P-selectin and C-reactive protein in relation to home blood pressure and coronary calcification: a SCAPIS substudy.

BACKGROUND: Soluble P-selectin (sP-selectin) and high-sensitivity C-reactive protein (hsCRP) have previously been associated with hypertension, but the relation with out-of-office blood pressure (BP) and coronary artery calcification score is unknown. We aimed to examine the relationship between sP-selectin, hsCRP and home BP, as well as coronary artery calcification score and carotid artery plaques. METHODS: In the Swedish CArdioPulmonary bioImage Study (SCAPIS), 5057 randomly selected participants were evaluated with office and home BP using the semi-automatic Omron M10-IT device. For this cross-sectional study, participants with sP-selectin <4 standard deviations above mean and hsCRP <5 mg/l, representing low-grade inflammation, were included. Using generalized linear models, these inflammatory markers were evaluated in relation to BP classifications, as well as coronary artery calcification score and carotid artery plaques. RESULTS: Of participants, 4548 were included in the analyses. The median age was 57.2 (53.4-61.2) years, and 775 (17.0%) reported taking medication for hypertension. Participants in the highest quartile of sP-selectin [odds ratio (OR) 1.67, 95% confidence interval (CI) 1.40-1.98, P  < 0.001] and hsCRP [OR 2.25, (95% CI 1.89-2.60), P  < 0.001] were more likely to have sustained hypertension. Participants in the highest quartile of hsCRP were also more likely to have masked hypertension, OR (95% CI) 2.31 (1.72-3.10), P  < 0.001 and carotid artery plaques, OR (95% CI) 1.21 (1.05-1.38), P  = 0.007. CONCLUSION: Increased sP-selectin and hsCRP were independently associated with sustained hypertension. These findings indicate an association between hypertension and platelet activity, as expressed by sP-selectin.

Proteomic profiling for biomarker discovery in heparin-induced thrombocytopenia.

ABSTRACT: New analytical techniques can assess hundreds of proteins simultaneously with high sensitivity, facilitating the observation of their complex interplay and role in disease mechanisms. We hypothesized that proteomic profiling targeting proteins involved in thrombus formation, inflammation, and the immune response would identify potentially new biomarkers for heparin-induced thrombocytopenia (HIT). Four existing panels of the Olink proximity extension assay covering 356 proteins involved in thrombus formation, inflammation, and immune response were applied to randomly selected patients with suspected HIT (confirmed HIT, n = 32; HIT ruled out, n = 38; and positive heparin/platelet factor 4 [H/PF4] antibodies, n = 28). The relative difference in protein concentration was analyzed using a linear regression model adjusted for sex and age. To confirm the test results, soluble P-selectin was determined using enzyme-linked immunosorbent assay (ELISA) in above mentioned patients and an additional second data set (n = 49). HIT was defined as a positive heparin-induced platelet activation assay (washed platelet assay). Among 98 patients of the primary data set, the median 4Ts score was 5 in patients with HIT, 4 in patients with positive H/PF4 antibodies, and 3 in patients without HIT. The median optical density of a polyspecific H/PF4 ELISA were 3.0, 0.9, and 0.3. Soluble P-selectin remained statistically significant after multiple test adjustments. The area under the receiver operating characteristic curve was 0.81 for Olink and 0.8 for ELISA. Future studies shall assess the diagnostic and prognostic value of soluble P-selectin in the management of HIT.

Comprehensive investigation of platelet function in patients with cirrhosis.

