Platelet Activation Markers Research Articles

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

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

Upregulation of Cortisol in Sepsis and Its Association with Markers of Hemostatic Dysregulation

Introduction:Sepsis is a life-threatening condition in which the host's response to a systemic infection contributes significantly to mortality. Patients with sepsis are at risk of developing a coagulation disorder leading to disseminated intravascular coagulation (DIC). In DIC, widespread activation of the coagulation cascade leads to microvascular thrombosis and organ failure. This consumes platelets and coagulation factors, putting the patient at risk for bleeding. Cortisol is a steroid hormone released in response to stress. Cortisol levels have been found to be increased in sepsis patients, and high cortisol levels have been correlated with increased mortality. However, the association of cortisol levels with the severity of coagulopathy in patients with DIC remains unclear. Cortisol levels may also be relevant to disease progression as a link between the stress response and the immune system. The purpose of this project is to assess the association of plasma cortisol levels with severity of coagulopathy in a population of patients with sepsis and clinically confirmed DIC. Furthermore, the association of cortisol with markers of hemostatic status, inflammation, and platelet function were also assessed.Materials and Methods:Citrated, de-identified plasma samples were collected from 52 adults with sepsis and suspected DIC at the time of ICU admission. Samples were collected at the University of Utah and transferred to LoyolaUniversity Chicago under an IRB approved protocol. Plasma samples from healthy individuals were acquired from a commercial source (George King Biomedical, Overland, KS). Samples were stored at -80˚C prior to analysis. Platelet count was determined as part of standard clinical practice. PT/INR and fibrinogen were measured using standard techniques on the ACL-ELITE coagulation analyzer. Cortisol, D-dimer, PAI-1, CD40L, inflammasomes (NLRP3), and microparticles were measured using commercially available ELISA kits and were performed according to the manufacturer's instructions. DIC score was calculated using the ISTH scoring algorithm. A score of 0-2 was classified as no DIC, 3-4 was classified as non-overt DIC, and 5 or greater was classified as overt DIC. Results were analyzed using GraphPad Prism.Results:Cortisol showed significant variation based on DIC status (Kruskal-Wallis ANOVA, p < 0.0001) . Patients with non-overt DIC and overt DIC exhibited significantly elevated cortisol levels compared to healthy controls (p < 0.0001 for both groups). Furthermore, cortisol levels showed significant variation amongst sepsis patients based on DIC score. Patients with sepsis and overt DIC had elevated cortisol compared to patients with sepsis and no DIC (p = 0.0069). Data is shown in Figure 1.Correlations were evaluated between cortisol and the hemostatic markers platelets, fibrinogen, INR, D-Dimer, and PAI-1 as well as with the inflammatory marker, inflammasomes (NLRP3) and the platelet markers CD40L and microparticles. Cortisol showed significant correlations with D-Dimer, PAI-1, and INR. (D-Dimer Spearman r = 0.480, p = 0.001; PAI-1 Spearman r = 0.415, p = 0.002; INR Spearman r = 0.305, p = 0.037). No significant correlations were observed between cortisol and the markers of inflammation or platelet activation.Conclusions:Cortisol showed a significant association with hemostatic status in a population of patients with sepsis and well-defined coagulopathy. Cortisol levels were significantly elevated in patients with overt or non-overt DIC compared to healthy individuals and in patients with overt DIC compared to those with sepsis without DIC. Cortisol also showed significant correlations with markers of hemostatic activation but not with markers of inflammation and platelet activation. This suggests that elevated levels of cortisol may play a specific role in the progression of hemostatic dysregulation observed in sepsis and DIC. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A CRISPR-Cas9 Generated Etv6 mutant Mouse Exhibits Decreased Platelet Counts and Functional Platelet Defects

