(1) Background: Prostacyclin analogues (epoprostenol, treprostinil, and iloprost) induce vasodilation in pulmonary arterial hypertension (PAH) but also inhibit platelet function. (2) Objectives: We assessed platelet function in PAH patients treated with prostacyclin analogues and not receiving prostacyclin analogues. (3) Methods: Venous blood was collected from 42 patients treated with prostacyclin analogues (49.5 ± 15.9 years, 81% female) and 38 patients not receiving prostacyclin analogues (55.5 ± 15.6 years, 74% female). Platelet reactivity was analyzed by impedance aggregometry using arachidonic acid (AA; 0.5 mM), adenosine diphosphate (ADP; 6.5 µM), and thrombin receptor-activating peptide (TRAP; 32 µM) as agonists. In a subset of patients, concentrations of extracellular vesicles (EVs) from all platelets (CD61⁺), activated platelets (CD61⁺/CD62P⁺), leukocytes (CD45⁺), and endothelial cells (CD146⁺) were analyzed by flow cytometry. Platelet-rich thrombus formation was measured using a whole blood perfusion system. (4) Results: Compared to controls, PAH patients treated with prostacyclin analogues had lower platelet reactivity in response to AA and ADP (p = 0.01 for both), lower concentrations of platelet and leukocyte EVs (p ≤ 0.04), delayed thrombus formation (p ≤ 0.003), and decreased thrombus size (p = 0.008). Epoprostenol did not affect platelet reactivity but decreased the concentrations of platelet and leukocyte EVs (p ≤ 0.04). Treprostinil decreased platelet reactivity in response to AA and ADP (p ≤ 0.02) but had no effect on the concentrations of EVs. All prostacyclin analogues delayed thrombus formation and decreased thrombus size (p ≤ 0.04). (5) Conclusions: PAH patients treated with prostacyclin analogues had impaired platelet reactivity, EV release, and thrombus formation, compared to patients not receiving prostacyclin analogues.

Keywords: extracellular vesicles; platelet reactivity; prostacyclin analogues; pulmonary arterial hypertension; thrombus formation.

Mass cytometry is a next generation flow cytometry technology which analyzes cells one at a time (up to 1000/sec) using mass spectrometry to detect probes labeled with rare-earth metals. Rare-earth metals detected by mass spectrometry have extremely low backgrounds and can be identified with high resolution enabling the routine simultaneous detection of more than 45 probes on each cell without the need for complex compensation matrices. Here we describe a panel of 14 platelet-specific metal-conjugated 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 methods for staining and analysis of platelets by mass cytometry. High dimensional clustering algorithms, which take into account the levels of all 14 markers detected by mass cytometry on each cell, allow identification of platelet subpopulations not previously appreciated. We previously reported that platelet heterogeneity identified by mass cytometry appears similar across healthy donors and consistent over time. High dimensional analysis revealed the presence of a platelet subpopulation with significantly higher levels of surface expression of activated GPIIb-IIIa and P-selectin suggesting this subpopulation may play a greater role in thrombus formation than other platelet subpopulations. Thus, analysis by mass cytometry of platelet heterogeneity and subpopulations may suggest distinct biological roles for different platelet subpopulations and may be useful in evaluating inherited or acquired platelet disorders and platelet function in health and disease.

Platelets play an important role in diseases such as cardiovascular disease and cancer, especially through their release of extracellular vesicles (EVs) and role in thrombosis. The effects of the anti-inflammatory drug colchicine on platelets are not well understood. We investigated the effect of colchicine on the release of pro-coagulant EVs from platelets under low-level activation. Citrated platelet-rich plasma (PRP) from healthy donors was incubated with 2 mM colchicine or vehicle at 37°C for 30 minutes with gentle rotation. The incubation conditions caused mild platelet activation (expression of CD62P and increased surface lactadherin binding) and release of EVs expressing phosphatidylserine (PS, measured by binding of lactadherin), CD61 and CD62P, both of which were attenuated by colchicine. The incubation conditions shortened the delay to fibrin generation and this correlated with elevated levels of PS+/CD61+ EVs. Removal of EVs from plasma abrogated clot formation in the overall haemostatic potential (OHP) assay. Colchicine decreased levels of both PS+/CD61+ and CD62P+ EVs and abrogated the shortened delay to fibrin generation achieved by platelet activation. Similar results were observed after incubation of PRP with 200 µM vinblastine, suggesting a microtubular effect. An alternative method of platelet activation using platelet agonists 20 µM ADP or 10 µM epinephrine also increased CD62P+ EV levels, and this too was attenuated by prior incubation with colchicine. Our novel findings demonstrate procoagulant effects of low-level platelet activation and EV formation which are inhibited by colchicine.