Cirrhosis presents with thrombocytopenia and possibly thrombocytopathy. Previous studies exploring platelet function gave conflicting results and most controversies are explained by the variety of methods employed for investigation. We sought to assess in-vitro the overall platelet function in cirrhosis. We investigated 34 patients by using the following tests. (i)Aggregometry. (ii)Measurement of the content of platelet granules. (iii)Cytometric platelet activation. (iv)Plasmatic markers of in-vivo platelet activation. (v)Platelet procoagulant activity by thrombin generation (TG) in platelet-rich plasma (PRP). TG measured in PRP for patients and controls was similar. Platelets from patients with cirrhosis showed reduction of aggregation and secretion of ATP. Similar results were observed for platelet activation parameters such as P-selectin expression and PAC-1 platelet binding. Plasma levels of ßeta-thromboglobulin and soluble P-selectin, were increased in patients-vs-controls. In contrast, there were no patients-vs-controls differences for plasmatic platelet-factor-4. Results are consistent with a state of in-vivo platelet activation and decreased in-vitro aggregation. Since bleeding events following invasive procedures are uncommon in cirrhosis, we speculate that in-vitro aggregometry testing does not reflect the situation occurring in-vivo. Results of the study and pathophysiological considerations support the conclusion that platelet function in cirrhosis as determined by aggregometry, although somewhat impaired, may support the overall hemostatic potential, which is needed for most invasive interventions. These conclusions are in line with the recommendations of international guidelines, warning against indiscriminate use of prophylactic preprocedural administration of platelets before invasive procedures. Decision on platelet support should not be made based on in-vitro laboratory testing for platelet function.

A tight interplay between platelet activation and mitochondrial DNA release promotes platelet storage lesion in platelet concentrates.

BACKGROUND AND OBJECTIVES: Platelet storage lesion (PSL) adversely affects the quality of platelet concentrates (PCs). Platelets are prone to activation during storage. Moreover, elevated free mitochondrial DNA (mtDNA) levels in PCs are associated with a higher risk of adverse transfusion reactions. Therefore, we aimed to evaluate the correlation between platelet activation markers and mtDNA release during PC storage. MATERIALS AND METHODS: Six PCs prepared by the platelet-rich plasma method were assessed for free mtDNA copy number using quantitative real-time PCR and CD62P (P-selectin) expression by flow cytometry on days 0 (PC collection day), 3, 5 and 7 of storage. Lactate dehydrogenase (LDH) activity, pH, platelet count, mean platelet volume (MPV) and platelet distribution width (PDW) were measured as well. The correlation between free mtDNA and other PSL parameters, and the correlation between all parameters, was determined. RESULTS: Significant increases in free mtDNA, MPV and PDW, and a significant decrease in platelet count and pH were observed. CD62P expression and LDH activity elevated significantly, particularly on storage days 5-7 and 0-3, respectively. Moreover, a moderate positive correlation (r = 0.61) was observed between free mtDNA and CD62P expression. The r values between free mtDNA and LDH, pH, platelet count, MPV and PDW were 0.81, -0.72, -0.49, 0.81 and 0.77, respectively. CONCLUSION: The interplay between platelet activation and mtDNA release in promoting PSL in PCs may serve as a promising target for future research on applying additive solutions and evaluating the quality of PCs to improve transfusion and clinical outcomes.

Occult cancer in patients with unprovoked venous thromboembolism: A nested case-control study.

OBJECTIVES: Detecting occult cancer in patients with unprovoked venous thromboembolism (VTE) remains a significant challenge. Our objective was to investigate the potential predictive role of coagulation-related biomarkers in the diagnosis of occult malignancies. METHODS: We conducted a nested case-control study with a 1-year prospective cohort of 214 patients with unprovoked VTE, with a focus on identifying occult cancer. At the time of VTE diagnosis, we measured various biomarkers, including soluble P-selectin (sP-selectin), dimerized plasmin fragment D (D-dimer), platelets, leukocytes, hemoglobin, total extracellular vesicles (EVs), EVs expressing tissue factor on their surface (TF+EVs), and EVs expressing P-selectin on their surface (Psel+EVs) in all participants. RESULTS: We observed statistically significant increased levels of sP-selectin (P = .015) in patients with occult cancer. Despite an increase in Psel+EVs, TF+EVs, D-dimer, and platelets within this group, however, no significant differences were found. When sP-selectin exceeded 62 ng/mL and D-dimer surpassed 10,000 µg/L, the diagnosis of occult cancer demonstrated a specificity of up to 91% (95% CI, 79.9%-96.7%). CONCLUSIONS: The combination of sP-selectin and D-dimer can be a valuable biomarker in detecting occult cancer in patients with unprovoked VTE. Further research is necessary to ascertain whether easily measurable biomarkers such as sP-selectin and D-dimer can effectively distinguish between patients who have VTE with and without hidden malignancies.

