to acute thrombotic events. While the effect of aspirin on platelet-to-platelet aggregation is well established, the impact of the drug on pro-inflammatory platelet function remains equivocal. Thus we investigated the effect of aspirin on selected platelet-related inflammatory biomarkers in both acute ischaemic stroke patients and healthy volunteers.

METHODS

Using five-colour flow cytometry the platelet surface expression of CD62P and CD40L and subpopulations of leukocyte-platelet aggregates were assessed in 63 acute stroke patients and 40 healthy volunteers at baseline and after a 10-day period of aspirin intake at a daily dose of 150 mg. Simultaneously the plasma levels of soluble CD62P and CD40L, serum level of TxB(2), and whole blood impedance platelet aggregation under arachidonic acid (AA) stimulation were investigated.

RESULTS

No differences in values of studied platelet-related inflammatory biomarkers in both resting platelets and those activated with TRAP after 10-day treatment with aspirin were confirmed in stroke subjects. In healthy individuals the resting platelet expression of CD62P, plasma level of soluble CD62P and percentage of circulating monocyte-platelet aggregates were lower after the aspirin intake period (P=0.009; P=0.04; P=0.004, respectively). In both studied groups serum level of TxB(2) and platelet aggregation under AA stimulation were lower than before treatment (P<0.001).

CONCLUSIONS

Despite effective inhibition of COX-1-dependent platelet aggregation, aspirin does not influence the platelet α-granule-derived inflammatory mediators and monocyte-platelet aggregation in acute stroke subjects, although it does in healthy individuals.

Essential thrombocythemia (ET) is characterized by persistently elevated platelet counts and an increased risk of thromboembolic events. Dysregulated expression of small noncoding microRNAs (miRNAs) have been shown in ET and may influence platelet maturity and function in ET patients. In this study, we included 22 ET patients and 19 healthy controls to investigate the expression of 12 platelet miRNAs previously reported to be dysregulated in ET. Further, we investigated the correlation between the expression of selected miRNAs and platelet maturity and platelet function. Total RNA was isolated from platelets, and expression analyses were performed using TaqMan quantitative PCR (qPCR). Mean platelet volume (MPV) and immature platelet count and -fraction (IPC and IPF) were measured using the Sysmex XE-5000 automated haematology system. Platelet function was investigated by multiple electrode aggregometry (agonists: arachidonic acid (AA), thrombin-receptor-activating-peptide (TRAP) and adenosine diphosphate (ADP)), while platelet activation was determined by multi-colour flow cytometry (antibodies: bound-fibrinogen, CD63 and P-selectin (CD62p), agonists: AA, TRAP and ADP). We showed that miR-9 and miR-490 were significantly upregulated in ET patients compared with healthy controls (p-values < 0.01), while miR-10a, miR-28, miR-126, miR-155, miR-221, miR-222, miR-223 and miR-431 were significantly downregulated in ET patients (all p-values < 0.001). A significant positive correlation was observed between miR-431 and MPV, IPC and IPF (all p-values < 0.05). The expression of miR-126 was negatively correlated with platelet aggregation induced by AA and TRAP (p < 0.05). In addition, we found the expression of miR-9 and miR-490 to be negatively correlated with the percentage of fibrinogen-, CD63- and P-selectin- positive platelets using TRAP as agonist (p < 0.05). In conclusion, our data indicate that platelet microRNAs may play a role in ET and that specific microRNAs are correlated with platelet maturity and platelet function.

Platelets express key proteins of the proteasome system, but its functional role in the regulation of platelet integrity, however, is not fully understood yet. Therefore, this study evaluated activating and inhibitory platelet signalling pathways using the potent and selective proteasome inhibitor bortezomib. In washed platelets, the effect of bortezomib on viability and on aggregation was assessed. In addition, fibrinogen binding and CD62P expression were determined. The influence on activating and inhibitory signalling was detected by phosphorylation levels of essential messenger molecules. Platelet viability was maintained after incubation with 0.01 μM to 1 μM bortezomib, but tampered with 100 μM bortezomib. Agonist-induced aggregation was only reduced under 100 μM bortezomib and with weak induction by 10 μM adenosine diphosphate. Similarly, phosphorylated kinase levels of the activating signalling pathways were not affected by 0.01 μM to 1 μM bortezomib. In contrast, proteasome inhibition resulted in the reduction of inhibitor-induced vasodilator-stimulated phosphoprotein phosphorylation, accompanied with the partial decrease of induced inhibition of fibrinogen binding and CD62P expression. In conclusion, platelet activation and aggregation are not dependent on proteasome activity. Instead, inhibitory signalling is partially attenuated under proteasome inhibition. Supramaximal inhibitory concentrations of bortezomib (above 1 μM) lead to heterogeneous effects on activating or inhibitory systems, probably caused by decreasing platelet viability.

