CD62P Expression Research Articles

Antibody‐mediated procoagulant platelet formation in COVID‐19 is AKT dependent

BackgroundThromboembolic events are frequently reported in patients infected with the SARS‐CoV‐2. Recently, we observed that platelets from patients with severe COVID‐19 infection express procoagulant phenotype. The molecular mechanisms that induce the generation of procoagulant platelets in COVID‐19 patients are not completely understood. ObjectivesIn this study, we investigated the role of AKT (also known as Protein Kinase B), which is the major downstream effector of PI3K (phosphoinositid‐3‐kinase) (PI3K/AKT) signaling pathway in platelets from patients with COVID‐19. Patients and MethodsPlatelets, Sera and IgG from COVID‐19 patients who were admitted to the intensive care unit (ICU) were analyzed by flow cytometry as well as western blot and adhesion assays. ResultsPlatelets from COVID‐19 patients showed significantly higher levels of phosphorylated AKT, which was correlated with CD62p expression and phosphatidylserine (PS) externalization. In addition, healthy platelets incubated with sera or IgGs from ICU COVID‐19 patients induced phosphorylation of PI3K and AKT and were dependent on Fc‐gamma‐RIIA (FcγRIIA). In contrast, ICU COVID‐19 sera mediated generation of procoagulant platelets was not dependent on GPIIb/IIIa. Interestingly, the inhibition of phosphorylation of both proteins AKT and PI3K prevented the generation of procoagulant platelets. ConclusionsOur study shows that pAKT/AKT signaling pathway is associated with the formation of procoagulant platelets in severe COVID‐19 patients without integrin GPIIb/IIIa engagement. The inhibition of PI3K/AKT phosphorylation might represent a promising strategy to reduce the risk for thrombosis in patients with severe COVID‐19.

Differential Expression of Platelet Activation Markers, CD62P and CD63, after Exposure to Breast Cancer Cells Treated with Kigelia Africana, Ximenia Caffra and Mimusops Zeyheri Seed Oils In Vitro

Cancer patients, including breast cancer patients, live in a hypercoagulable state. Chemo- and hormone- therapy used in the treatment of breast cancer increases the risk of thrombosis. Due to differences in health care services between developed and developing countries, the survival rate of women with breast cancer in developing countries is low. Consequently, ethnomedicines are used and their efficacy as potential alternatives are being scientifically explored. The seed oils of Kigelia africana, Ximenia caffra and Mimusops zeyheri have anti-proliferative effects on hormone-dependent (MCF-7) and cytotoxic effects on hormone-independent (MDA-MB-231) breast cancer cells. In this study, we determined if these seed oils reduce the thrombogenic ability of breast cancer cells by measuring the platelet surface expression of the activation-specific antigens CD62P and CD63. MDA-MB-231 and MCF-7 cells were pretreated with the seed oils before being exposed to whole blood of human female volunteers. An increase in CD62P and CD63 expression following whole blood exposure to untreated breast cancer cells was observed. Treated MDA-MB-231 cells reduced CD62P and CD63 expression while treated MCF-7 cells increased CD62P and decreased CD63 expression. Kigelia africana, Ximenia caffra and Mimusops zeyheri seed oils are able to reduce the thrombogenic ability of MDA-MB-231 breast cancer cells.

Sorting nexin 24 is required for α-granule biogenesis and cargo delivery in megakaryocytes.

Germline defects affecting the DNA-binding domain of the transcription factor FLI1 are associated with a bleeding disorder that is characterized by the presence of large, fused α-granules in platelets. We investigated whether the genes showing abnormal expression in FLI1-deficient platelets could be involved in platelet α-granule biogenesis by undertaking transcriptome analysis of control platelets and platelets harboring a DNA-binding variant of FLI1. Our analysis identified 2,276 transcripts that were differentially expressed in FLI1-deficient platelets. Functional annotation clustering of the coding transcripts revealed significant enrichment for gene annotations relating to protein transport, and identified Sorting nexin 24 (SNX24) as a candidate for further investigation. Using an induced pluripotent stem cell-derived megakaryocyte model, SNX24 expression was found to be increased during the early stages of megakaryocyte differentiation and downregulated during proplatelet formation, indicating tight regulatory control during megakaryopoiesis. CRISPR-Cas9 mediated knockout (KO) of SNX24 led to decreased expression of immature megakaryocyte markers, CD41 and CD61, and increased expression of the mature megakaryocyte marker CD42b (P=0.0001), without affecting megakaryocyte polyploidisation, or proplatelet formation. Electron microscopic analysis revealed an increase in empty membrane-bound organelles in SNX24 KO megakaryocytes, a reduction in α-granules and an absence of immature and mature multivesicular bodies, consistent with a defect in the intermediate stage of α-granule maturation. Co-localization studies showed that SNX24 associates with each compartment of α-granule maturation. Reduced expression of CD62P and VWF was observed in SNX24 KO megakaryocytes. We conclude that SNX24 is required for α-granule biogenesis and intracellular trafficking of α-granule cargo within megakaryocytes.