Asymmetric Dimethylarginine Is Associated with the Phenomenon of Coronary Slow Flow in Patients with Nonvalvular Atrial Fibrillation.

INTRODUCTION: Coronary slow flow phenomena (CSFP) are associated with endothelial and blood component abnormalities in coronary arteries. Asymmetric dimethylarginine (ADMA) can damage the endothelium of the heart or blood vessels in patients with non-valvular atrial fibrillation (NVAF), causing changes in levels of biological indicators. Our aim was to analyze the relationship between ADMA and CSFP in NVAF patients. METHODS: We consecutively enrolled 134 patients diagnosed with NVAF and underwent coronary angiography, 50 control patients without a history of atrial fibrillation and with normal coronary angiographic flow were included at the same time. Based on the corrected TIMI frame count (CTFC), the NVAF patients were categorized into two groups, CTFC ≤27 frames and CTFC >27 frames. Plasma ADMA, P-selectin (p-sel), von Willebrand factor (vWF), D-dimer (D-Di), plasminogen activator inhibitor 1 (PAI-1), and nitric oxide (NO) were detected by ELISA in the different groups. RESULTS: We found that plasma ADMA levels were significantly higher among NVAF patients in the CTFC >27 grade group compared with the control or CTFC ≤27 group. In addition, the levels of blood cells and endothelium-related biomarkers (NO, P-selectin, vWF, D-Di, and PAI-1) were significantly altered and correlated with ADMA levels. Multifactorial analysis showed that plasma ADMA (odd ratio [OR; 95% CI]: 1.65 [1.21-2.43], p < 0.001) and left atrial internal diameter (OR [95% CI]: 1.04 [1.02, 1.1], p < 0.001) could be used as independent risk factors for the development of CSFP in patients with NVAF. The ROC curves of ADMA can predict the development of CSFP in NVAF patients. The minimum diagnostic concentration for the development of CSFP in patients was 2.31 µmol/L. CONCLUSION: Our study demonstrated that CSFP in NVAF patients was associated with high levels of ADMA and left atrial internal diameter. Therefore, aggressive preoperative detection and evaluation of ADMA and left atrial internal diameter can help deal with the intraoperative presence of CSFP.

Elevated Selenoprotein P Levels in Thalassemia Major Patients.

INTRODUCTION: Previous studies have measured selenium levels and glutathione peroxidase 3 (GPX3) activity in patients with thalassemia major (TM). However, Selenoprotein P (SEPP), which is responsible for the storage and transport of selenium, has not been studied in thalassemia patients. This study aims to correlate thyroid functions of TM patients with their SEPP and GPX3 levels. MATERIALS AND METHODS: Eighty subjects (40 controls, 40 TM patients) were included in this study. GPX3 and SEPP concentrations were measured in all subjects using sandwich ELISA. Iron, ferritin, urinary iodine, thyroxine (T4), triiodothyronine (T3), thyrotropin (TSH), anti-thyroid peroxidase (anti-TPO), and anti-human thyroglobulin (anti-hTG) concentrations were also measured. RESULTS: Mean SEPP concentration was higher in the TM group compared to the control group. A slight elevation in GPX3 levels was also observed in thalassemia patients, yet it was not statistically significant. In both TM patients and controls, ferritin was inversely correlated with free T4 concentration and GPX3 was inversely correlated with free T4 and T3 concentrations. There was also a negative correlation between SEPP and TSH concentrations in healthy subjects. CONCLUSION: This is the first study, which has measured SEPP concentrations in thalassemia patients. SEPP levels were higher in TM patients compared to controls. Correlations between thyroid hormones and selenoproteins may indicate that selenium is necessary for thyroid function. Detailed studies are required to elaborate the role of SEPP in thyroid metabolism in thalassemia patients.

Comparative Study on the Chronic Vascular Responses Induced by Regular Versus Occasional Waterpipe Smoke Inhalation in Mice.

