Clinical and Molecular Characteristics of Megakaryocytes in Myelodysplastic Syndrome.

Objective Myelodysplastic syndrome (MDS) is a malignant clonal disorder of hematopoietic stem cells which is characterized by morphologic dysplasia. However, the pathological characteristics of megakaryocytes (MKs) in MDS patients with gene mutation are not well established. Methods Bone marrow MK specimens from 104 patients with primary MDS were evaluated, and all patients were distributed into two groups according to gene mutation associated with functional MKs. The morphologic and cellular characteristics of MKs and platelets were recorded and compared. Results The more frequently mutated genes in MDS patients were TUBB1 (11.54%), VWF (8.65%), NBEAL2 (5.77%), and the most common point mutation was TUBB1 p.(R307H) and p.(Q43P). Patients with MK mutation showed a decrease in adenosine diphosphate-induced platelet aggregation, high proportion of CD34 + CD61 + MKs (10.00 vs. 4.00%, p = 0.012), and short overall survival (33.15 vs. 40.50 months, p = 0.013). Further, patients with a higher percent of CD34 + CD61 + MKs (≧20.00%) had lower platelet counts (36.00 × 10 9 /L vs. 88.50 × 10 9 /L, p = 0.015) and more profound emperipolesis ( p = 0.001). By analyzing RNA-sequencing of MKs, differentially expressed mRNA was involved in physiological processes including platelet function and platelet activation, especially for MDS patients with high percent of CD34 + CD61 + MKs. The high levels of expression of CD62P, CXCL10, and S100A9 mRNA, shown by RNA sequencing, were validated by PCR assay. Conclusion High proportion of CD34 + CD61 + MKs was a poor prognostic factor in MDS patients with MK mutation. CD62P, CXCL10, and S100A9 may be the potential targets to evaluate the molecular link between gene defects and platelet function.

Decrease of exosomal miR-21-5p and the increase of CD62p+ exosomes are associated with the development of sepsis in polytraumatized patients.

Sepsis as a severe systemic inflammation leads oftentimes to organ dysfunction and subsequently to death. In polytrauma patients, septic complications represent with 45% the predominant cause of late death and are responsible for extremely high costs in the healthcare system. Therefore, clinicians have to detect as early as possible the begin of sepsis to improve the patient's outcome. One new promising diagnostic tool to diagnose septic complications in polytraumatized patients are exosomes. Plasma samples from polytraumatized patients (Injury Severity Score (ISS) ≥16) which developed sepsis (n = 10) and without sepsis (n = 10), were collected at emergency room (ER), 24h and 5 days after trauma. The EVs subpopulations were investigated by a bead-based multiplex flow cytometry measurement of surface epitopes and were compared with plasma EVs from healthy controls (n = 10). Moreover, exosomal cytokine concentrations were measured via high-sensitive ELISA and were correlated with systemic concentrations. For miRNA cargo analysis, we analysed the miRNAs miR-1298-5p, miR-1262, miR-125b-5p, miR-92a-3p, miR-93-5p, miR-155-5p and miR-21-5p and compared their exosomal concentrations by means of RT-qPCR. CD62p + exosomes were significantly increased in septic polytrauma-patients (p ≤ 0.05), while CD40+exosomes, as well as CD49e + exosomes were diminished (p ≤ 0.05). Furthermore, we observed that the exosomal IL-6 concentration reflects the systemic IL-6 concentration (r2 = 0.63) and did not significantly alter between patients with and without sepsis. The exosomal IL-10 concentration seemed to be constant in all patients and healthy controls. We observed that a decrease of miR-21-5p in exosomes was associated with the development of sepsis (p ≤ 0.05), while exosomal miR-93-5p, miR-155-5p and miR-92a-3p were not specifically altered in septic patients. Taken together, the present study in polytraumatized patients demonstrated that the development of sepsis is associated with an increase of CD62p + exosomes. Furthermore, the exosomal cargo was changed in septic patients: miR-21-5p was diminished.

Differential expression of serum TM, PAF, and CD62P in patients with autologous arteriovenous fistula and the correlation with vascular access function.