Evaluation of platelet concentrates prepared from whole blood donations with collection times between 12 and 15 min: The BEST Collaborative study.

In many countries, whole blood (WB) donations with collection times between 12 and 15 min are not allowed to be used for platelet concentrates (PC). Since the development of guidelines, many process-related changes have been introduced. We aimed to determine the effect of WB with long collection times on PC quality. Five participating centres tested buffy coat (BC)-derived PC in platelet additive solution type E prepared from only WB collections lasting <12 min (control) versus similar PC including one BC from a collection lasting >12 min (study group, n= 8). One centre produced platelet-rich plasma (PRP)-derived PC from single donations (<10 or >12 min). All PC were stored at 22 ± 2°C and sampled on Days 1, 6 and 8 post-collection for in vitro quality determination. Average collection time was significantly longer in the study group compared to controls (8.9 ± 2.6 vs. 7.3 ± 1.3min, p < 0.001). There were no differences in volume, platelet concentration, basal CD62P expression, soluble-CD62P and CCL5 levels, or nucleotide content between the groups. Stimulation with TRAP-6 resulted in comparable levels of cell surface CD62P. On Day 8, all PC fulfilled requirements for pH. The findings from single PRP-derived PC centre were similar. PC with one BC and single PRP derived from collections lasting >12 min had equivalent in vitro quality to controls during storage. This study provides evidence that 12-15 min donations should not be excluded for PC preparation and justifies to readdress the guidelines to <15 min instead of <12 min of collection in line with current practice in some countries.

Detection of Sepsis in Platelets Using MicroRNAs and Membrane Antigens.

The present study proposes to legitimize in sepsis a characteristic found in platelets that suffer storage lesions in blood banks, which is the increased expression of miRNA miR-320a in relation to miR-127. Under physiologically normal conditions, an inverse relationship is observed. The aim of this study was to verify whether the analysis of miR-320a and miR-127 expression in platelets could detect a decrease in their viability and function due to the presence of pathogens in the blood of patients hospitalized in the Intensive Care Unit. We also investigated the expression of membrane antigens sensitive to platelet activation. Of the 200 patients analyzed, only those who developed sepsis (140) were found to have a higher relative quantity of miR-320a than that of miR-127. This characteristic and the increased expression of membrane antigens P2Y12, CD62P, CD41, and CD61 showed a significant association (p < 0.01) with all types of sepsis evaluated in this study. Additionally, 40% of patients hospitalized for sepsis had negative results for the first cultures. We conclude that analysis of miR-127 and miR-320a expression combined with membrane antigens evaluation, in association with the available clinical and diagnostic parameters, are important tools to detect the onset of sepsis.

Evaluating apheresis platelets at reduced dose as a contingency measure for extreme shortages.

The COVID19 pandemic highlights the need for contingency planning in the event of blood shortages. To increase platelet supply, we assessed the operational impact and effect on platelet quality of splitting units prior to storage. Using production figures, we modeled the impact on unit numbers, platelet counts, and volumes of splitting only apheresis double donations into three units (yielding ⅔ doses), or all standard dose units in half. To assess quality, eight pools of three ABO/Rh-matched apheresis (Trima Accel) double donations in plasma were split to ⅔ and ½ volumes in both Terumo and Fresenius storage bags. These were irradiated and subject to maximal permitted periods of nonagitation (3 × 8 h) before comparing platelet quality markers (including pH, CD62P expression) to Day 9 of storage. Splitting all double donations into three predicted inventory expansion of 23% overall whereas halving all standard dose units clearly doubles stock. In our study, ⅔ and ½ doses contained 153 ± 15 × 109 (~138 ml) and 113 ± 11 × 109 (~102 ml) platelets respectively. Following storage, higher pH was observed in ⅔ than in ½ doses and in Terumo compared to Fresenius bags. The higher pH was reflected in better quality markers, including lower CD62P expression. Despite the differences, on Day 8 (of pH monitoring at expiry) all ⅔ doses and most ½ doses were ≥pH6.4. A strategy to split apheresis platelets in plasma to lower doses is feasible, maintains acceptable platelet quality, and should be considered by blood services in response to extreme shortages.