We and others recently described families with autosomal dominant thrombocytopenia, variable platelet function defects, and a predisposition to leukemia caused by heterozygous germline mutations in ETV6 . One of these mutations in ETV6, encoding an amino acid change at position 214 (P214L), was described in five independent families. The P214L mutation, located in the central domain of ETV6, was shown to disrupt nuclear localization of the ETV6 protein and to block megakaryocyte maturation of human cells overexpressing the mutant protein in vitro . Additionally, RNA seq of platelets from patients with the ETV6-P214L mutation showed downregulation of cytoskeletal transcripts and transcripts involved in platelet production and function. To further study the effect of the P214L mutation on ETV6 and on platelet production and function, we have generated a CRISPR-Cas9 knock-in mouse with the equivalent mutation to the human P214L in the mouse Etv6 ortholog (in the corresponding amino acid P216 in mice). CRISPR-Cas9 knock-in mice were generated by designing short guide RNAs specific to the site of Etv6 encoding the proline to be mutated. Additionally, a ssDNA oligo was designed encoding the mutation (cca>cta, P>L) and flanking homologous sequence to provide a template for recombination. Heterozygous and homozygous mice were viable. Heterozygous by heterozygous crosses yielded ratios not significantly different from expected Mendelian ratios: wild type (WT) 18.0%, heterozygous 63.0%, and homozygous 19.1% (p=0.18, Chi-squared test). Complete blood counts were obtained by retro-orbital bleeds and showed a mild but significant decrease in platelet counts in homozygous mice (WT 967.3 x 103/uL, heterozygous 840 x 103/uL, homozygous 807.5 x 103/uL, p=0.03). Western blot indicated normal expression of Etv6 protein in mouse bone marrow. Immunofluorescence and confocal imaging of megakaryocytes from these mice showed an increased percentage of small, immature megakaryocytes in homozygous mice compared to WT mice. Flow cytometry analysis suggested normal distribution of the hematopoietic stem cell compartment. These data are consistent with the patient phenotype and in vitro data, which showed that the P214L mutation resulted in variable thrombocytopenia, immature megakaryocytes, and normal ETV6 protein expression.Considering the downregulation of transcripts related to platelet function in patients with the P214L mutation and the variable platelet function defects reported in patients with germline ETV6 mutations, we characterized the platelet function of Etv6 mutant mice. Platelets from homozygous mice showed decreased platelet activation upon high (10 ug/ml) and low (2 ug/ml) collagen stimulation as measured by light transmission aggregometry (p=0.041 and 0.019, respectively). Additionally, homozygous mice had decreased markers of platelet activation (thrombin and convulxin) including granule secretion (P-selectin exposure) and aIIbb3 activation (JONA binding).In conclusion, we have generated a CRISPR-Cas9 knock-in mouse with the orthologous mutation to the human P214L recurrent germline mutation. Mice homozygous for this mutation recapitulate the human phenotype of decreased platelet counts and impaired megakaryocyte maturation. Additionally, mice homozygous for this mutation show a significant defect in platelet activation, further supporting that Etv6 is important for platelet function. Although complete knock-outs of Etv6 are embryonic lethal and Etv6 is expressed in the hematopoietic stem cell, we did not find a significant hematopoietic stem cell defect in our mutant mice. This finding suggests that the most important function of Etv6 is further downstream in hematopoiesis. The Etv6 P214L mouse represents a reliable model for studying the role of Etv6 in megakaryocyte biology, platelet function, and potentially future studies in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Pulmonary Hypertension in Sickle Cell Disease: The Role of Activated Blood Coagulation and Inflammation.

