CD62P Expression Research Articles

Platelet activation through CD62P and the formation of platelet-monocyte complexes are associated with the exacerbation of mucosal inflammation in patients with ulcerative colitis.

Ulcerative colitis (UC) is a refractory, chronic inflammatory bowel disease of unknown etiology. Although platelets are activated in UC, their relevance in pathophysiology remains unclear. We analyzed the correlation of platelet activation and platelet-monocyte complexes (PMCs) with severity of mucosal inflammation using the Mayo endoscopic subscore (MES). Platelet activation marker, CD62P was upregulated in patients with UC compared with that in healthy controls (P < 0.05). CD62P expression was significantly higher in patients with MES3 (severe inflammation) than in those with MES ≤ 2 (endoscopic remission to moderate inflammation) (P < 0.001). The concentration of sCD62P in patients with MES0 (endoscopic remission) was significantly higher than in those with MES ≥ 1 (P < 0.01). The expression of CD40L, CD63, PAC-1, annexin V, and CD36, and the concentrations of sCD40L, PF4, and RANTES did not correlate with MES. The proportion of PMCs in patients with MES3 was higher than in those with MES ≤ 2 (P < 0.05). CD16 expression on monocytes with platelets was significantly higher than in monocytes without platelets (P < 0.001). Patients with complete remission after treatment showed significant reduction in PMCs 3 months after treatment (P < 0.05) but had no change in CD62P and sCD62P. Our data suggest that platelet activation via the CD62P-PMC axis is involved in UC pathophysiology.

Cleavable Novel Thienopyridine Derivatives as Potent Antithrombotic Agents

AbstractThienopyridine derivatives are specifically designed for utilization in antithrombotic therapy to help combat cardiovascular disorders linked to thrombosis. This study introduces new thienopyridine derivatives with superior therapeutic efficacy through synergies by dual functional compounds, developed as antithrombotic agents. Through computational design, we discovered 32 compounds out of a library of 100 compounds that exhibit efficacy. The compounds are synthesized by multiple steps and subsequently purified using chromatographic procedures to achieve a purity level of &gt;99%. Using the ADP‐induced platelet aggregation assay, we have found 4 active test compounds (ACG‐0173‐19, ACG‐0173‐37, ACG‐A‐04, and ACG‐B‐03) out of a total of 32 compounds. On the other hand, ACG‐0173‐19, ACG‐0173‐37, ACG‐A‐04, and ACG‐B‐03 have demonstrated the highest percentage activity on factor Xa (FXa) inhibition. The ACG‐A‐04 chemical exhibited the highest hydrophilicity, specifically in terms of its aqueous solubility. It had a solubility of 0.44 mg/mL in pH 5.8, 0.29 mg/L in pH 6.2, and 0.145 mg/L in pH 7.4 buffers. In comparison, the standards APX‐01 (apixaban) and PRG‐01(prasugrel) had lower hydrophilicity. Based on cytotoxicity investigations, it was determined that the test substances do not exhibit harmful effects. The results indicated that the test chemicals can undergo hydrolysis in blood plasma rather than in other organs. Among the four test drugs, ACG‐1073‐19 exhibited an unbound fraction of 44.1%, which is twice as high as the standards (apixaban). ACG‐1073‐37 demonstrates superior plasma protein binding (33.2%) compared to all other test compounds. The compounds resulted in a higher expression of CD61, CD42b, and CD62P in platelets compared to the control. The ACG‐1073‐37 molecule had a Caco‐2 permeability of 51%, which is extremely close to the Caco‐2 permeability of the control medicines (55% for APX‐01 and 62% for PRG‐01). Through the assessment of microsomal stability, it was determined that ACG‐1073‐37 and ACG‐A‐04 exhibited metabolic stability for 42.4 and 34.14 min, respectively.

Prospective Quantitative and Phenotypic Analysis of Platelet-Derived Extracellular Vesicles and Its Clinical Relevance in Ischemic Stroke Patients