Palladium‐Based Metallic Glass with High Thrombogenic Resistance for Blood‐Contacting Medical Devices

AbstractAdvancements in the design of mechanical blood circulatory devices have greatly improved patient survival rates, but currently employed metals still provoke a thrombosis response upon contact with blood, with potential life‐threatening consequences. While coating strategies have been developed to address this limitation, they possess inherent drawbacks such as susceptibility to crack formation and delamination. Herein, an amorphous metal based on palladium (Pd) is scrutinized and reveals substantial thrombogenic resistance compared to a state‐of‐the‐art titanium alloy. In vitro assessment with human whole blood shows that the Pd glass provokes reduced platelet activation (lower expression of CD62P, CD41/CD61) and greatly retarded fibrin formation, but pronounced platelet spreading therewith challenging the dogma that platelet spreading equals activation. Mechanistic analysis of the early biomaterial–blood interactions reveals that conformational changes of adhered fibrinogen, and modified αIIbβ3 integrin expression and distribution across adhered platelets, underlay the superior performance of Pd glass. The study is accompanied by structural and thermophysical bulk investigations and physicochemical surface characterization to link the materials properties with the observed blood response. The results reveal a remarkable potential of Pd‐based glass as direct blood‐contacting bulk material without the need for coating.

A comparison of platelet function in cold-stored whole blood and platelet concentrates.

There is renewed interest in the use of whole blood (WB) for the resuscitation of trauma patients. Platelet function in stored WB compared to platelet concentrates is not well established and was assessed in vitro in this study. Leucocyte-depleted cold-stored WB (CS-WB) was prepared using a Terumo WB-SP Imuflex kit and held at 2-6°C alongside: (A) UK standard pooled platelets stored at 20-24°C (RT-PLTS), (B) pooled platelets stored at 2-6°C (CS-PLTS), and (C) platelet-rich plasma produced using the Terumo kit (CS-PRP), for 21 days. A series of in vitro assays were assessed platelet function. Platelet count was retained to 57 ± 14% of starting number at day 21 in CS-WB. Over time, CS-WB platelets become more activated, with increased CD62P expression (day 1: 7± 3.7% vs. day 21: 59 ± 17.1%) and annexin V binding (day 1: 2± 0.2% vs. day 21: 21 ± 15.1%). For comparison, 18.6± 6% of platelets in RT-PLTS demonstrated CD62P expression at day 7, whereas annexin V binding in RT-PLTS at day 7 was 2.6± 0.5%. Over storage, aggregatory response to agonists decreased in all arms. Functional platelet microparticles increased steadily in CS-WB throughout storage. During storage, platelet count reduced in CS-WB, whereas CD62P expression and annexin V binding increased. This was accompanied by a reduced aggregatory response, although compared to 7-day-old RT-PLTS, CS-WB maintained a maximal response to agonists for longer, suggesting that the shelf life for CS-WB can be considered for up to 21 days.

Evaluation of platelets activity and reactivity as risk factors for acute ischemic non-embolic stroke in young adults

BackgroundIschemic stroke (IS) constitutes a relevant health concern recently in younger population causing permanent cognitive and function-limiting disability and ranks as the 3rd cause of death in Egypt after cardiac and hepatic diseases. Platelet activation has a crucial mechanism in arterial thrombogenesis, thus in pathophysiology of IS. Surface expression of P-selectin (CD62P) reflects platelet activation and measured by flowcytometry. The purpose of the study is to evaluate whether platelet activity and reactivity are considered risk factors for IS so more restrict antiplatelet protocols could be implemented for management and recurrence prevention.ResultsStudy population was 60 IS patients and 60 apparently healthy age and gender-matched controls. Patients were subdivided into 37 patients without classical risk factors, aged 46.1 ± 8.2, and 23 patients with > 1 vascular risk factors, aged 52 ± 9.9. The percentage of platelets expressing CD62P reflecting ex vivo baseline activity was significantly higher in stroke patients to controls (p = 0.001), also platelet reactivity (CD62P expression after ADP provocation) was statistically significantly elevated in patients than in controls (p < 0.0001) and was significantly higher in IS patients with vascular risk factors compared to patients without risk factors (p = 0.02).ConclusionBoth baseline platelet activity and reactivity were significantly higher in IS patients, and were also higher in IS patients with other vascular risk factors than in cryptogenic stroke and considered risk factors for IS.

Early platelet dysfunction in patients receiving extracorporeal membrane oxygenation is associated with mortality