BACKGROUND: End-stage renal disease (ESRD) is the final stage of chronic kidney disease (CKD). AIMS: We aimed to analyze the expression differences of serum thrombomodulin (TM), platelet-activating factor (PAF), and P-selectin (CD62P) in patients with autologous arteriovenous fistula (AVF) and the correlation with vascular access function. METHODS: The case data were retrospectively analyzed. Moreover, 160 patients with AVF maintenance hemodialysis were selected as the AVF group, and 150 healthy participants were selected as the healthy control group. According to the function of vascular access, patients in the AVF group were divided into Group A (n = 50, after the first establishment of AVF), Group B (n = 64, normal vascular access function after hemodialysis treatment), and Group C (n = 46, vascular access failure). Pearson analysis was conducted to explore the correlation between serum TM, PAF, CD62P content, and vascular pathological examination indicators, to evaluate the value of TM, PAF, and CD62P levels in predicting vascular access failure in patients with AVF. RESULTS AND DISCUSSION: The serum levels of TM, PAF, and CD62P were positively correlated with the expressions of CD68 and MCP-1, respectively (p < .001). Serum TM was positively correlated with the levels of PAF and CD62P (p < .001), and PAF was positively correlated with the levels of CD62P (p < .001), respectively. Serum levels of TM, PAF and CD62P were risk factors for vascular access failure in AVF patients (p < .05). The area under the curve of serum TM, PAF and CD62P levels in predicting vascular access failure in AVF patients was 0.879. CONCLUSION: The serum levels of TM, PAF, and CD62P in AVF patients were correlated with the vascular access function of AVF patients, which was very important for maintaining the stability of vascular access function, and had certain value in predicting vascular access failure/disorder in AVF patients, and could be popularized and applied.

A comparative study of platelet storage lesion in platelet-rich plasma under cryopreservation.

Platelet-rich plasma (PRP) has significant potential for various applications and holds clinical value in regenerative medicine. Cryopreservation is used to extend the preservation period of PRP, facilitating its clinical application. However, the potential negative effects of long-term cryopreservation on platelet storage lesion are still uncertain. In this study, PRP was stored at - 30 °C or - 80 °C. Platelet count, apoptosis, reactive oxygen species (ROS) content, and CD62P expression were assessed on the 14th and 28th days. The study also evaluated platelet mitochondria morphology and function, serotonin (5-HT) secretion by platelets, and the inflammatory activating effect of cryopreserved platelets in PRP. The results showed that there were no significant differences in platelet count, the content of 5-HT, and inflammatory effects between fresh PRP and PRP cryopreserved at both - 30 °C and - 80 °C. However, there was an increase in ROS level, apoptosis, and CD62P level after cryopreservation at both temperatures. Additionally, the levels of ROS, apoptosis, and CD62P in platelets were similar after storage at - 30 °C and - 80 °C. The main difference observed was that the morphology and function of mitochondria were severely damaged after storage at - 30 °C, while they were less affected at - 80 °C. Based on these findings, it can be concluded that storing PRP at - 80 °C is more suitable for achieving a better therapeutic effect in clinical applications, but cryopreservation could not replace the current standard.

Impact of P-selectin-PSGL-1 Axis on Platelet-Endothelium-Leukocyte Interactions in Fatal COVID-19.

In critically ill patients infected with SARS-CoV-2, early leukocyte recruitment to the respiratory system was found to be orchestrated by leukocyte trafficking molecules accompanied by massive secretion of proinflammatory cytokines and hypercoagulability. Our study aimed to explore the interplay between leukocyte activation and pulmonary endothelium in different disease stages of fatal COVID-19. Our study comprised 10 COVID-19 postmortem lung specimens and 20 control lung samples (5 acute respiratory distress syndrome, 2 viral pneumonia, 3 bacterial pneumonia, and 10 normal), which were stained for antigens representing the different steps of leukocyte migration: E-selectin, P-selectin, PSGL-1, ICAM1, VCAM1, and CD11b. Image analysis software QuPath was used for quantification of positive leukocytes (PSGL-1 and CD11b) and endothelium (E-selectin, P-selectin, ICAM1, VCAM1). Expression of IL-6 and IL-1ß was quantified by RT-qPCR. Expression of P-selectin and PSGL-1 was strongly increased in the COVID-19 cohort compared with all control groups (COVID-19:Controls, 17:23, P < .0001; COVID-19:Controls, 2:75, P < .0001, respectively). Importantly, P-selectin was found in endothelial cells and associated with aggregates of activated platelets adherent to the endothelial surface in COVID-19 cases. In addition, PSGL-1 staining disclosed positive perivascular leukocyte cuffs, reflecting capillaritis. Moreover, CD11b showed a strongly increased positivity in COVID-19 compared with all controls (COVID-19:Controls, 2:89; P = .0002), indicating a proinflammatory immune microenvironment. Of note, CD11b exhibited distinct staining patterns at different stages of COVID-19 disease. Only in cases with very short disease course, high levels of IL-1ß and IL-6 mRNA were observed in lung tissue. The striking upregulation of PSGL-1 and P-selectin reflects the activation of this receptor-ligand pair in COVID-19, increasing the efficiency of initial leukocyte recruitment, thus promoting tissue damage and immunothrombosis. Our results show that endothelial activation and unbalanced leukocyte migration play a central role in COVID-19 involving the P-selectin-PSGL-1 axis.

