CD62P Expression Research Articles

Donor variation in stored platelets: Higher metabolic rates of platelets are associated with mean platelet volume, activation and donor health.

Platelets (PLTs) differ in glycolytic activity, resulting in rapid acidification of 'poor' storing PLT concentrates (PCs) in plasma, or depletion of glucose when stored in PLT additive solution (PAS). We aimed to understand why PLT glycolysis rates vary between donors and how this affects storage performance. Buffy coats from donors <45, 45-70 and >70 years were selected and single-donor PCs in plasma or PAS-E were prepared. PCs were stored for 8 days at 22 ± 2°C and sampled regularly for analysis. Mitochondrial activity was analyzed with an Oroboros oxygraph. Age groups, or subgroups divided into quartiles based on glucose consumption, were analyzed with ANOVA. In each comparison, PCs of the different groups were not different in volume and cellular composition. PLTs with the highest glucose consumption had a higher initial mean platelet volume (MPV) and developed higher CD62P expression and Annexin A5 binding during storage. Higher glycolytic activity in these PLTs was not a compensation for lower mitochondrial ATP production, because mitochondrial ATP-linked respiration of fresh PLTs correlated positively with MPV (R2 = 0.71). Donors of high glucose-consuming PLTs had more health-related issues. Storage properties of PCs from donors over 70 were not significantly different compared to PCs from donors younger than 45 years. High glucose-consuming PCs developing higher activation levels, not only displayed enhanced mitochondrial activity but were also found to contain larger PLTs, as determined by MPV. Storage performance of PLTs was found to be associated with donor health, but not with donor age.

Knock-out of N-glycolylneuraminic acid attenuates antibody-mediated rejection in xenogenically perfused porcine lungs.

Antibody-mediated rejection has long been known to be one of the major organ failure mechanisms in xenotransplantation. In addition to the porcine α1,3-galactose (α1,3Gal) epitope, N-Glycolylneuraminic acid (Neu5Gc), a sialic acid, has been identified as an important porcine antigen against which most humans have pre-formed antibodies. Here we evaluate GalTKO.hCD46 lungs with an additional cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene knock-out (Neu5GcKO) in a xenogeneic ex vivo perfusion model METHODS: Eleven GalTKO.hCD46.Neu5GcKO pig lungs were perfused for up to 6h with fresh heparinized human blood. Six of them were treated with histamine (H) blocker famotidine and 1-thromboxane synthase inhibitor Benzylimidazole (BIA) and five were left untreated. GalTKO.hCD46 lungs without Neu5GcKO (n=18: eight untreated and 10 BIA+H treated) served as a reference. Functional parameters, blood, and tissue samples were collected at pre-defined time points throughout the perfusion RESULTS: All but one Neu5GcKO organs maintained adequate blood oxygenation and "survived" until elective termination at 6h whereas two reference lungs failed before elective termination at 4h. Human anti-Neu5Gc antibody serum levels decreased during the perfusion of GalTKO.hCD46 lungs by flow cytometry (∼40% IgM, 60% IgG), whereas antibody levels in Neu5GcKO lung perfusions did not fall (IgM p=.007; IgG p<.001). Thromboxane elaboration, thrombin generation, and histamine levels were significantly reduced with Neu5GcKO lungs compared to reference in the untreated groups (p=.007, .005, and .037, respectively); treatment with BIA+H masked these changes. Activation of platelets, measured as CD62P expression on circulating platelets, was lower in Neu5GcKO experiments compared to reference lungs (p=.023), whereas complement activation (as C3a rise in plasma) was not altered. MCP-1 and lactotransferin level elevations were blunted in Neu5GcKO lung perfusions (p=.007 and .032, respectively). Pulmonary vascular resistance (PVR) rise was significantly attenuated and delayed in untreated GalTKO.hCD46.Neu5GcKO lungs in comparison to the untreated GalTKO.hCD46 lungs (p=.003) CONCLUSION: Additional Neu5GcKO in GalTKO.hCD46 lungs significantly reduces parameters associated with antibody-mediated inflammation and activation of the coagulation cascade. Knock-out of the Neu5Gc sialic acid should be beneficial to reduce innate immune antigenicity of porcine lungs in future human recipients.