Extracorporeal membrane oxygenation (ECMO) is used for patients with cardiopulmonary failure and is associated with severe bleeding and poor outcome. Platelet dysfunction may be a contributing factor. The aim of this prospective observational study was to characterize platelet dysfunction and its relation to outcome in ECMO patients. Blood was sampled from thirty ECMO patients at three timepoints. Expression of CD62P, CD63, activated GPIIb/IIIa, GPVI, GPIbα and formation platelet-leukocyte aggregates (PLA) were analyzed at rest and in response to stimulation. Delta granule storage-pool deficiency and secretion defects were also investigated. Fifteen healthy volunteers and ten patients with coronary artery disease served as controls. Results were also compared between survivors and non-survivors. Compared to controls, expression of platelet surface markers, delta granule secretion and formation of PLA was reduced, particularly in response to stimulation. Baseline CD63 expression was higher and activated GPIIb/IIIa expression in response to stimulation was lower in non-survivors on day 1 of ECMO. Logistic regression analysis revealed that these markers were associated with mortality. In conclusion, platelets from ECMO patients are severely dysfunctional predisposing patients to bleeding complications and poor outcome. Platelet dysfunction on day 1 of ECMO detected by the platelet surface markers CD63 and activated GPIIb/IIIa is associated with mortality. CD63 and activated GPIIb/IIIa may therefore serve as novel prognostic biomarkers, but future studies are required to determine their true potential.

Effect of Vitamin D3 to Platelet Activation Mediated by Tumor Cell Culture Medium

To investigate the effect of vitamin D3 to platelet activation by tumor cell culture medium. The peripheral blood platelets of BALB/c mice were isolated. The platelets were activated in 4T1 culture fluid for 24 h. The platelets were divided into 7 groups: control group, activation group, 1 nmol/L vitamin D3 group, 10 nmol/L vitamin D3 group, 50 nmol/L vitamin D3 group, 100 nmol/L vitamin D3 group, and positive drug (0.1 μmol/L eptifibatide) group. CCK-8 assay was used to detect the platelet proliferation at 24, 48 and 72 h. Flow cytometry was used to detect the expression of CD61 and CD62p and receptor for advanced glycation end products (RAGE) at 24, 48 and 72 h. ELISA was used to detect the level of platelet-endothelial cell adhesion molecule-1 (PECAM-1) at 24, 48 and 72 h. The CD41+/CD61+ and CD41+/CD62p+ ratio, PECAM-1 content and RAGE expression of platelets in activated group were all significantly increased as compared with those in control group (P<0.05). Compared with the activated group, the platelet proliferation, proportion of CD41+/CD61+ and CD41+/CD62p+, content of PECAM-1 and RAGE expression in vitamin D3 groups were all decreased (P<0.05). Vitamin D3 shows antiplatelet effect and can inhibit platelet proliferation and activation.

Exogenously Added Cofilin Increases Ex Vivo Production of Functional Platelets By Stimulating Proplatelet Formation

Platelet transfusion is essential for the treatment of thrombocytopenia. Platelet supply depends on blood donation and the ability to store platelets before use is limited by a short shelf-life. Furthermore, such supplied-platelets have the risk of bacterial or viral infection. Ex vivo platelet production could overcome these problems. Therefore, many methods of ex vivo platelet production from some stem cell sources such as umbilical cord blood (UCB) stem cells, human embryonic stem cells and induced pluripotent stem cells have been developed. However, the platelet yields achieved by these methods have not been sufficient for clinical use. The processes of platelet production are thought to involve differentiation into megakaryocytes (Mks) from hematopoietic stem cells, Mk polyploidization, proplatelet formation, and finally platelet release. Large-scale ex vivo production of functional platelets requires a system to control all these steps. Hematopoietic progenitor cell and Mk levels can be increased by the numerous hematopoietic growth factors; however, factors to stimulate proplatelet formation in Mks have not been identified.To find factors to stimulate proplatelet formation in Mks, we evaluated the effects of various conditioned media using Mks induced from UCB-CD34+ cells. The supernatants of COS cells transfected with cDNA coding for non-muscle cofilin stimulated proplatelet formation in Mks, whereas those of mock-transfected COS cells did not. Cofilin is an actin-binding protein; recently, endogenous non-muscle cofilin was reported to be associated with proplatelet formation in Mks, but that activity has not been reported for exogenous cofilin. The effects of cofilin on proplatelet formation in Mks in the presence or absence of TPO were compared with that in the presence of TPO alone. Cofilin alone increased the number of proplatelet-bearing Mks and enhanced proplatelet extension compared to TPO alone; moreover, a combination of cofilin and TPO had synergistic effects on both activities.The influences of cofilin on megakaryopoiesis from UCB-CD34+ cells were compared with those of TPO. The addition of cofilin to serum-free culture containing SCF and Flt3-L increased the size of both Mks and Mk colonies compared to SCF/Flt3-L/TPO. Next, the effects of cofilin on the platelet production in the serum-free culture system containing SCF and Flt3L were evaluated; the addition of coflin dramatically increased the number of platelets compared to that for SCF/Flt3-L/TPO. Thus, we established a suitable system for ex vivo platelet production by using cofilin. To determine whether the platelets produced using this system were functional; platelet activation in response to in vitro activation with thrombin was tested. Thrombin induces platelet granule secretion, resulting in CD62P (P-selectin) expression on the platelets surface membrane. The expression of CD62P was increased after thrombin stimulation of the platelets produced in cofilin culture. These results indicate that the platelets produced with cofilin were functional.We show here that exogenously adding cofilin to the serum-free culture system enhanced proplatelet formation in Mks, resulting in increased platelet yields. At present, many methods are under study for ex vivo platelet production for clinical use; we believe that our studies will provide the important information for the improvement of these methods. DisclosuresNo relevant conflicts of interest to declare.