Platelet Reactivity and Cardiovascular Mortality Risk in the LURIC Study.

BACKGROUND: The clinical and prognostic implications of platelet reactivity (PR) testing in a P2Y12-inhibitor naïve population are poorly understood. OBJECTIVES: This explorative study aims to assess the role of PR and explore factors that may modify elevated mortality risk in patients with altered PR. METHODS: Platelet ADP-induced CD62P and CD63 expression were measured by flow-cytometry in 1520 patients who were referred for coronary angiography in the Ludwigshafen Risk and Cardiovascular Health Study (LURIC). RESULTS: High- and Low-platelet reactivity to ADP were strong predictors of cardiovascular and all-cause mortality and risk equivalent to the presence of coronary artery disease. (High platelet reactivity 1.4 [95% CI 1.1-1.9]; Low platelet reactivity: 1.4 [95% CI 1.0-2.0]). Relative weight analysis indicated glucose control (HbA1c), renal function ([eGFR]), inflammation (high-sensitive C-reactive protein [hsCRP]) and antiplatelet therapy by Aspirin as consistent mortality risk modifiers in patients with Low- and High-platelet reactivity. Pre-specified stratification of patients by risk modifiers HbA1c (<7.0%), eGFR (>60 mL/min/1.73 m2) and CRP (<3 mg/L) was associated with a lower mortality risk, however irrespective of platelet reactivity. Aspirin treatment was associated with reduced mortality in patients with high platelet reactivity only (p for interaction: 0.02 for CV-death [<0.01 for all-cause mortality]. CONCLUSIONS: Cardiovascular mortality risk in patients with High- and Low platelet reactivity is equivalent to the presence of coronary artery disease. Targeted glucose control, improved kidney function and lower inflammation are associated with reduced mortality risk, however independent of platelet reactivity. In contrast, only in patients with High-platelet reactivity was Aspirin treatment associated with lower mortality.

Effect of mirasol pathogen reduction technology system on immunomodulatory molecules of apheresis platelets.

Pathogen inactivation for platelets by riboflavin system (MIRASOL) efficiently reduces transfusion related pathogen transmission. However little is known about its impact on platelets' immunomodulatory biochemical profile. We aimed was to assess the effects of MIRASOL treatment on platelet quality parameters and immunomodulatory molecules CD62P, RANTES, and CD40L in Single Donor Platelets (SDPs) resuspended in plasma (SDP-P) or T-PAS and additive solution (SDP-A). Twenty nine SDPs (15 SDP-P and 14 SDP-A) were included in the study. Samples were collected before, after MIRASOL treatment and just before transfusion. P-selectin (CD62P), RANTES, and CD40L were tested by ELISA. Platelet products quality assays were also performed. Platelet count/unit decreased after Mirasol treatment by 13 %. The pH of all units decreased over the 5-day storage period but remained above expected limits and the swirling test was positive throughout storage. P-selectin levels were not different between the three different time points in both SDPs-P and SDPs-A while RANTES levels were found to differ statistically significantly at the three different time points in all units and in the SPD-A subgroup. CD40L levels in all SDP products increased slightly during storage but this was not statistically significant. CD62P, RANTES, and CD40L in all time points were elevated in SDPs-A compared to SDPs-P but not at a statistically significant level. In conclusion MIRASOL treatment apart from RANTES increase does not seem to substantially affect platelets associated other cytokines and immunomodulatory molecules namely P-selectin and sCD40L which are implicated in immune transfusion reactions.