Cryopreservation can slow down the metabolism and decrease the risk of bacterial contamination. But, chilled platelets (PLTs) show a reduced period in circulation due to the rapid clearance by hepatic cells or spleen macrophages after transfusion. The deleterious changes that PLTs undergo are mainly considered the result of PLT protein variation. However, the basis for proteomic variation of stored PLTs remains poorly understood. Besides count, activation markers (CD62P and Annexin V), and aggregation, we used quantitative mass spectrometry to create the first comprehensive and quantitative human PLT proteome of samples stored at different temperatures (22°C, 10°C and -80°C). We found different conditions caused different platelet storage lesion (PSL). PLT count was decreased no matter at what temperature stored. PLTs viability at low temperature dropped by 21.78% and 11.21%, respectively, as compared 10.26% at room temperature, there were no significant differences between the storage methods. Membrane expression of CD62P gradually increased in all groups especially stored at 22°C up to 40% and 10°C up to 30%. However, exposure of PS on the PLT membrane was below 1% in every group. The PLT proteome showed there were 575 and 454 potential proteins identified by general iTRAQ analysis and phosphorylation iTRAQ a nalysis, respectively, among them, 33 common differentially expressed proteins caused by storage time and 44 caused by storage temperature Especially, membrane-bound proteins (such as FERMT3, STX4, MYL9 and TAGLN2) played key roles in PLT storage lesion. The pathways "Endocytosis", "Fc gamma R-mediated phagocytosis" and "Regulation of actin cytoskeleton" were affected predominantly by storage time. And the pathways "SNARE interactions in vesicular transport" and "Vasopressin-regulated water reabsorption" were affected by cold storage in our study. Proteomic results can help us to understand PLT biochemistry and physiology and thus unravel the mechanisms of PSL in time and space for more successful PLT transfusion therapy.

BACKGROUND

Donated platelets are stored at 22°C and discarded within 5 days because of diminished function and risk of bacterial contamination. Decline of platelet function has been attributed to decreased mitochondrial function and increased oxidative stress. Resveratrol (Res) and cytochrome c (Cyt c), in combination with hypothermic storage, may extend platelet viability.

METHODS

Platelets from 20 donors were pooled into four independent sets and stored at 22°C or 4°C in the absence or presence of Res (50 μM) or Cyt c (100 μM) for up to 10 days. Sequential measurement of platelet counts, coagulation function (thromboelastography), oxygen consumption, lipid peroxidation, glucose-lactate levels, pH, TCO2, and soluble platelet activation markers (CD62P/PF-4) was performed.

RESULTS

Platelet function diminished rapidly over time at 22°C versus 4°C (adenosine diphosphate, day 10 [0.6 ± 0.5] vs. [7.8 ± 3.5], arachidonic acid: day 10 [0.5 ± 0.5] vs. [30.1 ± 27.72]). At 4°C, storage treatment with Res or Cyt c limited deterioration in platelet function up to day 10, an effect not observed at 22°C (day 10, 4°C, Con [7.8 ± 3.5] vs. Res [37.3 ± 24.19] vs. Cyt c [45.83 ± 43.06]). Mechanistic analysis revealed oxygen consumption increased in response to Cyt c at 22°C, whereas neither Cyt c or Res affected oxygen consumption at 4°C. Lipid peroxidation was only reduced at 22°C (day 7 and day 10), but remained unchanged at 4°C, or when Res or Cyt c was added. Cytosolic ROS was significantly reduced by pretreatment with Res at 4°C. Total platelet count and soluble activation markers were unchanged during storage and not affected by Res, Cyt c, or temperature. Glucose concentration, pH and TCO2 decreased while lactate levels increased during storage at 22°C but not 4°C.

CONCLUSIONS

Platelet function is preserved by cold storage for up to 10 days. This function is enhanced by treatment with Res or Cyt c, which supports mitochondrial activity, thus potentially extending platelet shelf life.

OBJECTIVE

Cold-stored platelets may be an alternative to conventional room temperature (RT) storage. However, cold-stored platelets are cleared more rapidly from circulation, reducing their suitability for prophylactic transfusion. To minimise wastage, it may be beneficial to store platelets conventionally until near expiry (4 days) for prophylactic use, transferring them to refrigerated storage to facilitate an extended shelf life, reserving the platelets for the treatment of acute bleeding.

METHODS

Two ABO-matched buffy-coat-derived platelets (30% plasma/70% SSP+) were pooled and split to produce matched pairs (n = 8 pairs). One unit was stored at 2-6°C without agitation (day 1 postcollection; cold); the second unit was stored at 20-24°C with constant agitation until day 4 then stored at 2-6°C thereafter (delayed-cold). All units were tested for in vitro quality periodically over 21 days.