The levels of platelet-derived extracellular vesicles (pEVs) have been reported as elevated in acute ischemic stroke (IS). However, the results of studies remain equivocal. This prospective, case-control study included 168 patients with IS, 63 matched disease controls (DC), and 21 healthy controls (HC). Total pEVs concentration, the concentration of phosphatidylserine-positive pEVs (PS+pEVs), the percentage of PS+pEVs (%PS+pEVs) and the concentration of pEVs with expression of CD62P+, CD40L+, CD31+, and active form of GPIIb/IIIa receptor (PAC-1+) were assessed on days 1, 3, 10, and 90 with the Apogee A50-Micro flow cytometer. The concentrations of pEVs, PS+pEVs, and %PS+pEVs were significantly higher after IS vs. HC (p &lt; 0.001). PS+pEVs were higher after stroke vs. controls (p &lt; 0.01). The concentrations of pEVs with expression of studied molecules were higher on D1 and D3 after stroke vs. controls. The concentration of pEVs after platelet stimulation with ADP was significantly diminished on D3. IS most notably affects the phenotype of pEVs with a limited effect on the number of pEVs. Ischemic stroke moderately disturbs platelet microvesiculation, most notably in the acute phase, affecting the phenotype of pEVs, with a limited impact on the number of pEVs.

Reduced platelet activation in triple-negative essential thrombocythemia compared with JAK2V617F-mutated essential thrombocythemia.

Triple-negative (TN) essential thrombocytopenia (ET) is characterized by the absence of driver mutations while retaining histological and phenotypic characteristics sufficient for an ET diagnosis. Our understanding of TN-ET and its platelet activation remains incomplete. We carried out a large-scale multi-center clinical analysis to analyze the clinical and molecular characteristics, thrombotic complications of TN-ET. We also related the above characteristics to platelet activation to further explore the thrombosis mechanism of TN-ET. A retrospective multicenter study was conducted on 138 TN-ET and 759 ET patients with driver mutations from 1 March 2012 to 1 December 2021. The clinical and molecular characteristics of the TN-ET were summarized. Additionally, platelet activation, apoptosis, and reactive oxygen species (ROS) levels were analyzed in 73 TN-ET patients from this cohort and compared to 41 age- and sex-matched healthy donors. Compared to patients with the JAK2V617F mutation, those with triple-negative mutation were younger (P < 0.001) and exhibited fewer thrombotic events before diagnosis (P < 0.001) and during follow-up (P = 0.039). Patients with triple-negative mutation also presented with significantly reduced CD62P expression in platelets (P = 0.031), slightly reduced calcium concentration in platelets (P = 0.063), increased mitochondrial membrane potential (P = 0.011), reduced phosphatidylserine exposure (P = 0.015), reduced levels of ROS (P = 0.043) and MitoSOX in platelets (P = 0.047). In comparison to JAK2V617F-mutated ET, TN-ET is associated with lower platelet ROS levels, which leads to reduced platelet activation and consequently a lower risk of thrombosis.

Storage properties of platelet concentrates from umbilical cord blood prepared using three different methods.

Thrombocytopenia, common in preterm newborns, may increase bleeding risk and is often treated with transfusions. Recent studies reveal that transfusing platelets at a high threshold worsens outcomes, possibly due to a "developmental mismatch" between adult-derived platelets and neonatal hemostatic system. Cord blood-derived platelet concentrates (CBPCs) could be an alternative for newborns. Our study aims to produce and evaluate the quality parameters of CBPCs during storage. Cord blood was collected from placentas after near-term and full-term pregnancies. Several production methods were attempted to obtain CBPCs, varying centrifugation settings, filtration, and dilution procedures. Adult-derived platelet concentrates (PCs) processed with the same methods, and standard PCs from five buffy-coats were used as controls. Storage tests were performed on days 2, 4-5, 7 from the collection. CBPCs parameters were compared with adult-derived PCs, and no significant differences were found for mean platelet volume (MPV), swirling, morphology, glucose, lactate, pCO2, and pO2. pH and bicarbonate were lower in CBPCs. Some significant differences between methods in CD62P expression and JC-1 ratio were observed. Compared with standard PCs, CBPCs showed lower platelet concentration, pH, and JC-1. Additionally, both in CBPCs as well as in control PCs, the apoptosis marker phosphatidylserine was elevated. CBPCs were of comparable quality to control PCs during storage. However, apoptosis markers in both groups were elevated, suggesting processing and storage of low volumes of PCs require further optimization. Also, filtration of low volumes leads to significant platelet loss, an issue that requires remedy.