BACKGROUND/AIMS: Waterpipe smoke (WPS) is the second most prevalent form of smoking in the world. There are ample evidences about the vascular alterations caused by regular WPS (Reg-WPS). Nonetheless, comparison of the chronic vascular response induced by regular versus occasional WPS (Occ-WPS) exposure is very scarce. METHODS: We investigated, in BALB/c mice, the effects of Occ-WPS (30 minutes/day, 1 day/week) versus Reg-WPS (30 minutes/day, 5 days/week) for 6 months on thrombogenicity and platelet aggregation in vivo and in vitro. Moreover, various markers of endothelial integrity, inflammation and oxidative stress were assessed by enzyme-linked immunosorbent assay and colorimetric assay. Control mice were exposed to air. RESULTS: Our results showed that either Occ-WPS or Reg-WPS exposure shortened the thrombotic time in pial microvessels in vivo. Moreover, in pial venules, this effect was more marked in Reg-WPS group (-47%) compared with Occ-WPS (-34%). Similarly, exposure to either Occ-WPS or Reg-WPS reduced the prothrombin time and activated partial thromboplastin time. Platelet count was increased only in Reg-WPS exposure. Exposure to either Occ-WPS or Reg-WPS induced platelet aggregation in vitro. In addition, there was a statistically significant difference between Occ-WPS and Reg-WPS groups in platelet count and aggregation. Plasma concentration of tissue factor (+98%), P-selectin (+14%) and E-selectin (+16%) were significantly increased in Occ-WPS group compared with air exposed group. Likewise, compared with air group Reg-WPS caused an increase in concentration of tissue factor (+193%), P-selectin (+21%) and E-selectin (+42%). Nevertheless, only Reg-WPS induced a decrease (-38%) in the plasma concentration of tissue plasminogen activator. Notably, our results showed a statistically significant difference between Occ-WPS and Reg-WPS groups in the concentration of tissue factor. Erythrocyte numbers, hemoglobin concentration, hematocrit and lactate dehydrogenase activity were augmented only in Reg-WPS group compared with either control or Occ-WPS groups. Likewise, only Reg-WPS induced an increase in proinflammatory cytokines, tumor necrosis factor-α and interleukin-1ß compared with either control or Occ-WPS groups. However, markers of oxidative stress including 8-isoprostane and total antioxidants were enhanced in both Occ-WPS and Reg-WPS compared with control group. CONCLUSION: Our data confirm the vascular toxicity of the chronic Reg-WPS exposure and shows that even occasional chronic exposure to WPS caused thrombosis, platelet aggregation, endothelial alterations and oxidative stress. The latter findings are an additional cause of concern about the long-term toxicity of occasional waterpipe smoking.

Platelets mediate inflammatory monocyte activation by SARS-CoV-2 spike protein.

Infection with SARS-CoV-2, the causative agent of COVID-19, causes mild to moderate disease in most patients but carries a risk of morbidity and mortality. Seriously affected individuals manifest disorders of hemostasis and a cytokine storm, but it is not understood how these manifestations of severe COVID-19 are linked. Here, we showed that the SARS-CoV-2 spike protein engaged the CD42b receptor to activate platelets via 2 distinct signaling pathways and promoted platelet-monocyte communication through the engagement of P selectin/PGSL-1 and CD40L/CD40, which led to proinflammatory cytokine production by monocytes. These results explain why hypercoagulation, monocyte activation, and a cytokine storm are correlated in patients severely affected by COVID-19 and suggest a potential target for therapeutic intervention.

Serum thromboxane B2 but not soluble P-selectin levels identify ischemic stroke patients with persistent platelet reactivity while on aspirin therapy.