RESULTS

During storage, cold and delayed-cold platelets maintained a similar platelet count. While pH and HSR were significantly higher in delayed-cold platelets, other metabolic markers, including lactate production and glucose consumption, did not differ significantly. Furthermore, surface expression of phosphatidylserine and CD62P, release of soluble CD62P and microparticles were not significantly different, suggesting similar activation profiles. Aggregation responses of delayed-cold platelets followed the same trend as cold platelets once transferred to cold storage, gradually declining over the storage period.

CONCLUSIONS

The metabolic and activation profile of delayed-cold platelets was similar to cold-stored platelets. These data suggest that transferring platelets to refrigerated storage when near expiry may be a viable option for maximising platelet inventories.

High residual platelet reactivity (HRPR) assessed by multiple tests has been associated with worse clinical outcomes. However, the clinical impact of HRPR assessed by flow cytometry is unknown. The aim of this study was to validate the predictive value of HRPR measured by flow cytometry for clinical outcomes in ischemic stroke patients during clopidogrel therapy. Overall, 198 consecutive patients with ischemic stroke taking clopidogrel underwent platelet function testing on flow cytometer including adenosine diphosphate (ADP)-induced platelet aggregation (PAg) and platelet activation markers (CD62P, CD63, and PAC-1). Poor outcome was defined as poor prognosis and ischemic events during 12-month follow-up. By receiver operating characteristic curve analysis, residual platelet reactivity assessed by flow cytometry was able to distinguish between patients with and without poor outcomes, when platelet inhibition was evaluated with ADP-PAg (area under the curve [AUC], .77; 95% confidence interval [CI], .69-.84; P < .001), CD62P (AUC, .73; 95% CI, .64-.81; P < .001), CD63 (AUC, .72; 95% CI, .64-.80; P < .001), and PAC-1 (AUC, .70; 95% CI, .62-.78; P < .001). The prevalence of HRPR was 25.8% for ADP-PAg, 32.8% for CD62P, 41.4% for CD63, and 56.1% for PAC-1. The multiple logical regression analysis demonstrated that HRPR was an independent predictor of poor outcomes (ADP-PAg: odds ratio [OR] 13.03, 95% CI 5.66-29.98, P < .001; CD62P: OR 8.55, 95% CI 3.94-18.57, P < .001; CD63: OR 8.74, 95% CI 3.89-19.64, P < .001; PAC-1: OR 4.23, 95% CI 1.98-9.08). In conclusion, HRPR, assessed by flow cytometry, is able to detect ischemic stroke patients at increased risk of 12-month poor outcomes on clopidogrel treatment.

BACKGROUND

Platelet additive solutions (PASs) facilitate improved recovery of plasma and may reduce the severity and/or frequency of plasma-associated transfusion reactions. Current apheresis platelet (PLT) PAS products contain approximately 30 to 40% residual plasma. In an effort to further decrease the residual plasma, two in vitro studies were conducted with PLTs suspended in 5% plasma and a reformulated PAS-3, named PAS-5, that contains additional salts, glucose, and bicarbonate.

METHODS

In Study 1, PLTs suspended in 5% plasma/95% PAS-5 were prepared directly on a separator (Amicus, Fenwal, Inc.) without additional centrifugation or washing. In Study 2, a double unit of hyperconcentrated Amicus PLTs in plasma was collected, divided, and centrifuged to prepare a control unit in 100% plasma and a paired test unit in 5% plasma/95% PAS-5. The in vitro properties of PLTs were assessed in both studies during 7-day storage at 20 to 24°C with continuous agitation.

RESULTS

In Study 1, PLT concentration, pH, mean PLT volume (MPV), HCO(3)(-), pCO(2), pO(2), lactate dehydrogenase, and hypotonic shock response (HSR) did not significantly change during storage. By Day 7, glucose levels and morphology scores modestly decreased (17.6 and 14.4%, respectively) and lactate levels modestly increased (to 7.2 mmol/L). In Study 2, MPV, pH, glucose, pO(2), HSR, and morphology were comparable in control and test PLTs during 7-day storage. Glucose consumption and lactate production were significantly less in test versus control PLTs (p≤0.0015). Extent of shape change and %CD62P-positive test PLTs were less than those of controls (p<0.001).

CONCLUSIONS

Apheresis PLTs suspended in 5% plasma/95% PAS-5 maintained in vitro properties during 7-day storage.

OBJECTIVE

In mice, loss of sialic acid resulting in shedding of glycoprotein (GP) Ibα and GPV has been linked to platelet survival. The aim of this study was to determine whether loss of sialic acid and the GPIb-IX-V complex contributes to development of the platelet storage lesion (PSL) in human platelet concentrates (PCs).