Platelets and Sera from Donors of Convalescent Plasma after Mild COVID-19 Show No Procoagulant Phenotype.

Coronavirus disease-2019 (COVID-19) is associated with increased thromboembolic complications. Long-term alteration in the coagulation system after acute COVID-19 infection is still a subject of research. Furthermore, the effect of sera from convalescent subjects on platelets is not known. In this study, we investigated platelet phenotype, coagulation, and fibrinolysis in COVID-19 convalescent plasma (CCP) donors and analyzed convalescent sera-induced effects on platelets. We investigated CCP donors who had a history of mild COVID-19 infection and donors who did not have COVID-19 were used as controls. We analyzed phosphatidylserine (PS) externalization, CD62p expression, and glycoprotein VI (GPVI) shedding both in platelet-rich plasma (PRP) and after incubation of washed healthy platelets with donors' sera using flow cytometry. Coagulation and fibrinolysis systems were assessed with thromboelastometry. Forty-seven CCP donors (22 males, 25 females; mean age (±SD): 41.4 ± 13.7 years) with a history of mild COVID-19 infection were included. Median duration after acute COVID-19 infection was 97 days (range, 34-401). We did not find an increased PS externalization, CD62p expression, or GPVI shedding in platelets from CCP donors. Sera from CCP donors did not induce PS externalization or GPVI shedding in healthy platelets. Sera-induced CD62p expression was slightly, albeit statistically significantly, lower in CCP donors than in plasma donors without a history of COVID-19. One patient showed increased maximum clot firmness and prolonged lysis time in thromboelastometry. Our findings suggest that procoagulant platelet phenotype is not present after mild COVID-19. Furthermore, CCP sera do not affect the activation status of platelets.

Automated preparation of washed platelet concentrates through spinning-membrane filtration.

Washed platelet concentrates (WPC), prepared with an automated system cell processor (ACP), have recently been approved to be manufactured and marketed in Japan. From the perspective of risk management, it is preferable to secure alternative technologies for ACP. Here, we conducted a study to evaluate the quality of WPC prepared using an automated membrane filtration-based system, Lovo. Replaced PCs prepared from apheresis PCs were equally divided into control and test units, and subsequently washed using ACP and Lovo respectively. Work and operational efficiencies were evaluated by in vitro analyses, including total handling time, platelet recovery, and plasma protein removal rate. Product quality, including a set of biochemical and physiological indicators of platelets and supernatants, were assessed before and 3 days after washing. In vitro platelet recovery rates and plasma protein removal rates were >85% and >95%, respectively, in both groups. The pH values on day 0 were significantly high (6.97 vs. 6.86) due to low pCO2 in the test group, while no significant differences in glucose consumption and lactate production were observed between the two groups. The levels of hypotonic shock responses, aggregation response, platelet shape, CD62P expression, and sCD62P concentration were similar in both groups during the 3-day storage period. Platelet washing with Lovo provides platelet quality equivalent to, or better than, conventional washing with ACP. Thus, the new automated system, Lovo, can be considered as an alternative to ACP for WPC preparation.

Hemostatic Effect of 20(S)-Panaxadiol by Induced Platelet Aggregation Depending on Calcium Signaling Pathway.

Panax notoginseng (Burk.) F.H. Chen is the most traditional hemostatic herb in China. Our previous research found that 20(S)-protopanaxadiol showed the hemostatic effect. And 20(S)-panaxadiol (PD) has a similar structure to 20(S)-protopanaxadiol with a dammarane skeleton. So, this article mainly studies the hemostatic effect of PD. The mouse tail amputation and liver scratch models were used to detect the hemostatic effect of PD. Blood routine and plasma coagulation parameters were measured by using a blood analyzer. The platelet aggregometer analyzed the platelet aggregation rate and adenosine triphosphate (ATP) concentration. Moreover, the intracellular calcium concentration ([Ca2+]i), P-selectin (CD62P), PAC-1 (GP IIb/IIIa receptor marker), and cyclic adenosine monophosphate (cAMP) of platelets were also detected. The results showed that PD obviously shortened the bleeding time of the model mouse, affected the RBC and PLT parameters of rats, reduced APTT and TT, elevated FIB concentration, and promoted human/rat-washed platelet aggregation in vitro. PD promoted the release of ATP and [Ca2+]i and slightly increased the expression of CD62P and PAC-1 of platelets without 1 mM Ca2+. After adding 1 mM Ca2+, PD obviously increased ATP releasing and CD62P and GP IIb/IIIa expression rate and decreased the cAMP level of platelets. These parameter changes of PD-caused platelet were inhibited by vorapaxar. Besides, PD increased the phosphorylation of phosphoinositide 3-kinase/protein kinase B/glycogen synthase kinase 3β (PI3K/Akt/GSK3β) of human platelets. PD is an important hemostatic ingredient in Panax notoginseng, which induced platelet aggregation by affecting the calcium signaling and activating the PI3K/Akt/GSK3β signaling pathway.