BACKGROUND: Aspirin non-response due to persistent platelet reactivity has been associated with adverse vascular events. Light transmission aggregometry (LTA), the 'gold standard' for measuring the platelet response to aspirin therapy, is a cumbersome procedure and a simple and reliable alternative is required. Our aim was to explore whether serum thromboxane B2 (sTXB2) and soluble P-selectin can be used to identify patients who are at risk of increased platelet reactivity while on aspirin. METHODS AND RESULTS: We recruited 293 ischemic stroke patients, taking aspirin for more than seven days, and performed LTA to classify them. Based on therapeutic serum salicylate levels, 63 patients were excluded due to suspected non-compliance, followed by ELISA measurement of TXB2 and P-selectin in serum. Accordingly, patients were classified into 'Responders' (n = 122, 53%), 'Semi-responders' (n = 76, 33%) and 'Non-responders' (n=32, 14%) by LTA. Patients who had platelet aggregation of ≥70% with 10µM ADP and ≥20% with 0.5mM AA were defined as 'Non-responders'. In comparison with 'Responders', 'Non-responders' had 8.63-fold increased risk of secondary vascular events (p = 0.008). ROC curve analysis revealed that sTXB2, at a cut-off level of >4.15 ng/mL, could distinguish the patient group with elevated platelet reactivity with a sensitivity of 84.3% (AUC = 0.84), and was in fair agreement with the LTA-based classification of patients. Soluble P-selectin levels, on the other hand, had no discriminatory ability. CONCLUSION: We suggest sTXB2 measurement as an alternative to the LTA approach for identifying aspirin-treated ischemic stroke patients who are at risk of enhanced platelet reactivity and subsequent vascular events.

Further Findings Concerning Endothelial Damage in COVID-19 Patients.

Systemic vascular damage with micro/macro-thrombosis is a typical feature of severe COVID-19. However, the pathogenesis of this damage and its predictive biomarkers remain poorly defined. For this reason, in this study, serum monocyte chemotactic protein (MCP)-2 and P- and E-selectin levels were analyzed in 204 patients with COVID-19. Serum MCP-2 and P-selectin were significantly higher in hospitalized patients compared with asymptomatic patients. Furthermore, MCP-2 increased with the WHO stage in hospitalized patients. After 1 week of hospitalization, MCP-2 levels were significantly reduced, while P-selectin increased in patients in WHO stage 3 and decreased in patients in WHO stages 5-7. Serum E-selectin was not significantly different between asymptomatic and hospitalized patients. The lower MCP-2 levels after 1 week suggest that endothelial damage triggered by monocytes occurs early in COVID-19 disease progression. MCP-2 may also predict COVID-19 severity. The increase in P-selectin levels, which further increased in mild patients and reduced in severe patients after 1 week of hospitalization, suggests that the inactive form of the protein produced by the cleavage of the active protein from the platelet membrane is present. This may be used to identify a subset of patients that would benefit from targeted therapies. The unchanged levels of E-selectin in these patients suggest that endothelial damage is less relevant.

Hemostatic Biomarkers and Venous Thromboembolism Are Associated With Mortality and Response to Chemotherapy in Patients With Pancreatic Cancer.

Objective: Pancreatic cancer activates coagulation and increases risk of venous thromboembolism (VTE). We aimed at characterizing the association of hemostatic biomarkers and VTE with mortality and chemotherapy response. Approach and Results: Pancreatic cancer patients (N=145) were included in a prospective, observational cohort study (CATS [Vienna Cancer and Thrombosis Study]). Hemostatic biomarkers (D-dimer, extracellular vesicle-tissue factor activity, prothrombin fragment 1+2, fibrinogen, factor VIII, PAI-1 [plasminogen activator inhibitor 1], sP-selectin [soluble P-selectin], thrombin generation assay) were measured at inclusion. The impact of VTE on overall survival/progression-free survival (OS/PFS) was evaluated by multistate modeling. The association of biomarkers with OS was analyzed by Cox-regression and with PFS and disease control rate in patients initiating palliative chemotherapy (n=95) by Cox-regression and logistic regression. Multivariable analysis included stage, grade, sex, age, performance status, VTE (time-dependent), vascular infiltration/compression, and tumor marker levels (carbohydrate-antigen 19-9, carcinoembryonic antigen). VTE occurrence was associated with shorter OS (transition hazard ratio, 3.40 [95% CI, 2.05-5.64]) and shorter PFS (transition hazard ratio, 2.10 [1.16-3.79]). Median post-VTE OS/PFS in months was 5.5 [2.2-6.5] and 3.0 [1.5-3.9], compared with 13.4 [9.7-16.6] and 7.5 [5.9-9.8] in patients without VTE (both P<0.001). D-dimer, extracellular vesicle-tissue factor activity, PAI-1, and sP-selectin were associated with increased mortality (hazard ratio per doubling, 1.27 [1.00-1.61]; 1.63 [1.14-2.36]; 1.25 [1.06-1.47]; 1.52 [1.05-2.20]). In patients initiating palliative chemotherapy, higher D-dimer predicted shorter PFS (hazard ratio per doubling, 1.27 [1.01-1.60]) and lower disease control rate (odds ratio per doubling, 0.59 [0.36-0.98]). Conclusions: VTE diagnosis is associated with shorter OS and PFS. Higher baseline levels of D-dimer, extracellular vesicle-tissue factor activity, PAI-1, and sP-selectin were independently prognostic for increased mortality, and D-dimer predicted response to palliative chemotherapy.