METHODS

PCs (stored in plasma (with or without Mirasol treatment); PAS-C or PAS-E) were stored at room temperature. Flow cytometry was used to monitor membrane expression of the GPIb-IX-V complex, CD62P, surface glycans and PS exposure. The functionality of stored platelets was determined employing aggregometry and ristocetin-induced VWF binding.

RESULTS

Storage time of PCs in blood banks is limited to 7 days. During this time period, a minor but gradually increasing subpopulation of GPIbα-negative platelets was observed. Also, ristocetin-induced VWF binding was impaired in a small population of platelets. Mean surface expression of GPIbα and GPV remained stable until day 9, whereas CD62P expression increased; also a rapid decrease in ADP-induced aggregation was observed for PAS-C, PAS-E and Mirasol-treated PCs. Upon prolonged storage (>9 days), a slow decline in surface expression of GPIbα and GPV was observed; no major changes were observed in surface sialylation with the exception of Mirasol-treated platelets.

CONCLUSIONS

In a small population of stored platelets, changes in GPIbα occur from day 2 onwards. Loss of sialic acid and subsequent shedding of GPIbα and GPV is not an early event during the development of the PSL.

Biomaterial-induced human platelet activation remains one of the most crucial factors to determine the procoagulant properties of the biomaterial. In this experiment, a new type of biomacromolecule complex film (hyaluronic acid-collagen (I)/chitosan, HCC) was prepared using the electrostatic self-assembly method. Then the procoagulant properties of this complex film were characterized. Based on the nano-resolution of atomic force microscopy, the platelet-derived microparticles (PMPs) that present the activation of platelets were clearly visualized on the membrane surface of platelets for the first time, and the measurement indicated that the size of PMPs is around 50-110 nm. Furthermore, the results of AFM measurement were confirmed by flow cytometry analysis. The expression of CD62P (P-selectin) dramatically increased after the platelet-rich plasma interacted with the biomaterial solution. From the results, we could draw the conclusion that this biomacromolecule complex film has promising procoagulant properties, and has the potential to be practically used as procoagulant material.

High titers of HLA antibodies are associated with platelet refractoriness, causing poor platelet increments after transfusions in a subset of patients with HLA antibodies. Currently, we do not know the biological mechanisms that explain the variability in clinical responses in HLA alloimmunized patients receiving platelet transfusions. Previously we showed that a subset of anti-HLA IgG-antibodies induces FcγRIIa-dependent platelet activation and enhanced phagocytosis. Here we investigated whether anti-HLA IgG can induce complement activation on platelets. We found that a subset of anti-HLA IgG induced complement activation via the classical pathway, causing C4b and C3b deposition and formation of the membrane-attack complex. This resulted in permeabilization of platelet membranes and increased calcium influx. Complement activation also caused enhanced α-granule release, as measured by CD62P surface exposure. Blocking studies revealed that platelet activation was caused by FcγRIIa-dependent signaling as well as HLA antibody induced complement activation. Synergistic complement activation employing combinations of monoclonal IgGs suggested that assembly of oligomeric IgG complexes strongly promoted complement activation through binding of IgGs to different antigenic determinants on HLA. In agreement with this, we observed that preventing anti-HLA-IgG hexamer formation using an IgG-Fc:Fc blocking peptide, completely inhibited C3b and C4b deposition. Our results show that HLA antibodies can induce complement activation on platelets including membrane attack complex formation, pore formation and calcium influx. We propose that these events can contribute to fast platelet clearance in vivo in patients refractory to platelet transfusions with HLA alloantibodies, who may benefit from functional-platelet matching and treatment with complement inhibitors.

OBJECTIVE

To investigate the effect of endovascular aneurysm repair (EVAR) on platelet (PLT) function and reveal possible associated factors.

METHODS

Fifty consecutive patients were included. PLT count and activation (CD62P-CD36), white blood cell (WBC) count, and high sensitivity C-reactive protein (hs-CRP) were measured preoperatively, at the first and third postoperative day.

RESULTS

EVAR elicited a significant reduction in PLT count from baseline to first day after EVAR (p < 0.001), while no significant difference was noted between the first and third day. Furthermore, CD62P expression was markedly elevated at the first day after EVAR (median % positive PLTs from 13.7 at baseline to 22.1, p = 0.05), but returned to baseline levels by the third day. Maximum abdominal aortic aneurysm diameter was the only factor that significantly affected the CD62P values (p = 0.005). Postoperative CD36 values were significantly correlated with total aneurysm volume (p = 0.05) and were higher in endografts made from polyester (p = 0.01). There were no correlation between PLT activation and hs-CRP, WBC, maximum temperature, and 30-day morbidity.