BACKGROUND

Vascular dementia is related to intracranial arteriosclerosis associated with deep white matter lesions (DWMLs). DWMLs have been linked to thrombogenesis due to sustained platelet activation; therefore, an accurate hematological marker is needed. This study was done to evaluate the usefulness of a new method to examine the function of activated platelets in order to assess DWMLs associated with cognitive decline.

METHODS

A total of 143 individuals (70.4 ± 6.1 years old) who underwent hospital-based health screening using head MRI were evaluated. DWLs were evaluated on T2-weighted and FLAIR images by semi-quantitatively grading them from Grade 0 (none) to Grade 3 (severe) using the Fazekas classification. Cognitive function was evaluated using the MMSE and the word fluency test. Platelet activation was assessed using fluorescence-labeled anti-human platelet monoclonal antibodies and semi-quantitatively determining PAC-1- and CD62P-positive rates by flow cytometry.

RESULTS

Significant increases in hypertension and CD62P levels were observed with increasing DWML grade (2.6% in Group 0, 3.1% in Group 1, 4.1% in Group 2, and 5.0% in Group 3). CD62P levels were defined as elevated when they were above the mean+2SD of the Grade 0 group, and the odds ratio of the Grade 2+3 group was 3.03. A significant negative correlation was observed between CD62P levels and word fluency tests or the MMSE score.

CONCLUSIONS

Elevations in CD62P levels, which reflect platelet function activation, were associated with white matter lesions accompanied by a decline in cognitive function. CD62P levels may be useful as a sensitive clinical marker for the early detection of DWMLs with cognitive decline.

BACKGROUND

Despite similar residual factor VIII activity, patients with haemophilia A (HA) show significant interindividual variability with regard to bleeding frequency and severity, suggesting that additional factors modulate thrombin generation and fibrin deposition. Protein disulphide isomerase (PDI) is an abundant oxidoreductase that exerts pleiotropic effects in primary and secondary haemostasis and contributes to thrombosis and vascular inflammation.

OBJECTIVE

We conducted a pilot study to explore a potential role of platelet PDI in patients with HA.

METHODS

Expression and release of platelet PDI were studied by flow cytometry and enzyme-linked immunosorbent assay, respectively.

RESULTS

Compared to healthy male controls (n = 12), patients with HA (n = 24) showed significantly increased expression of PDI antigen on ADP- or TRAP-6-, but not on buffer-treated platelets, a finding that could not be explained by enhanced platelet activation, as indicated by expression of the α-granule protein, CD62P (P-selectin). While platelet agonists did not affect PDI secretion in healthy male controls, increased levels of PDI antigen were found in supernatants of TRAP-6-treated platelets from patients with HA. Importantly, in two patients with exceedingly high TRAP-6-induced PDI release over baseline, findings were consistent when platelets were isolated and stimulated on a separate occasion. No obvious association was found between platelet PDI and bleeding phenotype in this patient cohort.

CONCLUSIONS

Agonist-induced expression and release of platelet PDI were increased in patients with HA. Larger studies are needed to clarify if variations in this platelet response contribute to the diversity in bleeding frequency and severity among patients with congenital factor VIII deficiency.

BACKGROUND

Platelets are main effector cells in haemostasis and also promote inflammation. Platelet-leukocyte complexes are key mediators in a variety of thromboinflammatory disorders and consecutive organ failure. Cell-specific epitopes and activation markers on platelets and leukocytes can be measured using flow cytometry. However, until recently a major restriction has been a paucity in antibody combinations and lack of detection strategies. We aimed to develop a six-colour flow cytometry method which depicts multiple aspects of platelet and leukocyte interactions in human whole blood.