Dolutegravir potentiates platelet activation by a calcium-dependent, ionophore-like mechanism.

Dolutegravir is a highly potent HIV integrase strand transfer inhibitor that is recommended for first-line anti-retroviral treatment in all major treatment guidelines. A recent study has shown that people taking this class of anti-retroviral treatment have a substantially higher risk of early-onset cardiovascular disease, a condition shown previously to be associated with increased platelet reactivity. To date, few studies have explored the effects of dolutegravir on platelet activation. Accordingly, the current study was undertaken with the primary objective of investigating the effects of dolutegravir on the reactivity of human platelets in vitro. Platelet-rich plasma, isolated platelets, or buffy coat cell suspensions prepared from the blood of healthy adults were treated with dolutegravir (2.5-10 µg/ml), followed by activation with adenosine 5'-diphosphate (ADP), thrombin, or a thromboxane A2 receptor agonist U46619. Expression of platelet CD62P (P-selectin), formation of heterotypic neutrophil:platelet aggregates, and calcium (Ca2+) fluxes were measured using flow cytometry and fluorescence spectrometry, respectively. Dolutegravir caused dose-related potentiation of ADP-, thrombin- and U46619-activated expression of CD62P by platelets, as well as a significant increases in formation of neutrophil:platelet aggregates. These effects were paralleled by a spontaneous, receptor-independent elevation in cytosolic Ca2+ that appears to underpin the mechanism by which the antiretroviral agent augments the responsiveness of these cells to ADP, thrombin and U46619. The most likely mechanism of dolutegravir-mediated increases in platelet cytosolic Ca2+ relates to a combination of lipophilicity and divalent/trivalent metal-binding and/or chelating properties of the anti-retroviral agent. These properties are likely to confer ionophore-type activities on dolutegravir that would promote movement of Ca2+ across the plasma membrane, delivering the cation to the cytosol where it would augment Ca2+-dependent intracellular signaling mechanisms. These effects of dolutegravir may lead to hyper-activation of platelets which, if operative in vivo, may contribute to an increased risk for cardiometabolic co-morbidities.

Neutrophils contribute to elevated BAFF levels to modulate adaptive immunity in patients with primary immune thrombocytopenia by CD62P and PSGL1 interaction.

Objectives: Immune thrombocytopenia (ITP) is an autoimmune disease characterised by impaired platelet production and increased platelet destruction. However, the involvement of neutrophils in ITP is yet to be explored. Methods: B-cell activating factor (BAFF) expression and activation markers of neutrophils, as well as activation of platelets in ITP patients and healthy controls were measured. The interaction of CD62P on platelets and BAFF in neutrophils was analysed by correlation analysis and verified by co-culture. The effects of neutrophils on apoptosis of acquired immune cells were evaluated in co-culture systems with or without belimumab. Results: The BAFF expression and activation of neutrophils were increased in active ITP patients. BAFF levels in neutrophils were positively correlated with CD62P+ platelets and neutrophils produced increased BAFF by interfering with CD62P on platelets. Neutrophils inhibited the apoptosis of CD4+, CD8+ and CD19+ cells dependent on BAFF levels, and belimumab could interrupt the effects of neutrophils. Conclusions: Neutrophils were overactivated in ITP patients and participated in the progression of disease by producing excessive BAFF, which could be regulated by CD62P on platelets. Targeting BAFF by belimumab may be a novel potential therapy for ITP.

Increased plasma level of soluble P-selectin in non-hospitalized COVID-19 convalescent donors.