Buyang Huanwu decoction ameliorates myocardial injury and attenuates platelet activation by regulating the PI3 kinase/Rap1/integrin α(IIb)β(3) pathway

BackgroundBuyang Huanwu Decoction (BYHWD) is a traditional Chinese medicine to treat the syndrome of qi deficiency and blood stasis. Platelets play an important role in regulating thrombus and inflammation after ischemic injury, studies have shown that BYHWD regulate myocardial fibrosis and exert anti-inflammatory effects through IL-17 and TLR4 pathways, but the mechanism of platelet activation by BYHWD in stable coronary heart disease is still unknown. In the present study, model of left anterior descending coronary artery ligation was applied to investigate the mechanisms of BYHWD on modulating platelets hyperreactivity and heart function after fibrosis of ischemic myocardial infarction (MI).MethodsMyocardial infarction model was constructed by ligation of the left anterior descending coronary artery. The rats were randomly divided into five groups: sham, model, MI with aspirin (positive), MI with a low dosage of BYHWD (BYHWD-ld) and MI with a high dosage of BYHWD (BYHWD-hd) for 28 days.ResultsCoronary artery ligation prominently induced left ventricle dysfunction, increased cardiomyocyte fibrosis, which was accompanied by platelets with hyperreactivity, and high levels of inflammatory factors. BYHWD obviously reversed cardiac dysfunction and fibrosis, increased the thickness of the left ventricular wall, and inhibited aggregation ratio and CD62p expression. BYHWD restored the mitochondrial respiration of platelets after MI, concomitant with an increased telomere expression and decreased inflammation. According to the result of transcriptome sequencing, we found that 106 differentially expressed genes compared model with BYHWD treatment. Enrichment analysis screened out the Ras-related protein Rap-1 (Rap1) signaling pathway and platelet activation biological function. Quantitative real-time PCR and Western blotting were applied to found that BYHWD reduced the expression of Rap1/PI3K-Akt/Src-CDC42 genes and attenuated the overactivity of PI3 kinase/Rap1/integrin α(IIb)β(3) pathway.ConclusionBYHWD reduced inflammation and platelet activation via the PI3 kinase/Rap1/integrin α(IIb)β(3) pathway and improved heart function after MI.Graphical

Effect of toxins from different periodontitis-associated bacteria on human platelet function.

Periodontitis is caused by a dysbiosis of oral bacteria resulting in alveolar bone destruction and teeth loss. The role of platelets in pathogenesis of periodontitis is a subject of research. The release of toxins from periodontitis-associated bacteria may influence platelet function and contribute to the modulation of hemostatic or inflammatory responses. Therefore, we explored platelet function upon exposure to defined toxins: leukotoxin A from Aggregatibacter actinomycetemcomitans (LtxA), a synthetic version of the C14-Tri-LAN-Gly peptide from Fusobacterium nucleatum (C14), and lipopolysaccharides from Porphyromonas gingivalis (LPS). Light transmission aggregometry was performed after the addition of toxins to platelet-rich plasma in different doses. Flow cytometry was used to identify inhibitory effects of toxins by measuring phosphorylation of the vaso-dilator-stimulated phosphoprotein or to identify activating effects by the detection of CD62P expression. The release of chemokines derived from washed platelets was determined by immunoassays. Collagen-induced threshold aggregation values were diminished upon incubation with LtxA and C14, accompanied with an increase of vaso-dilator-stimulated phosphoprotein (VASP) phosphorylation, indicating platelet inhibition. In contrast, LPS did not affect aggregation but slightly enhanced CD62P expression under co-stimulation with low-dose thrombin pointing to slight platelet activation. The three toxins did not relevantly influence the secretion of chemokines. Although weak, the investigated toxins differently influenced human platelet function. LtxA and C14 mediated inhibitory effects, whereas LPS contributed to a slight activation of platelets. Further analysis of specific cellular responses mediated by bacterial toxins may render novel targets and suggestions for the treatment of periodontitis.

Influence of short-term refrigeration on collagen-dependent signalling mechanisms in stored platelets

Platelet concentrates (PC) are used to treat patients with thrombocytopenia and hemorrhage, but there is still the demand to find the optimal strategy for temperature-dependent storage of PC. Recently, we could show that cold storage for 1 h (short-term refrigeration) is sufficient to induce enhanced platelet responsiveness. The aim of this study was to investigate effects of cold storage on collagen-dependent activating signalling pathways in platelets from apheresis-derived PC (APC).APC on day 1 or day 2 of storage, were either continuously kept at room temperature (RT, 22 °C), or for comparison, additionally kept at cold temperature (CT, 4 °C) for 1 h. CD62P expression was determined by flow cytometry. Western Blot technique was used to analyze collagen-induced phosphorylation of p38, ERK1/2 or Akt/PKB and its inhibition by prostaglandin E1 (PGE1) or nitric monoxide donor. Adhesion of platelets on collagen-coated surfaces and intracellular phosphorylation of vasodilator-stimulated phosphoprotein (VASP) was visualized by immune fluorescence microscopy.CD62P expression was increased after short-term refrigeration. CT exposition for 1 h induced an elevation of basal ERK1/2 phosphorylation and an alleviation of PGE1- or DEA/NO-suppressed ERK1/2 phosphorylation in APC on day 1 and 2 of storage. Similar, but more moderate effects were observable for p38 phosphorylation. Akt/PKB phosphorylation was increased only in APC on day 2. Refrigeration for 1 h promoted platelet adhesion and reduced basal VASP phosphorylation in adherent platelets.The attenuation of inhibitory signalling in short-term refrigerated stored platelets is associated with enhanced reactivity of activating signalling pathways, especially ERK1/2. Functionally, these processes correlate with increased adhesion of refrigerated platelets on collagen-coated surfaces. The results help to further optimize temperature-dependent strategies for platelet storage.