METHODS

Platelets, including microparticles and aggregates, were detected in flow cytometry using a platelet-specific anti-CD41-FITC antibody and size-defined regions. The morphology of platelet-leukocyte complexes (including granulocyte and monocyte content) were depicted using anti-CD45-PerCP, anti-CD66b-PE-Cy7, and anti-CD14-APC antibodies in a single sample. Expression of platelet and leukocyte activation markers P-selectin and CD11b were detected using anti-CD62P-PE and anti-CD11b-BV421 antibodies, respectively.

RESULTS

The sensitivity of this assay to detect the effects of various agonists (TRAP-6, ADP, collagen, epinephrine, TNF-α and LPS) is demonstrated. Furthermore, the assay is shown to detect platelet and leukocyte activation induced by extracorporeal circulation in vitro. The suitability of this assay for bedside analysis is demonstrated exemplarily in a patient treated with mechanical circulatory life support.

CONCLUSIONS

Using the concurrent assessment of multiple parameters, this method gives detailed insights into the complexity and dynamics of platelet-leukocyte interactions. This assay carries the potential to increase our understanding of the mechanisms and pathophysiology of platelet-leukocyte interaction in the research laboratory and clinical setting.

Concomitant antithrombotic therapy is essential for the prevention of ischaemic events in percutaneous coronary intervention (PCI) and stenting. With new anticoagulant medications being developed and applied in PCI, this raises the question of possible interactions with platelet and leukocyte activation. We therefore sought to investigate the influence of bivalirudin and heparin in platelet and leukocyte activation in patients undergoing elective PCI. Forty-six patients were recruited consecutively in the setting of the Intracoronary Stenting and Antithrombotic Regimen-Rapid Early Action for Coronary Treatment (ISAR-REACT)-3 trial and were randomly assigned to receive either unfactionated heparin or bivalirudin during elective PCI. Surface expression of CD62P (P-Selectin), CD42b (GPIbalpha), CD40L, PAC-1 on circulating platelets and CD11b, CD14 and CD15 on circulating leukocytes were evaluated by flow cytometry. Cytokine levels of IL-12p70, tumour necrosis factor (TNF), IL-8, IL-6, IL-1beta and IL-10 were determined by cytometric bead array. Platelet surface expression of PAC-1, P-Selectin and GPIbalpha was significantly reduced after PCI in patients receiving bivalirudin as compared to heparin. Similarly, CD11b expression on CD14+ monocytes was diminished after bivalirudin. However, no differences were observed in cytokine levels between the bivalirudin and the heparin group, before or after PCI. In conclusion, our data suggest that bivalirudin may reduce platelet and monocyte activation in patients undergoing elective PCI. Thereby, bivalirudin might reduce periinterventional thrombotic complications.

BACKGROUND

Preventing the activation of PLTs may ameliorate (or mitigate) the PLT storage lesion (PSL), which encloses all structural and biochemical changes caused by collection, processing, and storage of PLT concentrates (PCs). Partial inhibition of PLT function due to ingestion of aspirin (ASA) by blood donors reduces the functional activity of the collected PLTs, however, by preventing premature PLT activation, it might reduce the PSL as well.

METHODS

In a randomized crossover study, 10 healthy donors donated two single-donor PCs (SDPCs) each, taking 500 mg ASA 12 hours before one of the aphereses (Group A) and taking no medication before the other donation (Group B). In-vitro tests of PLT function were performed in donors before and after apheresis and in SDPCs during storage (Days 1, 3, and 5).

RESULTS

ASA ingestion resulted in a significant decrease of induced PLT aggregation in donors (p < 0.005) and SDPCs on Day 1 (p < 0.01). TRAP-6-induced expression of p-selectin (CD62p) was significantly reduced in Group A SDPCs only on Day 1 (p < 0.02). There were no significant differences of in-vitro function (LDH, lactate, pH, morphology score, CD62p expression, fibrinogen binding) between Group A and B (SDPCs and donors). Apheresis did not result in a significant activation of PLTs in donors or SDPCs.