CONCLUSIONS

EVAR has elucidated a significant reduction in PLT count and increase in PLT activation at the immediate postoperative period. The type of the endograft material and the aneurysm maximum diameter and volume appear to play an important role in PLT activation.

CYP2C19 genetic polymorphisms influence clopidogrel response and clinical outcomes of cardiovascular disease. However, data on their relationship in stroke patients are scarce. We aimed to investigate the influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. A total of 211 patients were enrolled. All patients were given clopidogrel treatment and underwent CYP2C19 genotyping and platelet function testing by flow cytometry including adenosine diphosphate-induced platelet aggregation (ADP-PAg) and platelet activation markers (PAC-1, CD62P and CD63). The modified Rankin Scale (mRS) was used and ischemic events were evaluated. A total of 129 (61.1%) of the 211 enrolled patients were carriers of CYP2C19 loss-of-function (LOF) alleles (*2, *3). After clopidogrel therapy for 7 days, the levels of ADP-PAg, PAC-1, CD62P and CD63 were higher in carriers than noncarriers. CYP2C19 carriage was associated with more frequent high residual platelet reactivity. CYP2C19 polymorphisms alone could explain 12.9%, 4.3%, 8.9% and 5.5% of the inter-individual variability of ADP-PAg, PAC-1, CD62P and CD63 after clopidogrel treatment, respectively. At 6-month follow-up, 38 (19%) patients were scored poor prognosis and 15 (7.6%) ischemic events were observed. Carriers had poorer prognosis than noncarriers (P=0.025). No significant association of CYP2C19 carriage with ischemic events was found. Multiple regression analysis showed that CYP2C19 carriage was an independent predictor of poor prognosis (odds ratio, 3.01; 95% confidence interval, 1.23-7.38; P=0.016). In conclusion, carriage of the CYP2C19 LOF allele has significant influence on clopidogrel response and prognosis in patients with ischemic stroke.

Previous in vitro studies have demonstrated that normal platelets and platelet-released mediators can alter in vitro characteristics of human acute myelogenous leukemia (AML) blasts. To further investigate whether platelets can be expected to adhere to and thereby affect AML blasts through their release of soluble mediators into a common microenvironment, we investigated (i) the effects on platelet activation by cytotoxic drugs commonly used in AML therapy; (ii) the occurrence of circulating activated platelets in acute leukemia patients; and (iii) the in vivo and in vitro adherence of platelets to AML blasts. The anthracyclins daunorubicin and idarubicin increased the expression of activation-associated membrane molecules (GPIIb/IIIa, CD62P, CD63) by normal platelets, daunorubicin then having the strongest effect. In contrast, cytarabine, epirubicin, doxorubicin and mitoxantrone had no significant effects. Although AML patients did not show increased levels of activated platelets in the circulation, adhesion of platelets to AML blasts was demonstrated both in vivo and in vitro. These results suggest that platelets and AML blasts may locate to common in vivo microenvironments, and platelet-derived soluble mediators may thereby affect the functional characteristics of the leukemia cells.

To investigate whether cancer stem cells (CSCs) more efficiently activating platelets and evading immune surveillance than non-CSCs thus promoting metastasis.

We enriched and identified sphere-forming cells (SFCs) and coincubated washed platelets with several platelet activators including collagen, 4T1 and SFCs. Platelet-coating tumor cells, platelet activation and TGF-β1 release were analyzed. Then natural kell cells (NK) were incubated with supernatants of different activated platelet samples what we called sample release (SR). The degranulation assay and NKG2D expression on NK cells were conducted by flow cytometry. Finally tissue factor (TF) expression of SFCs or 4T1 were evaluated by western blot.

Breast cancer cell line 4T1 could form spheres in serum-free medium at low adherence. Sphere-forming cells expressed high levels of the CD24-/lowCD44 + stem cell phenotype. Both sphere-forming cells or 4T1 were coated with abundant platelets while sphere-forming cells induced significantly higher expression of platelet activating receptor CD62p than 4T1 did (P < 0.01). And sphere-forming cells induced platelets to produce more TGF-β1 than 4T1 did (P < 0.01). Furthermore, sample releases induced by sphere-forming cells caused more vigorous inhibition of NK cells antitumor reactivity (P < 0.05) and reduced NKG2D expression (P < 0.01). The final results showed that sphere-forming cells expressed higher levels of TF than 4T1 (P < 0.05).

Our findings indicate that CSCs could efficiently activate platelets, induce platelets to secrete more TGF-β1, decrease NKG2D expression and inhibit antitumor activity of NK cell, compared with 4T1. And higher levels of TF expression of CSCs may account for this correlation of CSCs and platelets.