A Compact Differential Dynamic Microscopy-based Device (cDDM): An Approach Tool for Early Detection of Hypercoagulable State in Transfusion-Dependent-β-Thalassemia Patients.

β-Thalassemia especially transfusion-dependent thalassemia (TDT) associates with a hypercoagulable state, which is the main cause of thromboembolic events (TEE). Plasma viscosity and rheological parameters could be essential markers for determining hypercoagulable state in β-thalassemia patients. The traditional methods for measuring viscosity are often limited by large sample volumes and are impractical for routine clinical monitoring. The compact differential dynamic microscopy-based device (cDDM), an optical microscopy for quantitative rheological assessment, was developed and applied for prognosis of the hypercoagulable state in β-TDT with and without splenectomy. The device was performed plasma viscosity measurement using low plasma volume (8 μL) and revealed a value as modulus of complex viscosity |η(ω)| in 7 min. We also parallelly demonstrated the correlation of the viscosity and related-coagulable parameters: complete blood count, prothrombin time (PT), activated partial thromboplastin time (APTT), protein C (PC), protein S (PS), CD62P and CD63 expression, and platelet aggregation test. The thalassemia plasma exhibited a higher value of |η(ω)| than healthy plasma, which can represent a different viscoelastic property among the groups. Even all related-coagulable parameters indicated hypercoagulable state in both nonsplenectomies and splenectomies β-TDT patients when compared to control, only high platelet numbers significantly correlated to high plasma viscosity in the splenectomy group. However, the other coagulable parameters have shown a trend of positive relationship with high plasma viscosity in all β-1thalassemia TDT patients. The relative results suggested that our device would be an approach tool for early detection of hypercoagulable state in transfusion-dependent-β-thalassemia patients, which can help to prevent TEE and the critical consequent-complications.

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.

MEASUREMENT OF PLATELET ACTIVATION MARKERS CD62P AND CD63 TO DETERMINE THE EFFICIENCY OF DUAL THERAPY WITH ASPIRIN AND CLOPIDOGREL: A PRELIMINARY STUDY

Background: A dual antiplatelet therapy with aspirin and clopidogrel is advised to prevent stent thrombosis. However, the most appropriate assay for evaluating the antiplatelet effects remains undefined. Objective: This study aimed to assess the efficacy of measuring platelet surface activation markers, CD62P and CD63, using flow cytometry to determine the effectiveness of dual antiplatelet therapy. Methods: Thirty patients who received aspirin plus clopidogrel before cerebral artery stent implantation and thirty unrelated healthy controls were enrolled. The expression of CD62P and CD63 was measured using flow cytometry. The diagnostic performances were evaluated and compared with the conventional light-transmitted aggregometry (LTA) using 5.0 and 20.0 µM of adenosine di-phosphate (ADP). Results: The expression ratios of both markers were significantly lower in the patients receiving dual antiplatelet therapy than controls (p &lt; 0.001). The area under the receiver operating characteristic (ROC) curve (AUC) of CD62P using flow cytometry and the LTA using 5.0 and 20.0 µM of ADP were comparable (0.97 vs. 0.95 and 0.97 vs. 0.96, respectively), whereas those of CD63 was lower than the LTA (0.88 vs. 0.96). The sensitivity of CD62P and CD63 were 71.4% and 60.0%, and the specificity of CD62P and CD63 were 100.0% and 96.7%, respectively. Conclusion: Flow cytometry measurements of CD62P could be utilized to identify the efficacy of dual antiplatelet therapy. Additional studies are suggested to support this issue and its effects on clinical outcomes.