Increased platelet activation and platelet-inflammasome engagement during chikungunya infection

Chikungunya fever is a viral disease transmitted by mosquitoes of the genus Aedes. The infection is usually symptomatic and most common symptoms are fever accompanied by joint pain and swelling. In most cases symptoms subside within a week. However, severe prolonged and disabling joint pain, that may persist for several months, even years, are reported. Although the pathogenesis of Chikungunya infection is not fully understood, the evolution to severe disease seems to be associated with the activation of immune mechanisms and the action of inflammatory mediators. Platelets are recognized as inflammatory cells with fundamental activities in the immune response, maintenance of vascular stability and pathogenicity of several inflammatory and infectious diseases. Although the involvement of platelets in the pathogenesis of viral diseases has gained attention in recent years, their activation in Chikungunya has not been explored. The aim of this study was to analyze platelet activation and the possible role of platelets in the amplification of the inflammatory response during Chikungunya infection. We prospectively included 132 patients attended at the Quinta D’Or hospital and 25 healthy volunteers during the 2016 epidemic in Rio de Janeiro, Brazil. We observed increased expression of CD62P on the surface of platelets, as well as increased plasma levels of CD62P and platelet-derived inflammatory mediators indicating that the Chikungunya infection leads to platelet activation. In addition, platelets from chikungunya patients exhibit increased expression of NLRP3, caspase 4, and cleaved IL-1β, suggestive of platelet-inflammasome engagement during chikungunya infection. In vitro experiments confirmed that the Chikungunya virus directly activates platelets. Moreover, we observed that platelet activation and soluble p-selectin at the onset of symptoms were associated with development of chronic forms of the disease. Collectively, our data suggest platelet involvement in the immune processes and inflammatory amplification triggered by the infection.

Modelling the procoagulatory effect of Anastrozole relative to ERα and ERβ expression in breast cancer cells.

Anastrozole is commonly used for the treatment of oestrogen receptor (ER)-positive breast cancer but can increase thromboembolic risk. It is unclear if ER presentation is associated with platelet-mediated hypercoagulation. We investigated the relationship between hypercoagulation and ERα and ERβ expression in breast cancer cell lines under Anastrozole treatment. In Model 1, MCF-7 or T47D cancer cells were treated with Anastrozole, then exposed to whole blood and platelet-rich plasma, modelling platelet engagement in the tumour bed. In Model 2, blood components were treated with Anastrozole, then exposed to cancer cells, modelling circulatory effects in the vasculature. Hypercoagulation was assessed as a combined function of thrombin activity, platelet CD62P and CD63 expression, and corresponding platelet ultrastructure. Tumour ERα and ERβ were immunolocalised and following quantification assessed for correlation with hypercoagulatory parameters. Anastrozole enhanced hypercoagulation in both Models and cell lines. T47D cells induced more distinct features of hypercoagulation and responded by heightening ERβ expression and sustaining expression of ERα, indicative of a more aggressive phenotype. Post-exposure to cell lines, CD62P and CD63 expression correlated, but this was not maintained following Anastrozole treatment. Substantive correlations could not be found explaining the changes in ER expression and hypercoagulatory parameters, indicating unknown causative factors. These results provide basic science evidence showing that the hypercoagulatory effects induced by Anastrozole treatment may be related to the tumour subphenotype. Clinical studies are required to determine whether tracking of hypercoagulatory parameters may hold value in describing subphenotypic alterations or metastatic potential during tumour progression.

In vitro exposure of whole blood to a cannabinoid mixture impairs the quality of red blood cells and platelets.