Intermediate (CD14⁺⁺CD16⁺) monocytes have important pro-inflammatory and atherogenic features and are increased in patients with chronic kidney disease (CKD). The present study aims to elucidate the role of the uremic milieu and of platelet activation in monocyte differentiation. Monocyte subtypes were analyzed in CKD patients (n = 193) and healthy controls (n = 27). Blood from healthy controls (Ctrl; n = 8) and hemodialysis patients (HD; n = 8) was centrifuged, and plasma (pl) was exchanged between Ctrl and HD (Ctrlcells/HDpl and HDcells/Ctrlpl) or reconstituted as original (Ctrlsham and HDsham) and incubated for 24 h (T24). Monocyte differentiation and platelet aggregation to monocytes (MPA) was assessed by flow cytometry. Especially, a higher proportion of CD14⁺⁺CD16⁺ monocytes was found in hemodialysis (HD) patients (p < 0.01). In plasma exchange experiments, Ctrl cells/HD pl T24 showed an increased percentage of CD14⁺⁺CD16⁺ monocytes versus Ctrl sham (33.7% ± 15 vs. 15.7% ± 9.6; P < 0.005), comparable to the level of CD14⁺⁺CD16⁺ monocytes in the HD sham condition. The percentage of CD14⁺⁺CD16⁺ monocytes was lowered by suspending HD cells in Ctrl pl (18.4% ± 7.8 vs. 36.7% ± 15 in HD sham; P < 0.005) reaching the level of the Ctrl sham condition (15.7% ± 9.6). A mixture of uremic sulfates increased CD14⁺⁺CD16⁺ monocytes compared to control (19.8 ± 9.6% vs. 15.8 ± 10.9%; P < 0.05), paralleled by a rise MPA. Blocking MPA by abciximab, a potential therapeutic strategy, or anti-CD62P did not inhibit differentiation towards the CD14⁺⁺CD16⁺ monocytes. In conclusion, in the present cohort, CD14⁺⁺CD16⁺ monocytes are especially increased in HD patients and this can at least in part be attributed to the presence of the uremic milieu, with uremic sulfates inducing a reversible shift towards pro-inflammatory CD14⁺⁺CD16⁺ monocytes.

OBJECTIVE

The purpose of this study was to compare platelet activation and hepatic cell damage produced by 2 ultrasonographic contrast agents with flow cytometric and ultrastructural analysis.

METHODS

Suspension samples were made by mixing Levovist (SH U508A; Schering AG, Berlin, Germany) or DD-723 (Nycomed; Amersham Health, Princeton, NJ) with whole blood. The final concentrations of Levovist in citrated whole blood were 0, 15, and 75 mg/mL, and those of DD-723 were 0, 5, and 50 microL/mL. After exposure to ultrasound in vitro, flow cytometric analysis was performed to determine the concentration of the CD62P activation-specific antigen. To compare the hepatic cell damage associated with these 2 agents, we divided 15 rats into 5 groups as follows: group 1, sham operation; group 2, Levovist injection only; group 3, DD-723 injection only; group 4, Levovist injection (contrast agent) and ultrasound exposure; and group 5, DD-723 injection and ultrasound exposure. The ultrasonographic contrast agents Levovist and DD-723 were administered through the femoral vein and sonicated continuously for the first minute; this was followed by sweeping for 5 minutes 10 seconds after the contrast agent was injected. The rats were perfused via the heart with a fixative solution immediately after the sweeping, and then the liver was excised; the specimens were studied with electron and light microscopy.

RESULTS

The percentage of CD62P-expressing platelets increased in both contrast agent-ultrasound exposure groups, and the percentage of CD62P-expressing platelets was greater in the Levovist group. We observed vacuolation and round deposits in the hepatocytes in both contrast agent-ultrasound exposure groups. Microbubbles were observed in the rat Kupffer cells, and a few hepatocytes were seen unexpectedly in the DD-723 group but were found in neither the Kupffer cells nor the hepatocytes in the Levovist group.

CONCLUSIONS

Both contrast agents, Levovist and DD-723, produced platelet activation and structural change in the rat hepatic cells, but only the microbubbles of DD-723 were taken up by the Kupffer cells and a few hepatocytes.

UNASSIGNED

Circulating monocytes from active ulcerative colitis (UC) patients produced high levels of tumor necrosis factor-alpha(TNFα) and interleukin(IL)-6 after Toll-like receptors (TLR) stimulation. Since platelets (PLT) can bind to leukocytes, thereby decreasing inflammatory cytokine production, UC patients may exhibit different levels of monocyte-platelet complexes depending on disease activity.

UNASSIGNED

We compared among healthy donors, active (onset flare and relapse), and inactive UC patients the presence of circulating monocyte-platelet complexes (CD14+PLT+) and membrane CD162 expression by flow cytometry. Lipopolysaccharide- binding protein, TNFα, and IL-10 were compared by ELISA. Binding of CD14+PLT+ to human umbilical vein endothelial cells (HUVECs) were analyzed by immunofluorescence.