Evaluation of activation characteristics of a canine platelet concentrate produced by a commercial double centrifugation system

IntroductionIn veterinary medicine there are few readily available products for platelet transfusion to patients with thrombocytopenia. Commercial tabletop platelet concentrating systems have recently become available to veterinarians, primarily directed towards uses associated with regenerative medicine. These systems could potentially be used to produce fresh concentrated platelets for use in transfusion medicine. This study evaluated the concentration, activation, and sterility of a double centrifugation platelet concentrate (PC) produced by a commercial benchtop system.MethodsTen healthy dogs were studied. Whole blood was collected and mixed with ACD-A in a 1:7.6 ratio of ACD-A to whole blood. 12 mL of this mixture was processed into PC via single centrifugation, while 60 mL of the anticoagulated whole blood was processed via a commercial double centrifugation system. Both types of PC were evaluated for platelet concentration, CD62P expression with and without thrombin stimulation, and for sterility.ResultsMean platelet count in the double centrifuged PC was 863 ± 352 × 103/μL, with very low white blood cell contamination (median of 0.47 × 103 leukocyte/μL (range 0.15–2.18 × 103/μL)). The double-centrifuged PC had similar baseline activation characteristics (as determined by P-selectin expression) as the single centrifuge PC (0.76% vs. 0.72% unstimulated, 30.5% vs. 34.9% stimulated, p = 0.432).DiscussionThe benchtop PC system studied here did not cause activation of platelets during production and produced a sterile product that can be further investigated as a source of fresh platelet concentrates for transfusion purposes.

Platelet Activation is Upregulated in Cirrhotic Patients with Portal Vein Thrombosis.

Platelet plays a key role in thrombosis formation, especially that the alteration of platelet function may influence the thrombosis development. This study aimed to investigate platelet function alterations in the formation of portal vein thrombosis (PVT) in cirrhosis. Cirrhotic patients admitted to The First Affiliated Hospital of Soochow University between October 2021 and April 2023 were recruited and divided into PVT and non-PVT groups according to radiological results. Clinical parameters and prognosis were also collected and assessed to identify potential risk factors. Flow cytometry was used to detect the expression of CD62p, CD63, monocyte-platelet aggregates (MPAs), neutrophil-platelet aggregates (NPAs), and von Willebrand factor antigen (vWF-Ag) to evaluate platelet activation and adhesion function. A total of 145 subjects were enrolled in our study including 60 cirrhotic PVT patients, 60 cirrhotic non-PVT patients, and 25 healthy volunteers. Multivariate analysis suggested that esophageal gastric varices, splenectomy, and D-dimer were independent risk factors for PVT pathogenesis in cirrhosis. The vWF-Ag expression level was reduced in the PVT group compared with the non-PVT group (p = 0.046) but was not an independent risk factor for PVT formation pathogenesis. The expression of CD41+CD62p+ and CD41+CD63+ platelets in the PVT group was significantly elevated compared with that in the non-PVT group (p < 0.05). There were no significant differences in MPAs and NPAs between the two cirrhotic groups. Subgroup analysis showed that the mean fluorescence intensity (MFI) of CD62p and CD63 was associated with portal hypertension-related complications (p = 0.008, p < 0.001), and CD63 MFI was significantly associated with thrombosis burden (p = 0.019). CD41+CD62p+ and CD41+CD63+ platelets as well as MPAs and NPAs were highly expressed in the splenectomy group compared with those in the nonsplenectomy group in cirrhotic patients (p < 0.05). Positive correlations were found between CD62p MFI and CD63 MFI, MPAs and NPAs (r = 0.642, p < 0.001; r = 0.378, p = 0.003; r = 0.430, p < 0.001). In addition, platelet counts were also correlated with MPAs (r = 0.556, p < 0.001) and NPAs (r = 0.467, p < 0.001). Cirrhotic patients with PVT had higher mortality and were more likely to experience portal hypertension-related complications in the prognostic analysis (p < 0.05). Highly activated platelet function exists in patients with cirrhosis, and platelet activation was elevated during PVT formation, suggesting that activated platelets may participate in the formation of PVT in patients with cirrhosis.