Platelet features allow to differentiate immune thrombocytopenia from inherited thrombocytopenia.

Immune thrombocytopenia (ITP) is an acquired bleeding disorder, for which no specific diagnostic test exists. Inherited thrombocytopenia (IT) can mimic ITP and lead to unappropriated management with significant morbidity. Here, in small cohorts of these two disorders, we explored whether platelet sialylation and platelet activation could allow to discriminate the two conditions. We also aimed to confirm the value of immature platelet counts in this discrimination. Platelet sialylation and the expression level of P-selectin were assessed by multiparameter flow cytometry. Immature platelets were estimated on a Sysmex XN 9000 analyzer. No significant difference in platelet sialylation was observed between ITP and IT. Contrarily, platelet activation was significantly higher in ITP patients (p = 0.008). The immature platelet fraction, as previously demonstrated, was significantly lower in the ITP group compared to the IT group (p = 0.014). That statistical significance was achieved in this small pilot study suggests that the two easily available assays of immature platelet count and P-selectin expression could help physicians to reach the proper diagnosis in complex cases of thrombocytopenia.

The Role of P-Selectin in COVID-19 Coagulopathy: An Updated Review.

In severe COVID-19, which is characterized by blood clots and neutrophil-platelet aggregates in the circulating blood and different tissues, an increased incidence of cardiovascular complications and venous thrombotic events has been reported. The inflammatory storm that characterizes severe infections may act as a driver capable of profoundly disrupting the complex interplay between platelets, endothelium, and leukocytes, thus contributing to the definition of COVID-19-associated coagulopathy. In this frame, P-selectin represents a key molecule expressed on endothelial cells and on activated platelets, and contributes to endothelial activation, leucocyte recruitment, rolling, and tissue migration. Briefly, we describe the current state of knowledge about P-selectin involvement in COVID-19 pathogenesis, its possible use as a severity marker and as a target for host-directed therapeutic intervention.

Dose-dependent effects of anthocyanin supplementation on platelet function in subjects with dyslipidemia: A randomized clinical trial.

BACKGROUND: Dyslipidemia induces platelet hyperactivation and hyper-aggregation, which are linked to thrombosis. Anthocyanins could inhibit platelet function in vitro and in mice fed high-fat diets with their effects on platelet function in subjects with dyslipidemia remained unknown. This study aimed to investigate the effects of different doses of anthocyanins on platelet function in individuals with dyslipidemia. METHODS: A double-blind, randomized, controlled trial was conducted. Ninety-three individuals who were initially diagnosed with dyslipidemia were randomly assigned to placebo or 40, 80, 160 or 320 mg/day anthocyanin groups. The supplementations were anthocyanin capsules (Medox, Norway). Platelet aggregation by light aggregometry of platelet-rich plasma, P-selectin, activated GPⅡbⅢa, reactive oxygen species (ROS), and mitochondrial membrane potential were tested at baseline, 6 weeks and 12 weeks. FINDINGS: Compared to placebo group, anthocyanins at 80 mg/day for 12 weeks reduced collagen-induced platelet aggregation (-3.39±2.36%) and activated GPⅡbⅢa (-8.25±2.45%) (P < 0.05). Moreover, compared to placebo group, anthocyanins at 320 mg/day inhibited collagen-induced platelet aggregation (-7.05±2.38%), ADP-induced platelet aggregation (-7.14±2.00%), platelet ROS levels (-14.55±1.86%), and mitochondrial membrane potential (7.40±1.56%) (P < 0.05). There were dose-response relationships between anthocyanins and the attenuation of platelet aggregation, mitochondrial membrane potential and ROS levels (P for trend <0.05). Furthermore, significantly positive correlations were observed between changes in collagen-induced (r = 0.473) or ADP-induced (r = 0.551) platelet aggregation and ROS levels in subjects with dyslipidemia after the 12-week intervention (P < 0.05). INTERPRETATION: Anthocyanin supplementation dose-dependently attenuates platelet function, and 12-week supplementation with 80 mg/day or more of anthocyanins can reduce platelet function in individuals with dyslipidemia. FUNDING: None.