In a recent study, 13.8% of blood donors had reported cannabis use in the 72 hours preceding their donation, and these donors are not deferred under existing criteria in Canada. This high prevalence raises concerns about the potential impact of cannabis use on the quality of blood products. The current study assessed the impact of a cannabinoid mixture on the quality of red blood cells and platelets, from the time of collection and processing to their storage. To mimic pre-donation cannabis use, whole blood was collected and exposed (in vitro) to varying concentrations (range: 1-24 μg/mL) of a cannabinoid mixture (CM) overnight. Whole blood was then separated into red blood cells (RBCs) and platelets-rich plasma (PRP), which were stored at 4°C (for RBCs) or at room temperature (for PRP). Flow cytometry analyses, hemolysis measurements and biochemical analyses were performed during the processing stage and throughout storage. In the RBC fraction, free hemoglobin levels were increased in a dose-dependent manner after the addition of a cannabinoid mixture to whole blood. Hemolysis and methemoglobin levels were significantly higher in CM-exposed RBCs than CM-free controls, after processing and throughout storage. Furthermore, platelet counts and CD62P expression (on day 7 post-separation) were significantly lower in CM-exposed PRP than cannabinoid-free PRP controls. The aggregation potential of CM-exposed platelets was significantly lower than that of cannabinoid-free controls, after the processing and throughout storage. An in vitro exposure to a cannabinoid mixture hemolyzed RBCs, impaired oxygen transport by RBCs, reduced platelet counts, and impaired platelet function. These results suggest that pre-donation cannabis use might impair the quality of blood products.

Assessment of on-treatment platelet reactivity at high and low shear stress and platelet activation status after the addition of dipyridamole to aspirin in the early and late phases after TIA and ischaemic stroke

BackgroundData are limited on the ability of dipyridamole to additionally inhibit platelet function/reactivity in ischaemic cerebrovascular disease (CVD) patients on aspirin. AimsTo assess inhibition of platelet function/reactivity and platelet activation with dipyridamole in CVD. MethodsThis prospective, observational study assessed TIA/ischaemic stroke patients before (baseline; N = 60), at 14 ±7 days (14d, N = 39) and ≥ 90 days (90d, N = 31) after adding dipyridamole to aspirin. Platelet function/reactivity at high shear stress (PFA-100® C-ADP) and low shear stress (VerifyNow® P2Y12 and Multiplate® ADP assays), and platelet activation status (% expression of CD62P, CD63 and leucocyte-platelet complexes on whole blood flow cytometry) were quantified. ‘Dipyridamole-high on-treatment platelet reactivity (HTPR)’ was defined as failure to inhibit ADP-induced platelet aggregation +/- adhesion compared with the patient's baseline on aspirin monotherapy by more than twice the coefficient-of-variation of the assay after adding dipyridamole to aspirin. ResultsDipyridamole-HTPR was identified in 71.4–75% of patients on PFA-100 C-ADP, 83.9–86.8% of patients on VerifyNow P2Y12, and 81.5–83.3% of patients on Multiplate ADP assays. There were no changes in CD62P/CD63 expression (P ≥ 0.18), or consistent changes in leucocyte-platelet complexes in CVD patients overall at 14d or 90d vs. baseline after commencing dipyridamole. Monocyte-platelet complexes increased in the patient subgroup with dipyridamole-HTPR at 14d and 90d on PFA-100, and at 14d on VerifyNow (P ≤ 0.04), but not in those without dipyridamole-HTPR. DiscussionAdditional antiplatelet effects of dipyridamole are detectable under high and low shear stress conditions with user-friendly platelet function/reactivity tests ex vivo. Increasing circulating monocyte-platelet complexes over time are associated with dipyridamole-HTPR.

Effects of wasp venom on venous thrombosis in rats.