Transferrin Saturation and Its Association with Platelet Reactivity

Introduction. While a plethora of risk factors have been well established as predictors for atherosclerosis and coronary heart disease, the possible association between body iron status and the risk of cardiovascular disease has yielded conflicting results. Epidemiological studies have found a significant negative association of transferrin saturation and coronary heart disease or myocardial infarction suggesting that higher body iron stores possibly confer a protective effect towards developing cardiovascular diseases. Despite the pivotal role of platelets on the pathogenesis of thrombotic complications, little information is available about the relationships between transferrin saturation and platelet reactivity. Aim of this study was to evaluate the influence of transferrin saturation on platelet function.Methods. The study included two groups: 32 healthy volunteers (17M/15F, age: 55±5 years) and 45 hereditary hemochromatosis (HH) patients (34M/11F, age: 62±2 years). HH patients were characterized by high prevalence of C282Y and/or H63D mutations of the hemochromatosis gene (HFE) and showed, just before the last therapeutic bloodletting, serum transferrin saturation and ferritin levels of 51±4% and 176±34 ng/ml, respectively.In platelet samples from healthy subjects and HH patients, we evaluated the effects of a 15-min preincubation with holotransferrin (holoTf) or apotransferrin (apoTf) (10-80 nmol/l) on: i) maximal aggregation to ADP (10 µmol/l), collagen (4 mg/l), epinephrine (5 µmol/l) and arachidonic acid (AA)(1 µmol/l) (Born's method); ii) expression of platelet activation marker CD62P in response to collagen (4mg/l) (flow-cytometry); iii) activation of signaling pathways PI-3K/Akt and MAPK/Erk-1/2 in response to collagen (4mg/l) and AA (100 µmol/l) (Western Blot); iv) reactive oxygen species (ROS) stimulated by AA (100 µmol/l) (DCF-DA fluorescence).Results. In healthy subjects we found that platelet exposure to holoTf, but not apoTf, significantly decreased platelet reactivity. In particular, 40 µmol/l HoloTf: i) reduced maximal aggregation by 39±1% to ADP, 40±4% to collagen, 49±3% to epinephrine, and 48±4% to AA (versus each agonist alone: p<0.05); ii) reduced platelet membrane expression of CD62P by 50±3% to collagen (p<0.0001); iii) reduced pAkt levels by 40±5% with collagen (p<0.001), and 38±2% with AA (p<0.0001); reduced pErk-2 levels by 66±5% with collagen (p<0.0001), and 38±4% with AA (p<0.005); iv) reduced the AA-induced ROS production by 35±3% (p<0.001). Also in platelets from HH patients holoTf significantly attenuated platelet reactivity. In fact, 40 µmol/l HoloTf: i) reduced platelet aggregation by 45±4% to ADP, 38±9% to collagen, 57±4% to epinephrine, and 49±5% to AA (versus each agonist alone: p<0.05); ii) reduced platelet membrane expression of CD62P by 54±6% to collagen (p<0.0001); iii) reduced pAkt levels by 37±6% with collagen (p<0.005), and 41±7% with AA (p<0.0001); reduced pErk-2 levels by 59±7% with collagen (p<0.0001), and 42±5% with AA (p<0.005); iv) reduced the AA-induced ROS production by 37±5% (p<0.001). The levels of transferrin saturation, but not of ferritin, were inversely correlated with the main platelet reactivity parameters; in particular, we found negative and significant correlations with the maximal aggregation to ADP (r:-0.332, p=0.05), collagen (r=-0.511, p=0.002), epinephrine (r=-0.404, p=0.02), AA (r=-0.460, p=0.009) and the CD62P expression at both basal conditions (r=-0.456, p=0.02) and after stimulation with collagen (r=-0.459, p=0.05) or AA (r=-0.629, p= 0.001).Conclusions. In healthy subjects, platelet exposure to holoTf, but not apoTf, reduced platelet reactivity as showed by a significant decrease of: i) aggregation, ii) membrane expression of CD62P, iii) activation of PI3K and MAPK pathways, and iv) oxidative stress. The effects of holoTf on platelet response were preserved in HH patients were transferrin saturation levels inversely correlated with platelet aggregation and activation. These results induce to hypothesize that in the association of high transferrin saturations and lower risk of cardiovascular diseases a role could be exerted by transferrin saturation effects on platelets. DisclosuresNo relevant conflicts of interest to declare.