#1227 The role of P-selectin (CD62P) expression in renal tissue in the development of diabetic nephropathy

Abstract Background and Aims The prevalence of diabetic nephropathy (DN) is increasing in line with the increase in the incidence of diabetes mellitus (DM) worldwide. The latest data from the International Diabetes Federation (IDF) predicts that by 2045, more than 10.5% of adults worldwide will have diabetes. This will lead to kidney damage in 100-250 million people. DN is characterized by structural and functional changes. As a result of chronic hyperglycemia, pathogenic changes in the vascular wall develop, and the frequency of micro- and macrovascular complications associated with morbidity and mortality among patients with diabetes increases. However, the exact mechanism of vascular complications is not completely clear. Endothelial dysfunction plays a key role in triggering the development of inflammatory mechanisms that are associated with vascular complications in patients with type 2 diabetes mellitus (T2DM). Activation of the endothelium due to increased release of cytokines and expression of adhesion molecules promotes platelet activation and their adhesion to the activated endothelium. Selectins (E-selectin, L-selectin and P-selectin) play a major role in the development of microvascular complications. The aim of the study was to examine the role of P-selectin (CD62p) expression in the glomerular endothelium and peritubular capillaries in patients with different morphological classes of DN and to evaluate its effect on the development and progression of morphological changes in renal tissue. Method Was examined 49 patients with T2DM complicated by the development of DN. The average age of the patients was 66.58 ± 3.27 years. There were 35 women, 14 men. The duration of the disease was 17.70 ± 0.35 years. The duration of DN from the moment of detection of microalbuminuria to the morphological examination of the renal tissue and diagnosis was 1.65 ± 0.34 years. All patients underwent light and immunofluorescence microscopy of kidney tissue biopsies obtained by intravital puncture biopsy. Morphological changes in tissue were assessed in accordance with the latest international classification of diabetic nephropathy developed by the Scientific Committee of the Pathology Society, USA. According to light microscopy data, class IIa (mild mesangial expansion) was detected in 12 patients, class IIb (severe mesangial expansion) in 14 patients, class III (Kimmelstil–Wilson nodular lesions) in 19 patients, and class IV (advanced diabetic glomerulosclerosis) in 5 patients. In addition to light microscopy, the expression of CD62β (P-selectin) in the glomerular endothelium and in peritubular capillaries was determined in all patients. FITc-labeled monoclonal antibodies (FITC anti-human CD62p Antibody, clone AK4 Cat#304904), were used (Biolegend, USA). Results Analysis of biopsy tissue from DN patients showed that CD62p expression is detected both in the glomerular endothelium (1.6383 ± 0.4789 (95% CI: 0.9574-2.7234)) and in peritubular capillaries (1.1702 ± 0.1308 (95% CI: 0.9149–1.4250)). Using the linear regression method with the calculation of the coefficients of determination R2 (R Square), the values of the F criterion, significant regression models were obtained: Expression of CD62p in the glomerular endothelium promotes the development and progression of arteriolar hyalinosis (R2=0.213, F=12.476 p=0.001); expansion of the mesangial matrix (R2=0.362, F=26.09, p=0.0001); thickening of the basement membrane (R2=0.127, F=6.57, p=0.014); development and progression of nodular Kimmelstiel–Wilson lesions (R2=0.106, F=5.360, p=0.025). Expression of CD62p in peritubular capillaries promotes the development of interstitial sclerosis (R2=0.549, F=54.732, p=0.0001). The severity of CD62p expression in the glomerular endothelium depends on the morphological class of DN in patients (R2=0.216, F=12.684, p=0.001). Conclusion

#2191 Platelet function in chronic kidney disease; can flow cytometry guide in the difficult balance between risk for bleeding versus thrombotic events?

Abstract Background and Aims Increased risk of bleeding is a well-known complication of chronic kidney disease (CKD). However, these patients also have an increased risk of thrombotic events. Several studies have implicated both impaired and enhanced platelet function in patients with chronic kidney disease, but results vary between different studies and methods. Flow cytometry has emerged as a new method to study platelet function. Our aim was to investigate platelet function using flow cytometry in patients with chronic kidney disease. Method 20 patients were included between February and May 2023. Pre-dialytic patients with an eGFR between 8-20 mL/min/1,73 m2 were included, mean eGFR 14.7 mL/min/1,73 m2. Patients with platelet levels &amp;gt;100 × 109/L were included. Medication in the form of anticoagulants, NSAIDs, platelet inhibitors or SSRIs were used as exclusion criteria. Platelet activation assay was performed with flow cytometry. The expression of activation markers PAC-1 (activated GPIIb/IIIa), CD62P (P-selectin) and CD63 (dense granules) were analysed before and after activation with ADP, CRP-XL (collagen stimulating peptide, activates GPVI) or TRAP-6 (stimulates platelet activating receptor 1, PAR1). Platelets from 20 healthy control subjects were analysed for comparison. Results The level and size of platelets (MPV) were comparable in both groups. There were significantly higher levels of D-dimer in the CKD group vs healthy controls (1,17 mg/L FEU vs 0,27 mg/L FEU). Except for the decreased expression of activated GPIIb-IIIa receptor after activation with ADP and CRP-XL in one patient, the platelet activation did not significantly differ between CKD patients and healthy controls. Conclusion No significant differences in the activation of platelets were observed between the CKD population and the healthy controls when analysed by flow cytometry. Elevated levels of D-dimer in CKD patients have previously been described and are believed to be due to increased fibrin formation, lysis and decreased renal clearance. Further studies in a larger patient population, including thrombocytopenic patients are planned, with greater focus to be placed on dense granula quantities which may correlate with decreased levels in the CKD population.