Objective(s):This study aimed to investigate the potential effects of wasp venom (WV) from Vespa magnifica on antithrombosis in rats with inferior vena cava (IVC) thrombosis.Materials and Methods:The thrombosis rat model was established by improving the IVC stenosis, in which rats were subjected to IVC ligation for 75 min. Rats were administered argatroban (IP) or WV (s.c.) for 4 hr after IVC thrombosis. The weight, inhibition rate, and pathological morphology of the thrombosis induced by IVC ligation and the variation in four coagulation parameters, coagulation factors, and CD61+CD62P+ were simultaneously determined in IVC rats.Results:The thrombus formed as a result of IVC ligation was stable. Compared with the control group, the weight of the thrombus was significantly reduced in the argatroban group. Thrombus weight was reduced by treatment with 0.6, 0.2, and 0.05 mg/kg WV, with inhibition rates of 52.19%, 35.32%, and 28.98%, respectively. Inflammatory cells adhered to and infiltrated the vessel wall in the IVC group more than in the sham group. However, the pathological morphology and CD61+CD62P+ of the WV treatment groups tended to be normal. Conclusion:We improved the model of IVC thrombosis to be suitable for evaluation of antithrombotic drugs. Our findings demonstrated that WV could inhibit IVC thrombosis associated with reducing coagulation factors V and CD61+CD62p expression in rats.

Platelet Toll-Like-Receptor-2 and -4 Mediate Different Immune-Related Responses to Bacterial Ligands.

Background Like immune cells, platelets express toll-like receptors (TLRs) on their surface membrane. TLR2 and TLR4 are able to recognize bacterial antigens and have the potential to influence hemostatic functions and classical intracellular signaling pathways. This study investigated the role of TLR2 and TLR4 for immune-related functions in human platelets. Materials and Methods Washed platelets and neutrophils were prepared from fresh human peripheral blood. Basal-, Pam3CSK4- (as TLR2 agonist) and Lipopolysaccharides (LPS; as TLR4 agonist) -induced CD62P expression, fibrinogen binding and TLR2 or TLR4 expression, intracellular reactive oxygen species (ROS) production in H 2 DCFDA-loaded platelets and uptake of fluorescence-labeled TLR ligands, and fluorophore-conjugated fibrinogen were evaluated by flow cytometry. Analysis of platelet–neutrophil complexes was performed after coincubation of washed platelets and neutrophils in the presence and absence of TLR2 or TLR4 agonists on poly-L-lysine coated surfaces, followed by immunostaining and immunofluorescence imaging. Results Pam3CSK4 rapidly and transiently increased TLR2 and TLR4 expression. Over the course of 30 minutes after activation with Pam3CSK4 and LPS, the expression of both receptors decreased. Pam3CSK4-stimulated intracellular ROS production and the uptake of TLR ligands or fibrinogen much stronger than LPS. Besides, TLR4 activation led to a significant increase of platelet–neutrophil contacts. Conclusion Stimulation leads to rapid mobilization of TLR2 or TLR4 to the platelet surface, presumably followed by receptor internalization along with bound TLR ligands. After activation, platelet TLR2 and TLR4 mediate different immune-related reactions. In particular, TLR2 induces intracellular responses in platelets, whereas TLR4 initiates interactions with other immune cells such as neutrophils.

The Antibacterial Effect of Platelets on Escherichia coli Strains.

Platelets play an important role in defense against pathogens; however, the interaction between Escherichia coli and platelets has not been well described and detailed. Our goal was to study the interaction between platelets and selected strains of E. coli in order to evaluate the antibacterial effect of platelets and to assess bacterial effects on platelet activation. Washed platelets and supernatants of pre-activated platelets were incubated with five clinical colistin-resistant and five laboratory colistin-sensitive strains of E. coli in order to study bacterial growth. Platelet activation was measured with flow cytometry by evaluating CD62P expression. To identify the difference in strain behavior toward platelets, a pangenome analysis using Roary and O-antigen serotyping was carried out. Both whole platelets and the supernatant of activated platelets inhibited growth of three laboratory colistin-sensitive strains. In contrast, platelets promoted growth of the other strains. There was a negative correlation between platelet activation and bacterial growth. The Roary results showed no logical clustering to explain the mechanism of platelet resistance. The diversity of the responses might be due to strains of different types of O-antigen. Our results show a bidirectional interaction between platelets and E. coli whose expression is dependent on the bacterial strain involved.