CONCLUSIONS

These limited data do not show a detectable beneficial effect of ASA ingestion on the PSL but do suggest that ASA ingestion before apheresis may not be detrimental to the clinical effectiveness of the stored product.

Antiphospholipid syndrome (APS) is a systemic autoimmune disease, characterized by thrombosis, obstetric complications and the presence of antiphospholipid antibodies (aPL), which drive endothelial injury and thrombophilia. Extracellular vesicles (EVs) have been implicated in endothelial and thrombotic pathologies. Here, we characterized the quantity, cellular origin and the surface expression of biologically active molecules in small EVs (sEVs) isolated from the plasma of thrombotic APS patients (n = 14), aPL-negative patients with idiopathic thrombosis (aPL-neg IT, n = 5) and healthy blood donors (HBD, n = 7). Nanoparticle tracking analysis showed similar sEV sizes (110-170 nm) between the groups, with an increased quantity of sEVs in patients with APS and aPL-neg IT compared to HBD. MACSPlex analysis of 37 different sEV surface markers showed endothelial (CD31), platelet (CD41b and CD42a), leukocyte (CD45), CD8 lymphocyte and APC (HLA-ABC) cell-derived sEVs. Except for CD8, these molecules were comparably expressed in all study groups. sEVs from APS patients were specifically enriched in surface expression of CD62P, suggesting endothelial and platelet activation in APS. Additionally, APS patients exhibited increased CD133/1 expression compared to aPL-neg IT, suggesting endothelial damage in APS patients. These findings demonstrate enhanced shedding, and distinct biological properties of sEVs in thrombotic APS.

Aflibercept appears to accumulate in systemic circulation following intravitreal injections in therapy of neovascular age-related macular degeneration. This gives raise to the question of whether aflibercept affects platelets and their function such as activation and aggregation, which are substantial in the pathogenesis of an arterial thromboembolic event (ATE). In order to determine the effect of aflibercept in platelet activation, platelets from healthy volunteers were treated with aflibercept and its solvents at equal concentrations (0.04 μg/mL - 4 μg/mL - 40 μg/mL - 400 μg/mL - 4 mg/mL) for 10 and 30 min before addition of agonists. IgG1 antibody was used as a control. The surface expression of GPIIb/IIIa, P-selectin, and platelet-bound stromal-cell-derived factor-1, which are potential blood biomarkers for ATEs, was determined on resting and activated platelets by the multispectral imaging flow cytometry, combining the features of flow cytometry with fluorescence microscopy. Platelet aggregation was assessed with light transmission aggregometry. To determine whether aflibercept directly interacts with platelets, aflibercept was labeled with the fluorescence FITC. Co-treatment of platelets with thrombin or PAR-4-AP and aflibercept resulted in increased activation of the fibrinogen receptor GPIIb/IIIa in comparison to controls (P < 0.05). Interestingly, the expression of platelet-derived P-selectin and SDF-1 was not affected by aflibercept, except thrombin-activated CD62P with 0.04 μg/mL aflibercept (aflibercept vs. its solvent: MSI = 1.54, IC = 1.201-1.879 vs. MSI = 1.37, IC = 1.136-1.604 [P = 0.031]) and SDF-1 with 4 mg/mL aflibercept (aflibercept vs. its solvent: MSI = 1.971, IC = 1.206-2.737 vs. MSI = 1.200, IC = 0.738-1.662 [P = 0.041]). Although the levels of platelet-bound aflibercept-FITC were significantly increased in all activated platelets, no effect was observed in platelet aggregation. Albeit no impact of aflibercept was found on platelet aggregation under the studied experimental conditions, the increased activation of the fibrinogen receptor GPIIb/IIIa and the presence of a direct interaction between aflibercept and platelets may partially explain the risk of ATE in patients under aflibercept treatment due to FcγRIIa mediated αIIbβ3 outside-in integrin signaling and transport of aflibercept into platelets. Therefore, the Fc domain seems to be involved in interactions between aflibercept and platelets. Further research is needed to explain the role of Fc containing aflibercept in the pathogenesis of drug-associated vascular events involving platelets, coagulation cascade, extracellular matrix proteins and other cells.