Evaluation of coagulation and platelet activation state and function in heartworm-infected dogs.

Enhanced platelet responses have been demonstrated in heartworm-infected (HWI) dogs; however, the cause and clinical implications of altered platelet function have not been fully elucidated. This study evaluated platelet function in HWI dogs. Anticoagulated whole blood collected from eight HWI and eight uninfected dogs was evaluated using turbidometric platelet aggregometry, a platelet function analyzer (PFA-100), a total thrombus analysis system (T-TAS), tissue factor-activated and tissue plasminogen activator modified thromboelastography (TF- and tPA-TEG), CBC, von Willebrand Factor activity, and fibrinogen concentrations. Platelet activation state and the presence of reticulated platelets were assessed via flow cytometric expression of P-selection (CD-62P) and thiazole orange staining. Platelet aggregation responses to adenosine diphosphate (ADP, 10 μM) or collagen (20 μg/mL), PFA-100 closure times, and T-TAS occlusion times did not differ between groups. TEG values TF-R, tPA-R, TF-K, and TF-LY60 were decreased (P = .025, P = .047, P = .038, P = .025) and TF-MA, tPA-MA, TF-G, tPA-G and TF-alpha angle were increased (P < .04) in HWI dogs. HWI dogs had higher fibrinogen concentrations (465.6 ± 161 mg/dL vs 284.5 ± 38 mg/dL, P = .008) and eosinophil counts (0.686 ± 0.27 × 103/μL vs 0.267 ± 0.20 × 103/μL, P = .003). There was no difference in hematocrit, activation state, or percent of reticulated platelets. Non-activated reticulated platelets exhibited higher CD62P expression compared with mature platelets. Chronic canine heartworm disease was accompanied by hypercoagulability, hyperfibrinogenemia, and decreased fibrinolysis. Enhanced platelet activation was not identified in this group of HWI dogs.

The effect of near-infrared low-level light on the in vitro quality of platelets during storage.

Near-infrared (NIR) light has been successfully applied to improve the quality of mouse platelets during storage. Because it is suspected that the mitochondria contain the primary photon acceptor, we hypothesized that human platelets for transfusion may be affected similarly and could benefit from NIR light treatment. The optimal light dose was determined using portions of platelet concentrates (PCs) in PAS-E. A pool-and-split design was used to prepare PCs in PAS-E or plasma (n = 6). On day 1, one unit of both pairs was illuminated with 830 nm light (light-emitting diodes, 15 J/cm2). PCs were stored at 22°C and sampled regularly for analysis. Data were compared with their corresponding controls with a paired two-sided t-test. Illuminated platelets in PAS-E were less activated with significantly lower CD62P expression (day 8: 10.8 ± 1.8 vs. 12.2 ± 2.6, p < 0.05) and lower Annexin A5 binding (day 8: 11.8 ± 1.9 vs. 13.1 ± 2.4, ns). They produced significantly less lactate resulting in a higher pH (days 6-10). ATP content and mitochondrial membrane potential were not affected. Although these trends were also observed for PCs in plasma, the differences did not reach statistical significance as compared with the control group. Our study demonstrates that the glycolysis rate of human platelets can be modulated through the use of NIR, possibly through mitochondrial aerobic metabolism, but this requires confirmation. If NIR illumination can be further optimized, it may potentially become a useful tool in situations in which glycolysis and platelet activation are exacerbated.