Endothelial microparticle-associated protein disulfide isomerase increases platelet activation in diabetic coronary heart disease.

BACKGROUND: Endothelial microparticles (EMPs) carrying the protein disulfide isomerase (PDI) might play a key role in promoting platelet activation in diabetes. This study aimed to examine the activation of platelets, the amounts of MPs, PMPs, and EMPs, and the concentration and activity of PDI in patients with diabetic coronary heart disease (CHD) and non-diabetic CHD. METHODS: Patients with CHD (n=223) were divided as non-diabetic CHD (n=121) and diabetic CHD (n=102). Platelet activation biomarkers, circulating microparticles (MPs), the concentration of protein disulfide isomerase (PDI), and MP-PDI activity were determined. The effect of EMPs on platelet activation was investigated in vitro. Allosteric GIIb/IIIa receptors that bind to PDI were detected by a proximity ligation assay (PLA). RESULTS: Platelet activation, platelet-leukocyte aggregates, circulating MPs, EMPs, PDI, and MP-PDI activity in the diabetic CHD group were significantly higher than in the non-diabetic CHD group (P<0.05). Diabetes (P=0.006) and heart rate <60 bpm (P=0.047) were associated with elevated EMPs. EMPs from diabetes increased CD62p on the surface of the platelets compared with the controls (P<0.01), which could be inhibited by the PDI inhibitor RL90 (P<0.05). PLA detected the allosteric GIIb/IIIa receptors caused by EMP-PDI, which was also inhibited by RL90. CONCLUSIONS: In diabetic patients with CHD, platelet activation was significantly high. Diabetes and heart rate <60 bpm were associated with elevated EMPs and simultaneously increased PDI activity on EMP, activating platelets through the allosteric GPIIb/IIIa receptors.

Thrombopoietin Contributes to Enhanced Platelet Activation in Patients with Type 1 Diabetes Mellitus.

Atherosclerotic cardiovascular disease is the major cause of morbidity and mortality in patients with type 1 diabetes mellitus (T1DM). Enhanced platelet reactivity is considered a main determinant of the increased atherothrombotic risk of diabetic patients. Thrombopoietin (THPO), a humoral growth factor able to stimulate megakaryocyte proliferation and differentiation, also modulates the response of mature platelets by enhancing both activation and binding to leukocytes in response to different agonists. Increased THPO levels have been reported in different clinical conditions characterized by a generalized pro-thrombotic state, from acute coronary syndromes to sepsis/septic shock, and associated with elevated indices of platelet activation. To investigate the potential contribution of elevated THPO levels in platelet activation in T1DM patients, we studied 28 T1DM patients and 28 healthy subjects. We measured plasma levels of THPO, as well as platelet-leukocyte binding, P-selectin, and THPO receptor (THPOR) platelet expression. The priming activity of plasma from diabetic patients or healthy subjects on platelet-leukocyte binding and the role of THPO on this effect was also studied in vitro. T1DM patients had higher circulating THPO levels and increased platelet-monocyte and platelet-granulocyte binding, as well as platelet P-selectin expression, compared to healthy subjects, whereas platelet expression of THPOR did not differ between the two groups. THPO concentrations correlated with platelet-leukocyte binding, as well as with fasting glucose and Hb1Ac. In vitro, plasma from diabetic patients, but not from healthy subjects, primed platelet-leukocyte binding and platelet P-selectin expression. Blocking THPO biological activity using a specific inhibitor prevented the priming effect induced by plasma from diabetic patients. In conclusion, augmented THPO may enhance platelet activation in patients with T1DM, potentially participating in increasing atherosclerotic risk.