Flow cytometry based platelet activation markers and state of inflammation among subjects with type 2 diabetes with and without depression

Type 2 Diabetes Mellitus (T2DM) and Major Depressive Disorder (MDD) are highly disabling disorders associated with a multitude of vascular complications. Platelets are known to play a role in the pathogenesis of vascular complications in both T2DM and MDD. These complications could increase in patients with comorbid diabetes and depression. To quantify and compare flow cytometry based platelet activation markers and the inflammatory state between individuals of diabetes with depression, individuals of diabetes without depression and healthy controls. Out of 114 participants, each study group contained 38 participants in diabetic group, diabetics with depression group and matched control group. Diabetes was diagnosed with the American Diabetes Association (ADA) criteria. Screening of MDD was done with Patient Health Questionnaire 2 (PHQ2) and severity of depression assessed with Hamilton Depression Rating (HAM-D) scale. Platelet markers CD41, CD42b, CD62P and CD63 were assayed using flow cytometer. Platelet count, surface expression of platelet activation markers CD62P and CD63, hs-CRP, insulin and HOMA-IR score differed significantly between the groups. Post hoc analysis showed significantly high CD63 expression in patients with comorbid diabetes and depression compared to those having diabetes without depression. Patients with comorbid diabetes and depression have enhanced platelet hyperactivation and a pro inflammatory state which increases susceptibility to vascular complications.

Panax Notoginseng Saponins Combined with Dual Antiplatelet Drugs Potentiates Anti-Thrombotic Effect with Alleviated Gastric Injury in A Carotid Artery Thrombosis Rat Model

ObjectiveTo observe the combination effects of Panax notoginseng saponins (PNS)and dual antiplatelet drugs (DAPT), and to explore the mechanism via cyclooxygenase /prostaglandin pathway. MethodsRight carotid artery thrombosis was induced in Wistar rats by infiltration with 70% FeCl3, and the animals were randomly divided into sham group, model group, DAPT group and PNS + DAPT group, intragastrically treated for 4 weeks. The cerebral pia mater microcirculation was observed in vivo after anesthetizing by anatomical microscope. The wet weight of carotid artery thrombosis was measured. Gastric mucosal injury was observed by hematoxylin and eosin staining. Platelet aggregation rate was detected with adenosine diphosphate -induced turbidimetry. Platelet CD62p expression was detected by flow cytometry. Concentrations of 6-Ketoprostaglandin F1 alpha, prostaglandin E2 in gastric mucosa and thromboxane B2, 6-Ketoprostaglandin F1 alpha, tissue plasminogen activator, plasminogen activator inhibitor, and fibrin fragment D in the plasma were measured by radioimmunoassay. ResultsPNS and DAPT increased the blood flow volume of cerebral pia mater and decreased erythrocyte aggregation and leukocyte adhesion of model rats. Compared to DAPT, PNS and DAPT further reduced the weight of carotid artery thrombosis with enhanced inhibition of platelet aggregation, increased tissue plasminogen activator levels and decreased fibrin fragment D levels. PNS and DAPT alleviated gastric injury induced by dual antiplatelet drugs and upregulated the expression of 6-Ketoprostaglandin F1 alpha in the gastric mucosa compared with DAPT. ConclusionsPNS combined with DAPT increased anti-thrombosis effects of DAPT and mitigated DAPT-related gastric injury. The underlying mechanisms may be associated with enhanced antiplatelet aggregation and activation of the fibrinolytic system and up-regulation of 6-Ketoprostaglandin F1 alpha expression in gastric mucosa.

Platelet Activation and Platelet Indices as Markers for Disease Progression in Women with Breast Cancer

Background: Several studies reported the role platelet activation, platelet volume (MPV) and other Indices in breast cancer but the data is inconsistent and/or diverse. The aim of this study was to systematically evaluate the role of platelet activation and platelet volume indices in women with BC as predictors for cancer progression and poor prognosis. Methods: Patients were recruited from our local oncology center between 2019 to 2020 following ethics approval. 80 patients with locally invasive BC, 20 metastatic and 100 controls were recruited. ADP-induced platelet activation was assessed by light-transmission aggregometry. Platelet P-selectin (CD62P) expression with and without ADP stimulation was assessed by flow cytometry. A comprehensive analysis of platelet count and platelet volume indices (PVIs) (MPV, PDW, MPV/P and PDW/P) was conducted. Data were analyzed in relation to tumor pathology, hormone receptors (ER, PR, HER-2) and proliferation index Ki-67. Regression analyses were conducted for the prediction of poor prognosis, tumor aggression and metastatic potential. Results: We found a significant increase in platelet aggregation (MA), CD62P expression, CD62P+ADP, MPV, PDW, MPV/P and PDW/P in the metastatic group compared to the locally invasive group. Univariate regression analysis showed significance for ADP, MA, CD62P+ADP, MPV and PDW/P. Conclusion: MPV/P and PDW/P can be used as simple low-cost predictors for cancer progression and poor prognosis. We conclude platelet activation and specific platelet indices can help predict prognosis in females with BC.