BACKGROUND: The coronavirus disease-2019 (COVID-19) is a systemic disease with severe implications on the vascular and coagulation system. A procoagulant platelet phenotype has been reported at least in the acute disease phase. Soluble P-selectin (sP-sel) in the plasma is a surrogate biomarker of platelet activation. Increased plasma levels of sP-sel have been reported in hospitalized COVID-19 patients associated with disease severity. Here, we evaluated in a longitudinal study the sP-sel plasma concentration in blood donors who previously suffered from moderate COVID-19. METHODS: 154 COVID-19 convalescent and 111 non-infected control donors were recruited for plasma donation and for participation in the CORE research trial. First donation (T1) was performed 43-378 days after COVID-19 diagnosis. From most of the donors the second (T2) plasma donation including blood sampling was obtained after a time period of 21-74 days and the third (T3) donation after additional 22-78 days. Baseline characteristics including COVID-19 symptoms of the donors were recorded based on a questionnaire. Platelet function was measured at T1 by flow cytometry and light transmission aggregometry in a representative subgroup of 25 COVID-19 convalescent and 28 control donors. The sP-sel plasma concentration was determined in a total of 704 samples by using a commercial ELISA. RESULTS: In vitro platelet function was comparable in COVID-19 convalescent and control donors at T1. Plasma samples from COVID-19 convalescent donors revealed a significantly higher sP-sel level compared to controls at T1 (1.05 ± 0.42 ng/mL vs. 0.81 ± 0.30 ng/mL; p < 0.0001) and T2 (0.96 ± 0.39 ng/mL vs. 0.83 ± 0.38 ng/mL; p = 0.0098). At T3 the sP-sel plasma level was comparable in both study groups. Most of the COVID-19 convalescent donors showed a continuous decrease of sP-sel from T1 to T3. CONCLUSION: Increased sP-sel plasma concentration as a marker for platelet or endothelial activation could be demonstrated even weeks after moderate COVID-19, whereas, in vitro platelet function was comparable with non-infected controls. We conclude that COVID-19 and additional individual factors could lead to an increase of the sP-sel plasma level.

Evaluation of platelet activation marker expression and its correlation with tumorigenesis and tumor progression in patients with gastric cancer.

BACKGROUND AND OBJECTIVES: Gastric cancer (GC) remains responsible for over one million new cases in 2020. Activated platelets express the CD40 ligand (CD40L) and CD62P in the cytoplasmic membrane, and interaction with the vascular endothelium can induce the production of tumor growth factors and metastases. We aimed to characterize the soluble levels of sCD40L and sCD62P in GC patients. METHODS: A cross-sectional study was performed on 83 GC patients and 20 healthy controls. RESULTS: High levels of sCD40L were obtained in GC patients compared to healthy controls (p = 0.003) and in the I/II compared with III and IV stages (p < 0.0001 and p = 0.007, respectively). Low levels of sCD62P in the GC patients compared to healthy controls (p = 0.009). High soluble levels of sCD62P in I/II compared with III and IV stages (p = 0.002 and p = 0.01, respectively). There are no significant differences in the levels of sCD40L and sCD62P were observed between intestinal, diffuse, and mixed types. CONCLUSIONS: We concluded that sCD40L and sCD62P molecules may be predictive biomarkers since the increase in plasma levels was associated with disease progression and metastasis in GC. In addition, the serum sCD40L and sCD62P can potentially be used as an indicator of response to anticancer therapy.

P-selectin glycoprotein ligand-1 (PSGL-1/CD162) is incorporated into clinical HIV-1 isolates and can mediate virus capture and subsequent transfer to permissive cells.

BACKGROUND: P-selectin glycoprotein ligand-1 (PSGL-1/CD162) has been studied extensively for its role in mediating leukocyte rolling through interactions with its cognate receptor, P-selectin. Recently, PSGL-1 was identified as a novel HIV-1 host restriction factor, particularly when expressed at high levels in the HIV envelope. Importantly, while the potent antiviral activity of PSGL-1 has been clearly demonstrated in various complementary model systems, the breadth of PSGL-1 incorporation across genetically diverse viral isolates and clinical isolates has yet to be described. Additionally, the biological activity of virion-incorporated PSGL-1 has also yet to be shown. RESULTS: Herein we assessed the levels of PSGL-1 on viruses produced through transfection with various amounts of PSGL-1 plasmid DNA (0-250 ng), compared to levels of PSGL-1 on viruses produced through infection of T cell lines and primary PBMC. We found that very low levels of PSGL-1 plasmid DNA (< 2.5 ng/well) were necessary to generate virus models that could closely mirror the phenotype of viruses produced via infection of T cells and PBMC. Unique to this study, we show that PSGL-1 is incorporated in a broad range of HIV-1 and SIV isolates and that virions with incorporated PSGL-1 are detectable in plasma from viremic HIV-1-infected individuals, corroborating the relevance of PSGL-1 in natural infection. Additionally, we show that PSGL-1 on viruses can bind its cognate selectin receptors, P-, E-, and L-selectins. Finally, we show viruses with endogenous levels of PSGL-1 can be captured by P-selectin and transferred to HIV-permissive bystander cells, highlighting a novel role for PSGL-1 in HIV-1 infection. Notably, viruses which contained high levels of PSGL-1 were noninfectious in our hands, in line with previous findings reporting the potent antiviral activity of PSGL-1. CONCLUSIONS: Our results indicate that levels of PSGL-1 incorporation into virions can vary widely among model systems tested, and that careful tailoring of plasmid levels is required to recapitulate physiological systems when using pseudovirus models. Taken together, our data suggest that PSGL-1 may play diverse roles in the physiology of HIV-1 infection, particularly due to the functionally active state of PSGL-1 on virion surfaces and the breadth of PSGL-1 incorporation among a wide range of viral isolates.