Phenotype of Platelet Is Altered and Associated with Disease Activity of Rheumatoid Arthritis

Platelets play a major role in the hemostasis physiologically through their activation. Activated platelets acquire functional abilities such as adhesion, morphological changes, and releasing humoral factors or microparticles (MPs). Recent reports have been shown that platelets and MPs include unique humoral factors such as chemokines and cytokines. We hypothesized that platelets and MPs can be vectors for transferring these factors to the lesions through circulation and contribute to disease processes of systemic diseases. To clarify the role of platelets in the pathogenesis of connective tissue diseases (CTDs), especially in rheumatoid arthritis (RA), we examined the phenotype and activation status of circulating platelets and the association with clinical characteristics.Fifty-seven patients with RA were involved and 21 systemic sclerosis (SSc), which has different characteristic compared to RA in CTDs, and 16 healthy individuals (HI) were examined as controls. Platelet rich plasma was used to detect fractions of platelets and MPs by flow cytometry. Fraction of platelets and MPs were defined by the size using standardized Megamix® beads. For the phenotypic analysis of platelets, surface expression of CD62P (P-selectin), membrane-bound TGF (transforming growth factor)-beta, CD147 (emmprin), CD142 (tissue factor), CD31 (platelet endothelial cell adhesion molecule (PECAM)-1) on CD41+CD45-CD14- platelets was examined. For the activation status of platelets, the ratio of CD62P or PAC1-positive to -negative platelets for the activation and the ratio of MPs to platelets as a marker for the release of MPs. Clinical parameters at blood drawing were retrospectively obtained and correlation was examined.We first examined the phenotype of circulating platelets in RA and controls to have a general picture of the difference. Proportion of CD62P+ platelets were higher in both RA and SSc compared to HI (0.054 ± 0.032, 0.060 ± 0.030 versus 0.021 ± 0.006, P = 0.0005, P < 0.0002, respectively). Interestingly, CD147+ platelets were significantly higher in RA (0.61 ± 0.05 versus 0.29 ± 0.01, P = 0.0004), whereas not only proportion of CD147+ platelets (0.69 ± 0.11) but that of TGF-beta+ platelets (0.073 ± 0.021 versus 0.014± 0.006) were higher in SSc compared to healthy controls (P = 0.0001, P = 0.0001, respectively). Thus, phenotype of platelets is altered in RA compared to HI or SSc. Then, we focused on RA and activation status of circulating platelets was further examined by expression of activation markers and release of MPs. Proportion of activated platelets such as CD62P (0.047 ± 0.041 versus 0.012 ± 0.007) and PAC1 (0.075 ± 0.072 versus 0.013 ± 0.011) and that of MPs (0.034 ± 0.06 versus 0.21 ± 0.03) were higher in RA compared to HI (P < 0.001, P < 0.004, P < 0.00001, respectively). In patients with RA, proportion of activated platelets (CD62P+ or PAC1+ platelets) and that of MPs were correlated each other (r = 0.57, P < 0.0001; r = 0.36, P < 0.004, respectively). When activation status of platelets was compared to clinical parameters, ratio of CD62P+ platelets and that of MPs were positively correlated with clinical parameters associated with disease activity of patients with RA, such as serum CRP (r = 0.36, P < 0.006 and r = 0.29, P < 0.03), erythrocyte sedimentation rate (r = 0.31, P < 0.02 and r = 0.35, P < 0.009) and composite disease markers such as Simplified Disease Activity Index (SDAI) (r = 0.28, P < 0.04 and r = 0.31, P < 0.02) and Clinical Disease Activity Index (CDAI) (r = 0.28, P < 0.04 and r = 0.32, P < 0.02), but interestingly, there was no correlation between proportion of PAC1+ platelets and clinical parameters. No correlation was observed with age, sex, HAQ-DI, nor MMP-3. Furthermore, six patients with RA who were in active phase and treated with methotrexate and/or biologics and achieved improvement of disease activity at 12 week after the treatment were compared. The ratio of CD62+ platelets and that of MPs were decreased along with the improvement of disease. (P < 0.03, P < 0.03, respectively).In conclusion, circulating platelets in RA were activated and their activation status is correlated with the inflammatory markers and disease activity, suggesting that circulating platelets reflects and can contribute to the process of systemic chronic inflammation in RA. Phenotype of platelets can be a novel biomarker for the disease. DisclosuresNo relevant conflicts of interest to declare.