Divalent magnesium restores cytoskeletal storage lesions in cold-stored platelet concentrates

Cold storage of platelet concentrates (PC) has become attractive due to the reduced risk of bacterial proliferation, but in vivo circulation time of cold-stored platelets is reduced. Ca2+ release from storage organelles and higher activity of Ca2+ pumps at temperatures < 15 °C triggers cytoskeleton changes. This is suppressed by Mg2+ addition, avoiding a shift in Ca2+ hemostasis and cytoskeletal alterations. We report on the impact of 2–10 mM Mg2+ on cytoskeleton alterations of platelets from PC stored at room temperature (RT) or 4 °C in additive solution (PAS), 30% plasma. Deformation of platelets was assessed by real-time deformability cytometry (RT-DC), a method for biomechanical cell characterization. Deformation was strongly affected by storage at 4 °C and preserved by Mg2+ addition ≥ 4 mM Mg2+ (mean ± SD of median deformation 4 °C vs. 4 °C + 10 mM Mg2+ 0.073 ± 0.021 vs. 0.118 ± 0.023, p < 0.01; n = 6, day 7). These results were confirmed by immunofluorescence microscopy, showing that Mg2+ ≥ 4 mM prevents 4 °C storage induced cytoskeletal structure lesion. Standard in vitro platelet function tests showed minor differences between RT and cold-stored platelets. Hypotonic shock response was not significantly different between RT stored (56.38 ± 29.36%) and cold-stored platelets with (55.22 ± 11.16%) or without magnesium (45.65 ± 11.59%; p = 0.042, all n = 6, day 1). CD62P expression and platelet aggregation response were similar between RT and 4 °C stored platelets, with minor changes in the presence of higher Mg2+ concentrations. In conclusion, increasing Mg2+ up to 10 mM in PAS counteracts 4 °C storage lesions in platelets, maintains platelet cytoskeletal integrity and biomechanical properties comparable to RT stored platelets.

Triple apheresis platelet concentrate quality after pneumatic tube system, conveyor box, and courier transport: An observational study.

Background and AimsPlatelets are prone to activation from handling; they are therefore transported as gently as possible, most commonly by courier. Speedier methods like pneumatic tube system (PTS) transport could improve patient care but may subject platelets to mechanical stress. To test the impact of mechanical stress caused by transport, we compared a PTS with a conveyor box and courier transport on apheresis platelet function.MethodsFourteen apheresis platelet concentrate triple donations were analyzed by light transmission aggregometry (LTA), rotational thrombelastometry (ROTEM), and flow cytometry before and after indoor transport over 800 m by PTS, conveyor, and courier, respectively, while recording shocks and vibrations with a high‐frequency acceleration data logger. Shock index scores were calculated as shock intensity (g‐force) times frequency.ResultsThe shock index was 81 for courier, 6279 for conveyor, and 9075 for PTS. Flow cytometry revealed no significant difference in platelet surface expression of CD62p before (16%) and after transport via courier (15%), conveyor (14%), or PTS (16%). LTA with adenosine phosphate and thrombin receptor‐activating peptide‐6 resulted in comparable platelet aggregation for courier, conveyor, and PTS. ROTEM assays showed no relevant differences in coagulation time, clot formation time, and maximum clot firmness between transport modes.ConclusionThough the mechanical challenge was smallest with courier transport, there were no significant differences in platelet activation or aggregation between the three transport modes. These data contradict restrictions on the use of PTSs for platelet concentrate transport.