Cold-stored platelets do not potentiate detrimental monocyte and neutrophil proinflammatory responses in vitro

Abstract Cold-stored platelets (CSP) have increased hemostatic function compared to room temperature-stored platelets (RTP), leading to recent FDA conditional approval for CSP transfusion in bleeding patients. However, the immune consequences of CSP transfusion are not well defined. CD62P and CD40L expression is increased in CSP, potentially enhancing interaction with neutrophils (PMN, CD62P:PSGL-1) and monocytes (Mo, CD40L:CD40). We hypothesized CSP would form aggregates with leukocytes more readily than RTP, thereby increasing tissue factor/CD142 expression, inflammasome assembly, and neutrophil extracellular trap (NET) formation – functions potentiated by native platelets. We stored apheresis platelets in plasma (PLT) for 7 days at 22oC (RT) or 4oC (CS), and incubated PLT with freshly isolated PBMC or PMN at a 1:14 ratio. We measured CD142 and Apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC) expression by flow cytometry in Mo, and ASC specks and NET formation in PMN using fluorescent microscopy. We found no difference in CD142 or ASC GMFI expression in monocytes incubated with RT or CS PLT, and no difference in ASC specks or NET formation between neutrophils incubated with RT and CS PLT. Therefore, our in vitro study shows CSP do not potentiate detrimental acute proinflammatory responses by monocytes or neutrophils, further supporting CSP use in actively bleeding patients and paving the way for precision transfusion medicine.

Abstract 3094: Traditional Chinese Medicine Buyang Huanwu Decoction Regulates The Platelet Gpvi And Its Downstream Protein Signaling Pathways,inhibiting Thrombosis And Preventing Cardiovascular Disease.

Background: Coronary heart disease Qi-Deficiency and Blood Stasis Syndrome will lead to the body of platelets disorders, blood viscosity, conducive to the formation of thrombosis, Buyang Huanwu Decoction (BYHWD) is a classic Chinese medicine treatment of Qi-Deficiency and Blood Stasis Syndrome. Platelet glycoprotein VI (GPVI) plays a crucial role in collagen-induced activation and aggregation of platelets. GPVI has become the focus of new approaches to antithrombotic therapy. Methods: After 6 weeks of irregular sleep deprivation in rats with syndrome of deficiency of qi and blood stasis formed, the coronary heart disease model was established by ligation of the left anterior descending coronary artery in rats. The dosage of 1 mL/100 g was given by intragastric administration for 14 days after operation. Results: The purpose of this study was to investigate the effects of BYHWD on platelet granule secretion and platelet function in vitro. We found that BYHWD can significantly reduce the platelet aggregation induced by ADP and collagen, significantly reduce the median expression of CD62p and the ratio of activated platelets, and reduce the concentration of fluorescent calcium ions in platelets. Under the electron microscope observation found that under the action of BYHWD, the ultrastructure of rat platelets pseudopodia reduced, aggregation reduced, rat coronary inner wall platelets, thrombus mass adhesion reduced, and the inner wall was smoother. In addition, BYHWD can regulate GPVI and its downstream proteins. Compared with the coronary heart disease group with syndrome of blood stasis, BYHWD can significantly reduce the expression of GPVI. Significantly reduced the expression of Syk, CALDAG-GEFI, significantly reduced the expression of IP 3 and significantly reduced the degree of PLCγ 2 of rats. Decreased the degranulation reaction of the rat blood, significantly decreased the level of TXA 2 , 5-HT in the rat blood, and significantly decreased the content of the rat blood COX-1 in the rat serum. Conclusion: Therefore, BYHWD can regulate GPVI and its downstream protein signaling pathways, reduce platelet over-activation, degranulation reaction, thereby inhibiting thrombosis, protecting heart function and preventing cardiovascular disease.

Single molecule studies of dynamic platelet interactions with endothelial cells.

A biotechnological platform consisting of two-color 3D super-resolution readout and a microfluidic system was developed to investigate platelet interaction with a layer of perfused endothelial cells under flow conditions. Platelet activation has been confirmed via CD62P clustering on the membrane and mitochondrial morphology of ECs at the single cell level were examined using 3D two-color single-molecule localization microscopy and classified applying machine learning. To compare binding of activated platelets to intact or stressed ECs, a femtosecond laser was used to induced damage to single ECs within the perfused endothelial layer. We observed that activated platelets bound to the perfused ECs layer preferentially in the proximity to single stressed ECs. Platelets activated under flow were ∼6 times larger compared to activated ones under static conditions. The CD62P expression indicated more CD62P proteins on membrane of dynamically activated platelets, with a tendency to higher densities at the platelet/EC interface. Platelets activated under static conditions showed a less pronounced CD62P top/bottom asymmetry. The clustering of CD62P in the platelet membrane differs depending on the activation conditions. Our results confirm that nanoscopic analysis using two-color 3D super-resolution technology can be used to assess platelet interaction with a stressed endothelium under dynamic conditions.