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.

Evaluation of admission levels of P, E and L selectins as predictors for thrombosis in hospitalized COVID-19 patients.

Thromboembolic complications are the most reported cause of death in coronavirus disease-2019 (COVID-19). Hypercoagulability, platelets activation and endotheliopathy are well-recognized features in COVID-19 patients. The aim of this work was to evaluate circulating soluble selectins P, E and L at the time of hospital admission as predictors for upcoming thrombosis. This retrospective study included 103 hospitalized COVID-19 patients and 50 healthy volunteer controls. COVID-19 patients were categorized into two groups; group 1 who developed thrombosis during hospitalization and group 2 who did not. Soluble selectins were quantitated using ELISA technique. Higher levels of sP-selectin, sE-selectin and sL-selectin were detected in COVID-19 patients compared to controls. Furthermore, significantly higher levels were found in group 1 compared to group 2. Their means were [5.86 ± 1.72 ng/mL vs. 2.51 ± 0.81 ng/mL]; [50 ± 8.57 ng/mL vs. 23.96 ± 6.31 ng/mL] and [4.66 ± 0.83 ng/mL vs. 2.95 ± 0.66 ng/mL] for sP-selectin, sE-selectin and sL-selectin respectively. The elevated selectins correlated with the currently used laboratory biomarkers of disease severity. After adjustment of other factors, sP-selectin, sE-selectin and sL-selectin were independent predictors for thrombosis. At sP-selectin ≥ 3.2 ng/mL, sE-selectin ≥ 32.5 ng/mL and sL-selectin ≥ 3.6 ng/mL thrombosis could be predicted with 97.1%, 97.6% and 96.5% sensitivity. A panel of the three selectins provided 100% clinical sensitivity. Admission levels of circulating soluble selectins P, E and L can predict thrombosis in COVID-19 patients and could be used to identify patients who need prophylactic anticoagulants. E-selectin showed a superior clinical performance, as thrombo-inflammation biomarker, to the most commonly studied P-selectin.

Distribution and quantification of activated platelets in platelet-rich fibrin matrices.

Platelet-rich fibrin (PRF) has been widely applied in regenerative therapy owing to its simple preparation protocol. To date, the original protocol for preparing leukocyte-rich (L)-PRF has been modified to produce derivatives such as advanced (A)-PRF, concentrated growth factors (CGF), and horizontal (H)-PRF. However, these derivatives have not been rigorously compared to explore possible differences. We previously developed and validated a nondestructive near-infrared (NIR) imaging method to quantitatively examine the platelet distribution in PRF matrices. To further evaluate the characteristics of platelets in PRF, we herein examined the distribution of activated platelets. Four types of PRF matrices were prepared under different centrifugal conditions from blood samples obtained from the same healthy donors. After fixation and compression, the matrices were stained immunohistochemically without sectioning and visualized using an NIR imager. Qualitative morphological analysis revealed that whole platelets were distributed widely and homogeneously in H-PRF and A-PRF, but localized along the distal tube surface in L-PRF and CGF. Activated platelets were distributed as were whole platelets in A-PRF, L-PRF, and CGF, but localized mainly in the "buffy coat" region in H-PRF. Quantitative analysis revealed that there was no significant difference in the ratio of activated to whole platelets between PRF derivatives. These findings suggest that platelet activation is similarly induced in fibrin matrices regardless of centrifugal speed or rotor angulation. However, only the H-PRF group was distinguishable from the other PRF derivatives in terms of activated platelet distribution.