Activation Mediated By Peroxisome Proliferator-Activated Receptor (PPAR) Ligands and Brown Algae-Based Compounds: Different Mechanisms between Megakaryoblasts and Mature Platelets

Background: Nuclear receptors aretranscription factors that are activated by ligands and subsequently bind to regulatory regions in target genes. Recently ligand-activated transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) has been reported to promote the formation of platelets from megakaryocytes and accelerate platelet recovery after radiation-induced bone marrow injuries. The endogenous prostaglandin 15-deoxy-Δ12,14 prostaglandin J2 (15d-PGJ2) are ligands of PPARγ. Accumulating evidences also suggest that PPAR activation is involved in inflammation responses and atherosclerosis. In the present study, we analyzed how PPAR ligands and some brown algae-based compounds affect platelet differentiation and functions in vitro.Methods: Platelets were obtained from plateletpheresis products by centrifugation. DAMI and Meg-1 cells, both human megakaryocytic cell lines, were also obtained. Western blotting for PPARγ, PPARα and COX-2 was performed, and bands were visualized by chemiluminescence. The effects of 15d-PGJ2, GW9662, Fucoidan, Seanol, GW6471 and Celecoxib were evaluated. Morphology analysis, platelet flow cytometry, total thrombus formation analysis system (T-TAS) and microarray expression analysis were also used in order to analyze the effects of the treatment on platelets.Results: Megakaryoblasts and mature platelets showed different results in the expression of PPARα, COX-2, glycoproteins and platelet activation markers. PGJ2 induced morphological differentiation evidences in DAMI and Meg-1 cells. PGJ2 increased the levels of CD41, CD42b, CD62p and PAC-1 both in DAMI and Meg-1 cells. In mature platelets, Fucoidan and Seanol decreased the expression of PPARα and increased COX-2 expression. Fucoidan elevated the expression of CD62p and CD63 in mature platelets, suggesting that Fucoidan causes platelet activation. In microarray analysis, fucoidan showed early growth response (EGR)-1 gene upregulation. Unfortunately thrombogenesity by T-TAS was not evident from the study.Conclusions: The mechanism of platelet production and activation is not fully understood. Understanding the mechanisms underlying megakaryocyte maturation and platelet production will facilitate the development of methods to increase the production of platelets from precursor cells. Although platelets are non-nucleated cells, they contain transcription factors, especially PPAR and NF-κB. From the present study, our findings support the hypothesis that PPAR, both PPARγ and PPARα play a role in platelet differentiation and activation, and exact mechanisms need to be defined related to prostaglandin pathways. Fucoidan may activate platelets via different pathways than PGJ2. DisclosuresNo relevant conflicts of interest to declare.

Distinct phenotypes of platelet, monocyte, and neutrophil activation occur during the acute and convalescent phase of COVID-19

SARS-CoV-2 has spread rapidly worldwide, causing the COVID-19 pandemic. Platelet activation and platelet-leukocyte complex formation are proposed to contribute to disease progression. Here, we report platelet and leukocyte activation during acute and convalescent COVID-19 in patients recruited between May-July 2020. Blood samples were analyzed by flow cytometry and ELISA using paired comparison between inclusion (day 0) and 28 days later. The majority of patients were mildly or moderately ill with significantly higher cytokine levels (IL-6 and IL-10) on day 0 as compared with day 28. Platelet activation and granule release were significantly higher on day 0 compared with day 28, as determined by ADP- or thrombin-induced surface CD62P expression, baseline released CD62P, and thrombin-induced platelet-monocyte complex formation. Monocyte activation and procoagulant status at baseline and post activation were heterogeneous but generally lower on day 0 compared with day 28. Baseline and thrombin- or fMLF-induced neutrophil activation and procoagulant status were significantly lower on day 0 compared with day 28. We demonstrate that during the acute phase of COVID-19 compared with the convalescent phase, platelets are more responsive while neutrophils are less responsive. COVID-19 is associated with thromboembolic events where platelet activation and interaction with leukocytes may play an important role.

UV light-emitting diode (UV-LED) at 265 nm as a potential light source for disinfecting human platelet concentrates.

The risk of sepsis through bacterial transmission is one of the most serious problems in platelet transfusion. In processing platelet concentrates (PCs), several methods have been put into practice to minimize the risk of bacterial transmission, such as stringent monitoring by cultivation assays and inactivation treatment by photoirradiation with or without chemical agents. As another potential option, we applied a light-emitting diode (LED) with a peak emission wavelength of 265 nm, which has been shown to be effective for water, to disinfect PCs. In a bench-scale UV-LED exposure setup, a 10-min irradiation, corresponding to an average fluence of 9.2 mJ/cm2, resulted in >2.0 log, 1.0 log, and 0.6 log inactivation (mean, n = 6) of Escherichia coli, Staphylococcus aureus, and Bacillus cereus, respectively, in non-diluted plasma PCs. After a 30-min exposure, platelet counts decreased slightly (18 ± 7%: mean ± SD, n = 7); however, platelet surface expressions of CD42b, CD61, CD62P, and PAC-1 binding did not change significantly (P>0.005), and agonist-induced aggregation and adhesion/aggregation under flow conditions were well maintained. Our findings indicated that the 265 nm UV-LED has high potential as a novel disinfection method to ensure the microbial safety of platelet transfusion.