Antiplatelet Activity of Tetramethylpyrazine via Regulation of the P2Y12 Receptor Downstream Signaling Pathway

Background Tetramethylpyrazine (TMP) is an alkaloid in Chinese herbal medicine, which possesses antiplatelet activity. TMP inhibits platelet activation in many ways. The platelet P2Y12 receptor for adenosine 5′ diphosphate (ADP) plays a central role in platelet function, hemostasis, and thrombosis. Here, we investigated the inhibitory effect of TMP on P2Y12 receptor-related platelet function. Methods The inhibitory potential of TMP was assessed using agonist-induced platelet aggregation, flow cytometric analysis of CD62p expression, PAC-1 activation, and fibrin clot retraction. After the P2Y12 receptor-related signaling pathway was inhibited using the blocker, platelet activation was studied by platelet aggregation, CD62p expression, and PAC-1 activation. The secretion of cyclic adenosine monophosphate (cAMP) was measured using enzyme-linked immunosorbent assay (ELISA), and the expression of signaling pathway protein, phosphorylation of vasodilator-stimulated phosphoprotein, and phosphorylation of Akt were investigated using western blotting. The release of platelet inflammatory mediators was measured using ELISA. Results TMP had an antiplatelet effect by inhibiting ADP-induced aggregation, P-selectin secretion, and glycoprotein (GP) IIb/IIIa expression and reducing the release of atherosclerotic-related inflammatory mediators (sCD40L and IL-1β). TMP decreased the area of clot retraction, reflecting inhibition of GPIIb/IIIa activation. TMP inhibited adenosine diphosphate-induced platelet activation via increased cAMP production, VASPser157 phosphorylation, and Akt dephosphorylation. Conclusion TMP selectively inhibits ADP-induced platelet activation via P2Y12 receptor-related signaling pathways.

Saccharides from Arctium lappa L. root reduce platelet activation and thrombus formation in a laser injury thrombosis mouse model.

Arctium lappa L., also known as burdock, is a popular medicinal plant in traditional Chinese medicine due to its potential therapeutic properties. Saccharides from Arctium lappa L. root (ALR-S) have been extensively studied for their anti-inflammatory and anti-diabetes effects. Platelets play a pivotal role in thrombosis. The present study describes the effects of ALR-S on platelet activation and thrombosis using a laser injury thrombosis in vivo model. The study also measured the effects of ALR-S on platelet activation by analysing aggregation, ATP release, platelet spreading, adhesion and clot retraction in vitro. Specifically, the effects were ALR-S concentration-dependent inhibition of platelet aggregation and ATP release. Activated platelets pretreated with ALR-S showed diminished CD62P expression levels and fibrinogen binding, as measured by flow cytometry. ALR-S inhibited platelet spreading on fibrinogen and adhesion on collagen under shear. ALR-S attenuated platelet activation by decreasing oxidative stress and thrombus formation. These results demonstrated the antiplatelet effects of ALR-S, suggesting the antithrombotic and cardiovascular protective activities of ALR-S as a functional food.

Activated platelets and platelet-leukocyte aggregates in the equine systemic inflammatory response syndrome.

In humans, activated platelets contribute to sepsis complications and to multiple organ failure. In our prospective analytical study of cases of the equine systemic inflammatory response syndrome (SIRS), we adapted a standard human protocol for the measurement of activated platelets and platelet-leukocyte aggregates (PLAs) in equine platelet-leukocyte-rich plasma (PLRP) by flow cytometry, and we investigated the hypothesis that activated platelets and PLAs are increased in clinical cases of SIRS. We included 17 adult horses and ponies fulfilling at least 2 SIRS criteria, and 10 healthy equids as controls. Activation of platelets was determined by increased expression of CD62P on platelets. Activated platelets and PLAs were measured before and after in vitro activation of platelets with collagen. Median expression of CD62P on platelets was significantly increased after activation in the control group: 1.45% (interquartile range [IQR]: 1.08–1.99%) initially versus 8.78% (IQR: 6.79–14.78%, p = 0.002) after activation. The equids with SIRS had significantly more activated platelets and PLAs in native PLRP than controls: CD62P 4.92% (median, IQR: 2.21–12.41%) versus 1.45% in controls (median, IQR: 1.08–1.99%, p = 0.0007), and PLAs 4.16% (median, IQR: 2.50–8.58%) versus 2.95% in controls (median, IQR: 1.57–3.22%, p = 0.048). To our knowledge, increased platelet activation and PLAs have not been demonstrated previously with flow cytometry in clinical cases of equine SIRS.