Effect of trehalose on platelets stored at 4℃ in vitro / 中国输血杂志

【Objective】 To investigate the effect of trehalose added to M-Sol on platelet preservation at 4℃ in vitro. 【Methods】 Seven bags of ABO homotype concentrated platelets were randomly selected and divided into 7 groups according to storage temperature, preservation medium and trehalose concentration: 22℃+ PRP group, PRP group, M-Sol+ PRP group, M-Sol+ PRP+ 1 g/L trehalose group, M-Sol+ PRP+ 5 g/L trehalose group, M-Sol+ PRP+ 15 g/L trehalose group and M-Sol+ PRP+ 25 g/L trehalose group. PRP group and M-Sol preservation groups were stored at 4℃. Plt, PDW, MPV, maximum aggregation, CD62p positive rate, GLU and LAC concentration were detected on the 1st, 3rd, 5th and 7th day after preservation, and the changes of GLU and LAC concentration within 7 days were calculated. 【Results】 With the extension of preservation time, Plt decreased in all groups, and there was no significant difference among groups at the same time (P>0.05). PDW and MPV increased in all groups. When preserved to the 7th day, the PDW 10.22±0.43(fL) and MPV 8.74±0.40(fL) of M-Sol+ PRP+ 5 g/L trehalose group were the lowest, which was significantly different from that of 22℃+ PRP group (P0.05). During the preservation period, the maximum aggregation degree of each group decreased gradually. Except for the PRP group, the maximum aggregation degree of the M-Sol+ PRP+ 5g/L trehalose group was the highest(27.29±6.62), which was significantly higher than that of the 22℃+ PRP group on the 7th day after preservation (P<0.05). On the 7th day after preservation, the positive rate of CD62p in M-Sol+ PRP+ 5g/L trehalose group was the lowest(15.46±2.46), and there was significantly different compared with other groups (P<0.05). 【Conclusion】 Adding appropriate concentration of trehalose to M-Sol can inhibit platelet activation at 4℃ and reduce platelet storage lesion.

The Correlation Between Bronchopulmonary Dysplasia and Platelet Metabolism in Preterm Infants.

Background: Platelets play an important role in the formation of pulmonary blood vessels, and thrombocytopenia is common in patients with pulmonary diseases. However, a few studies have reported on the role of platelets in bronchopulmonary dysplasia. Objective: The objective of the study was to explore the relationship between platelet metabolism and bronchopulmonary dysplasia in premature infants. Methods: A prospective case-control study was performed in a cohort of premature infants (born with a gestational age <32 weeks and a birth weight <1,500 g) from June 1, 2017 to June 1, 2018. Subjects were stratified into two groups according to the diagnostic of bronchopulmonary dysplasia: with bronchopulmonary dysplasia (BPD group) and without bronchopulmonary dysplasia (control group). Platelet count, circulating megakaryocyte count (MK), platelet-activating markers (CD62P and CD63), and thrombopoietin (TPO) were recorded and compared in two groups 28 days after birth; then serial thrombopoietin levels and concomitant platelet counts were measured in infants with BPD. Results: A total of 252 premature infants were included in this study. Forty-eight premature infants developed BPD, 48 premature infants without BPD in the control group who were matched against the study infants for gestational age, birth weight, and admission diagnosis at the age of postnatal day 28. Compared with the controls, infants with BPD had significantly lower peripheral platelet count [BPD vs. controls: 180.3 (24.2) × 109/L vs. 345.6 (28.5) × 109/L, p = 0.001]. Circulating MK count in the BPD group was significantly more abundant than that in the control group [BPD vs. controls: 30.7 (4.5)/ml vs. 13.3 (2.6)/ml, p = 0.025]. The level of CD62p, CD63, and TPO in BPD group was significantly higher than the control group [29.7 (3.1%) vs. 14.5 (2.5%), 15.4 (2.0%) vs. 5.8 (1.7%), 301.4 (25.9) pg/ml vs. 120.4 (14.2) pg/ml, all p < 0.05]. Furthermore, the concentration of TPO was negatively correlated with platelet count in BPD group with thrombocytopenia. Conclusions: Our findings suggest that platelet metabolism is involved in the development of BPD in preterm infants. The possible mechanism might be through increased platelet activation and promoted TPO production by feedback.

Differences in human and sheep platelet adherence, aggregation and activation induced by glass beads in a modified chandler loop-system.