UNASSIGNED

Onset flare UC patients had the lowest levels of CD14+PLT+. Membrane CD162, crucial for the PLT binding, was downregulated only on monocytes from onset flare UC patients. Membrane CD162 expression on CD14+ cells inversely correlated with lipopolysaccharide binding protein levels. As an expected consequence, more CD14+PLT+ than CD14+PLT- from onset flare UC patients bound to activated HUVECs. TNFα tended to negatively correlate with CD14+PLT+ in relapse and inactive UC patients, whereas IL-10 positively correlated with CD14+PLT+ in all UC patients (r = -0.43, P = 0.1 and r = 0.61, P = 0.01, respectively). The anti-inflammatory role of PLT binding to monocytes was confirmed in cocultures of PLT and monocytes. These cocultures increased the percentage of CD14+PLT+ and IL-10 production, and decreased TNFα production. These anti-inflammatory effects were abolished when we blocked the binding of PLT with neutralizing anti-CD62P antibody.

UNASSIGNED

Decreased CD162 expression associated with endotoxemia reduced the binding of PLT to monocytes through membrane CD162-CD62P, favoring the inflammatory response of onset flare UC patients.

Sepsis is an organic dysfunction that puts at risk the life of patients suffering this disorder due to an exacerbated immunological response to the infection mediated by the host. Platelets have been largely researched on sepsis owing to its role in Disseminated Intracellular Coagulation (DIC) and because thrombocytopenia is an important clinical feature of these patients. Nevertheless, a great number of evidence shows that platelets have also an important role in immunological response since they have pattern recognition receptors, chemokine receptors and granules with stored soluble mediators. In this work, the immunological features of platelets in individuals with sepsis are described. The results show that platelets of these individuals have high levels of surfaces expression of TLR4, CD62P, CD32 and thrombin receptor 1 (PAR-1), these platelets have also greater capability to join Escherichia coli, and show a different profile of soluble mediators (IL-1β, CD40L and TNF-α). Platelets from patients with sepsis form aggregates with neutrophils in circulation, but are unable to induce the production of reactive oxygen species. This research describes important features of platelets to help the understanding of the immunological role of these cells in sepsis.

OBJECTIVE

To explore effects of Tongxinluo Capsule (TC) on platelet activating factor (PAF), vascular endothelial function, thrombolysis in myocardial infarction (TIMI) blood flow, and heart function in acute myocardial infarction (AMI) patients after delayed percutaneous coronary intervention (PCI).

METHODS

Totally 80 AMI inpatients were recruited at Department of Cardiology, People's Hospital of Jiangxi Province, from Jan. 2008 to Sep.2013. Those in line with inclusion criteria were randomly assigned to TC treatment group and the conventional treatment group by random digit table, 40 in each group. Besides, another 40 healthy subjects from examinees at Outpatient Department were recruited as a healthy control group. PCI was performed after 1-week treatment. Then blood samples were collected, and then blood contents of CD62P, CD63, GP II b/III a, ET-1, NO, and plasma von Willebrand factor (vWF) levels were detected. Coronary TIMI blood flow and corrected TIMI frame count (CTFC) were determined during PCI. Meanwhile, noninvasive blood pressure (BP) and heart rate (HR) were recorded before and after PCI, and cardiac function measured. They were compared with the healty control group.

RESULTS

Compared with the healthy control group, blood contents of CD62p, CD63, GP II b/IIIa receptor compound, vWF, and ET-1 significantly increased, but NO significantly decreased in AMI patients (all P < 0.05). After 1-week intervention of TC, blood contents of CD62p, CD63, GP II b/IIIa receptor compound, vWF, NO, and ET-1 significantly decreased (P < 0.05, P < 0.01). Compared with the conventional treatment group at the same time point, blood contents of CD62p, CD63, GP II b/IIIa receptor compound, vWF, and ET-1 decreased more significantly in the TC group (P < 0.05, P < 0.01), increased NO levels were also more obviously seen (P < 0.01). The aforesaid parameters changed more obviously at day 30, as compared with those changes at week 1 (P < 0.05, P < 0.01). The TIMI blood flow grade and CTFC were more obviously improved after PCI in the two treatment groups. Better TIMI blood flow was seen in the TC group. TIMI level 3 blood flow rate was higher in the TC group than in the conventional treatment group with statistical difference (P < 0.05). The left ventricular ejective factor (LVEF) after PCI was obviously elevated in the TC group and the conventional treatment group (P < 0.01), and the improvement was more obviously seen in the TC group (P < 0.05). There were 6 cases of recurrent angina, 3 cases of ventricular tachycardial (VT)/ventricular fibrillation (VF), 6 cases of heart failure (HF), 1 case of cardiac sudden death in the conventional treatment group, with the total incidence of cardiovascular events being 40% (16/40). There were 2 cases of recurrent angina, 2 cases of VT/VF, 2 cases of HF, no cardiac sudden death in the TC treatment group, with the total incidence of cardiovascular events being 15% (6/40). There was statistical difference in the recurrent rate of cardiovascular events between the two groups (χ² = 2.27, P < 0.05).