Platelet number and function in response to a single intravenous dose of vincristine.

BackgroundVincristine might increase circulating platelet numbers but the functional capacity of these newly released platelets is unknown.ObjectiveTo evaluate and compare the functionality of mature and immature (reticulated) platelets after a single intravenous dose of vincristine in dogs.AnimalsTen healthy purpose‐bred dogs.MethodsDogs prospectively received a single IV injection of 0.02 mg/kg vincristine or 0.9% saline. Before and after treatment on days 3, 5, and 7, platelets (resting and after thrombin stimulation) were assessed by flow cytometric determination of P‐selectin (CD62P) expression. Reticulated platelets were distinguished using thiazole orange (TO) staining.ResultsRelative to saline, vincristine administration increased platelet count from day 0 to day 7 (225 ± 58 to 273 ± 65 × 103/μL, vs 299 ± 76.4 to 214 ± 20 × 103/μL, P = .01) and increased percentage of reticulated platelets from day 0 to day 5 (3.9 ± 1.5% to 6.1 ± 1.6%, P = .02). On all days, reticulated platelets had greater resting expression of CD62P than did mature platelets (49.6 ± 4% vs 10.2 ± 1%, P ≤ .001). Across all days, CD62P expression by reticulated platelets in the vincristine and saline‐treated groups was not different when unstimulated (P = .7) or after thrombin stimulation (P = .33).Conclusions and Clinical ImportanceReticulated platelets released in response to vincristine administration function similarly to mature platelets.

Platelet and neutrophil responses to Porphyromonas gingivalis in human whole blood.

Porphyromonas gingivalis is a causative agent for periodontal disease. Binding of platelets to this gram-negative anaerobe can regulate host hemostatic (thrombus forming) and immune (neutrophil interacting) responses during bacterial infection. Additionally, in response to bacterial pathogens neutrophils can release their DNA, forming highly prothrombotic neutrophil extracellular traps (NETs), which then further enhance platelet responses. This study evaluates the role of P. gingivalis on platelet expression of CD62P, platelet-neutrophil interactions, and labeled neutrophil-associated DNA. Human whole blood was preincubated with varying P. gingivalis concentrations, with or without subsequent addition of adenosine diphosphate (ADP). Flow cytometry was employed to measure platelet expression of CD62P using PerCP-anti-CD61 and PE-anti-CD62P, platelet-neutrophil interactions using PerCP-anti-CD61 and FITC-anti-CD16, and the release of neutrophil DNA using FITC-anti-CD16 and Sytox Blue labeling. Preincubation with a high (6.25×106 CFU/mL) level of P. gingivalis significantly increased platelet expression of CD62P in ADP treated and untreated whole blood. In addition, platelet-neutrophil interactions were significantly increased after ADP stimulation, following 5-22min preincubation of blood with high P. gingivalis CFU. However, in the absence of added ADP, platelet-neutrophil interactions increased in a manner dependent on the preincubation time with P. gingivalis. Moreover, after ADP addition, 16min preincubation of whole blood with P. gingivalis led to increased labeling of neutrophil-associated DNA. Taken together, the results suggest that the presence of P. gingivalis alters platelet and neutrophil responses to increase platelet activation, platelet interactions with neutrophils, and the level of neutrophil antimicrobial NETs.

In Vitro Characterization and Metabolomic Analysis of Cold-Stored Platelets.

Platelet concentrates are currently stored at room temperature (RP) under constant agitation for up to 5-7 days depending on national regulations. However, platelet quality deteriorates during storage and room-temperature storage also increases the risk of bacterial growth. Previous studies have shown that cold-stored platelets (CPs) have higher hemostatic functions and can be stored for up to 3 weeks. While these studies have compared the metabolic phenotypes of CPs and RPs, they have neither compared the impact of storage temperature and cold agitation (CPAs) on platelet function nor identified metabolic correlates to such parameters. In vitro analysis showed that CPAs and CPs had reduced count, faster CD62P expression, and increased lactadherin binding. Furthermore, CPAs and CPs had higher maximal aggregation and a reduced aggregation lag phase compared to RPs. Metabolomic analysis revealed that CPAs and CPs exhibited lower oxidative stress shown by preserved glutathione and pentose phosphate pools. CPAs and CPs also had reduced markers of beta-oxidation and amino acid catabolism, demonstrating reduced needs for energy. Agitation did not significantly impact in vitro function or metabolomic parameters of cold-stored platelets. Correlation of in vitro and metabolomic results highlighted important metabolites that may contribute to stored platelet functions. Raw data are publicly available through Metabolomics Workbench with the study identifier ST001644.