A Single-Center Clinical Study to Evaluate Shenxiong Glucose Injection Combined with Edaravone in the Treatment of Acute Large-Area Cerebral Infarction.

OBJECTIVES: To investigate the clinical efficacy and safety of Shenxiong glucose injection combined with edaravone in the treatment of acute large-area cerebral infarction. METHODS: 156 patients with acute large-area cerebral infarction admitted to our hospital from July 2015 to January 2017 were included in the analysis. The patients were randomly divided into experimental (78 cases) and control (78 cases) groups. Patients in the experimental group were given a 30 mg injection of edaravone in 100 ml of 0.9% sodium chloride solution by intravenous drip, twice a day within 30 minutes and a daily 200 ml injection of Shenxiong glucose by intravenous drip. Patients in the control group were given a 30 mg edaravone injection in 100 ml of 0.9% sodium chloride solution by intravenous drip, twice a day, and the drip was completed within 30 minutes. Patients in both groups were treated for 2 weeks. The levels of fibrinogen (FIB), D-dimer, interleukin 6 (IL-6), P-selectin (CD62P), and hypersensitive C-reactive protein (hs-CRP) were evaluated in the two groups of patients. Neurological disability was evaluated using the modified Rankin scale (mRS) and the neurological deficit score (National Institute of Health Stroke Scale, NIHSS). Adverse reactions to the treatments were also recorded. RESULTS: No significant differences in age, gender, medical histories, and blood biochemical indices were observed between the two groups before treatment (P > 0.05). After treatment, the levels of FIB, D-dimer, IL-6, CD62P, and hs-CRP were significantly lower following treatment and compared to the control group (P < 0.05). Also, the mRS and NIHSS scores were significantly lower after treatment and compared with the control group (P < 0.05). The total effective rate of the treatment in the experimental group was significantly higher compared to the control group (P < 0.05). During the treatment period, no obvious adverse reactions were observed in the two groups of patients. CONCLUSIONS: In addition to the routine basic treatment of acute large-area cerebral infarction, the addition of Shenxiong glucose injection combined with edaravone injection can improve platelet aggregation and reduce inflammation by affecting P-selectin, D-dimer, and FIB. This treatment approach promotes the recovery of nerve defect function without obvious adverse reactions in patients with acute large-area cerebral infarction.

Left Atrial Appendage Morphology and Local Thrombogenesis-Related Blood Parameters in Patients With Atrial Fibrillation.

Background Left atrial appendage (LAA) morphology predicts stroke risk in patients with atrial fibrillation. However, it is not precisely understood how LAA morphology influences stroke risk. The present study aimed to investigate the relationship between LAA morphology and local thrombogenesis-related blood parameters in LAA. Methods and Results We enrolled 205 patients undergoing catheter ablation of atrial fibrillation. The prevalence of chicken wing-, cactus-, windsock-, and cauliflower-type LAAs were 23.9%, 32.7%, 29.3%, and 14.1%, respectively. Blood samples were collected from the femoral vein, left atrium, and LAA in each patient. The levels of blood parameters were tested for each blood sample. The cauliflower-type LAA was associated with elevated platelet P-selectin expression, and interleukin-6 levels and with lower NO levels in LAA blood samples (P<0.05) independent of LAA flow velocity and LAA volume. LAA flow velocity, which was lowest in the cauliflower-type LAA, was the only independent predictor of von Willebrand factor antigen and plasminogen activator inhibitor-1 levels in LAA blood samples. In femoral vein blood samples, no significant difference was detected in the above blood parameters among the four LAA morphological types. In all blood samples, the levels of thrombin-antithrombin complex, D-dimer, fibrinogen, and tissue plasminogen activator were comparable among the four LAA morphological types. Conclusions In patients with atrial fibrillation, LAA morphological types might be associated with local platelet activity, fibrinolysis function, endothelial dysfunction, and inflammation.