In human cardiovascular research, sheep in particular are used as a large animal model in addition to pigs. In these animals, medical products, developed and tested for human medical purposes, are almost exclusively used in interventional studies. Therefore, the extent to which platelets from human and ovine blood differ in terms of adherence, aggregation and activation after a 4- or 8-minutes exposure to glass was investigated. Testing was performed with platelet-rich plasma (PRP) and a modified chandler loop-system, with 4- and 8-minute blood-material exposure times corresponding to 20 and 40 test cycles, respectively, through the entire silicone tube loop of the test system.In sheep and human PRP, contact with the silicone tubing resulted in a decrease in platelet count after 4 minutes and 20 test cycles, respectively. Four more minutes (20 additional test cycles) caused a further decrease of the platelet count only in sheep PRP. When the silicon tube was partly filled with glass beads, these effects were more pronounced and stronger in sheep then in human PRP.The mean platelet volume, which was used as parameter for platelet aggregation, did not change over time in human PRP without glass exposure. With glass exposure in human and sheep PRP the mean platelet volume increased within 40 test cycles, but this increase was stronger in sheep than in human PRP.Regarding activation behavior, the activation markers CD62P and CD63 were detectable only in < 30% (sheep) and < 45% (human) of platelets, whereas after 8 min of glass exposure, the proportion of CD62P+ and CD63+ cells was more increased than before only in sheep. These results indicate that ovine platelets adhere more strongly to glass and show stronger aggregation behavior after glass contact than human platelets, but that ovine and human platelets differ only slightly in activability by glass.

Differences in human and minipig platelet number, volume and activation induced by borosilicate glass beads in a modified chandler loop-system.

The pig is the most widely used large animal model in Europe, with cardiovascular research being one of the main areas of application. Adequate refinement of interventional studies in this field, meeting the requirements of Russel and Burchs' 3 R concept, can only be performed if blood-contacting medical devices are hemocompatible. Because most medical devices for cardiovascular interventional procedures are developed for humans they are tested mostly for compatibility with human blood. The aim of this study was therefore to determine whether there are differences in behavior of porcine and human platelets when they come into contact with glass, which was used as an exemplary thrombogenic material. For this purpose changes of platelet count, platelet volume and platelet expression of the activation markers CD61, CD62P and CD63 were measured using a modified chandler loop-system simulating the fluidic effects of the blood flow. Minipig and human platelets showed significant differences in number and volume, but not in activation after 4-8 min exposure to glass.

CD62P (P-selectin) expression as a platelet activation marker in patients with liver cirrhosis with and without cholestasis.

AIM OF THE STUDY: P-selectin (CD62P) is a platelet activation marker that was claimed to mediate the accumulation of platelets induced by cholestasis. The nature of platelet dysfunction and hemostasis abnormalities in cholestatic liver disease needs to be more explored. The aim of this study was to assess platelet CD62P expression in cirrhotic patients with and without cholestasis, and to evaluate its relationship with a bleeding tendency. MATERIAL AND METHODS: 150 patients were included in this case-control study. Participants were divided into 84 patients with liver cirrhosis (group I), 44 of whom had cholestasis (Group Ia) and 40 patients were without cholestasis (group Ib); 36 patients who were cholestatic without liver cirrhosis (group II); and 30 healthy subjects who formed the control group (group III). Platelet CD62P expression was assessed by a flow cytometer. RESULTS: Platelets expressing CD62P were significantly increased in all patient groups compared to controls (p < 0.001). Platelets expressing CD62P were significantly increased in gastrointestinal (GIT) bleeders compared to non-bleeders in cirrhotic and cholestatic groups (p < 0.001 each). Among group I patients at cut-off > 12.4, up-regulation of platelet CD62P yielded 72% sensitivity and 44.1% specificity to discriminate bleeders from non-bleeders (p = 0.01), while among group II at cut-off > 12.9, it yielded 90% sensitivity and 80.8% specificity (p < 0.001). In cirrhotic patients, platelet CD62P expression was significantly increased in patients with an advanced Child-Pugh class (p < 0.001). Platelet expressing CD62P was shown as an independent risk factor for bleeding among cirrhotic cases with an odds ratio of 1.07 and CI 0.99-1.15. CONCLUSIONS: Up-regulation of platelet CD62P expression can serve as a GIT bleeding predictor in liver cirrhosis.