CONCLUSIONS

TC not only could prevent coronary embolism of AMI patients after delayed PCI, attenuate vascular endothelial injury, but also could improve TIMI blood flow, and strengthen cardiac systolic function.

Introduction: Microvesicles (MVs) are bioactive, submicron-sized (0.01-1000 nm) membrane vesicles released from various types of cells under normal physiological and pathophysiological conditions. MVs have emerged as important mediators of cell-to-cell communication in a diverse range of normal and pathological processes. MVs have been recognized as potential biomarkers in coagulation, inflammation, and cancer. However, for clinical use, minimizing factors which could affect enumeration and phenotypic characterization of MVs during pre-analytical steps is crucial. In this study, we used flow cytometry and nanoparticle tracking analysis (NTA) to investigate the impact of blood collection using with and without anticoagulant on the number and phenotype of MVs in blood samples.

Methods: Blood from 30 healthy volunteers was collected by venipuncture into 3.2% sodium citrate and clot activator tubes. MV subpopulations and their concentrations were investigated using flow cytometry and NTA. MV morphology was examined by transmission electron microscopy.

Results: Results showed that the concentration of MVs was significantly lower in serum than in plasma and that CD41⁺ MV, CD41⁺ /CD62P⁺ MV, CD45⁺ MV, and CD142⁺ MV levels from serum were significantly lower than those from plasma, whereas no significant differences in Annexin V (Anx V)⁺ MV, CD235a⁺ MV, and CD144⁺ MV levels were found. Interestingly, serum MVs had a higher proportion of small-sized MVs and lower proportion of large-sized MVs than did plasma MVs.

Conclusion: Although plasma samples are commonly used, our results suggest that serum can also be used in enumeration of MVs, but care must be taken if coagulation is an aspect of the research.

Keywords: flow cytometry; microvesicles; nanoparticle tracking analysis; plasma; serum.

Burn injury has severe impact on the physiologic homeostasis. Platelet counts show a distinct course post-burn injury, with a nadir at day 3 followed by a thrombocytotic period with at peak at day 15, with a gradual return to normal. So far, it is unknown how the functionality and activational status of platelets develop post burn. In this study, we investigated if the function, activation and growth factor content of platelets of burn patients are affected and how this evolves in time. Six burn patients with over 15% total burned surface area were followed during 1 month. Standard hematological and coagulation analyses, thromboelastography (TEG), platelet-function analyzer-100 (PFA), several platelet activation parameters (CD62P-CD63, AnnexinV) and growth factors (TGFb1, VEGF, PDGF-AB/BB, EGF, TGFb2, FGF-2, PDGF-AA) analyses were performed. TEG analyses showed procoagulant changes. PFA-100 analyses were nearly all within normal range. CD62P and CD63 and Annexin-V indicated no clear activation of platelets. Growth factor content followed the same course as the platelet count, reflecting a constant growth factor per platelet ratio. Concluding, platelets post burn-injury appears to be functional and not overly activated. However, burn patients seem to remain in a procoagulant state for an extensive period, which may impact their pathology.

Colchicine inhibits coronary and cerebrovascular events in patients with coronary artery disease (CAD), and although known to have anti-inflammatory properties, its mechanisms of action are incompletely understood. In this study, we investigated the effects of colchicine on platelet activation with a particular focus on its effects on activation via the collagen glycoprotein (GP)VI receptor, P2Y₁₂ receptor, and procoagulant platelet formation. Therapeutic concentrations of colchicine in vitro (equivalent to plasma levels) significantly decreased platelet aggregation in whole blood and in platelet rich plasma in response to collagen (multiplate aggregometry) and reduced reactive oxygen species (ROS) generation (H₂DCF-DA, flow cytometry) in response to GPVI stimulation with collagen related peptide-XL (CRP-XL, GPVI specific agonist). Other platelet activation pathways including P-selectin expression, GPIIb/IIIa conformational change and procoagulant platelet formation (GSAO⁺/CD62P⁺) (flow cytometry) were inhibited with higher concentrations of colchicine known to inhibit microtubule depolymerization. Pathway specific mechanisms of action of colchicine on platelets, including modulation of the GPVI receptor pathway at low concentrations, may contribute to its protective role in CAD.