Introduction: Pulmonary hypertension (PHT) is a common complication of sickle cell disease (SCD). The pathogenesis of PHT in this setting is likely multifactorial. There is an abundance of evidence to suggest that the coagulation system is activated in patients with SCD. In addition, autopsy series have shown that an in situ thrombotic arteriopathy is seen in the majority of SCD patients with PHT. The purpose of this study is to determine whether markers of thrombin generation, platelet activation and inflammation are associated with PHT in SCD patients.Methods: This cross-sectional study was performed using a cohort of patients followed at the Sickle Cell Clinic at UNC-Chapel Hill. The pulmonary artery systolic pressure (PASP) was determined with a Doppler echocardiogram by applying the modified Bernoulli equation (PASP = 4V2 + right atrial pressure). Pulmonary hypertension was defined using an age-, sex- and BMI-adjusted reference range. Laboratory studies, including a complete blood count, soluble CD40 ligand [sCD40L] (a marker of inflammation and platelet activation) and markers of thrombin generation (thrombin-anti-thrombin complex [TAT], prothrombin fragment 1+2 [F1+2], and D-dimers) were obtained on all SCD subjects and on six healthy, race-matched control subjects without SCD. Wilcoxon Rank Sum Tests were used to compare median values in SCD patients with and without PHT, as well as between SCD patients and the control individuals.Results: Of the 76 subjects with SCD (SS = 56, SC = 9, Sβ0 = 4, Sβ+ = 7), PHT was found in 26 (34%). Compared with SCD patients without PHT, those with PHT had higher WBC counts (10.4 X 103/μL vs. 8.7 X 103/μL; p = 0.05). In addition, while PHT patients had consistently higher levels of such parameters as: D-dimers (1603 ng/mL [FEU] vs. 1233 ng/mL [FEU]; p = 0.3), TAT (7.05 μg/L vs. 5.7 μg/L; p = 0.8) and F1+2 (1.15 nmol/L vs. 0.9 nmol/L; p = 0.3), they failed to achieve statistical significance. On the other hand, the sCD40L level was lower (6.7 ng/mL vs. 7.6 ng/mL; p = 0.6) in the PHT group, although this difference was also not significant. In a second analysis, SCD patients had significantly higher levels of D-dimers (1251.5 ng/mL [FEU] vs. 318 ng/mL [FEU]; p = 0.02), sCD40L (7.3 ng/mL vs. 2.8 ng/mL; p = 0.0003), WBC count (9.2 X 103/μL vs. 7.1 X 103/μL; p = 0.02) and platelet count (399 X 103/μL vs. 251 X 103/μL; p = 0.002) when compared to control individuals. The TAT and F1+2 levels were also higher in SCD patients, although the differences were not significant.Conclusion: Compared to controls, SCD patients have increased thrombin generation, platelet activation and inflammation. In our cohort, SCD patients with PHT have higher levels of various markers of thrombin generation compared to patients without PHT. The lack of statistical significance is likely a result of the baseline elevation of these markers in SCD patients in steady state and as such, much higher increases in the levels of these markers and/or much larger sample sizes would be required to reveal a significant difference between both groups. This evidence of further activation of coagulation in SCD patients with PHT provides a rationale for clinical trials exploring the use of anticoagulation therapy in these patients.

Significant difference of c-type lectin-like receptor 2 between colorectal cancer and polyp subgroups.

Platelets play a key role in tumor progression and metastasis. C-type lectin-like receptor 2 (CLEC-2) is the receptor expressed on platelets and the marker of platelet activation. This study aims to determine whether soluble CLEC-2 levels differ between patients with benign colorectal polyps and those with colorectal cancer (CRC). We measured plasma soluble CLEC-2 by enzyme-linked immunosorbent assay in 150 patients with colorectal polyps, 150 CRC patients without metastasis, 150 CRC liver metastasis, and 150 control subjects. The CRC patients had higher soluble CLEC-2 levels than patients with colorectal polyps (p< 0.001). Moreover, CRC patients with liver metastases displayed higher CLEC-2 levels than those in CRC patients without metastases (p< 0.001). In the CRC patients, CLEC-2 levels were correlated with lymph node metastasis and advanced stage. In the patients with polyps, there was a significant difference in CLEC-2 levels among patients with hyperplastic polyp, sessile serrated adenoma, and traditional serrated adenoma (p< 0.001). The ROC curve analysis revealed CLEC-2 had an optimal sensitivity of 77.3% and specificity of 94.6% for the screening of CRC, and sensitivity of 71.0% and specificity of 76.7% for the differential diagnosis of colorectal polyps and CRC. CRC patients have higher CLEC-2 levels than patients with colorectal polyps and healthy controls. Moreover, there is a significant difference in CLEC-2 levels among polyp subtypes. Further research is warranted.

The Molecular Aspects of Disturbed Platelet Activation through ADP/P2Y12 Pathway in Multiple Sclerosis.

Epidemiological studies confirm a high risk of ischemic events in secondary-progressive multiple sclerosis (SP MS) patients, directly associated with an increased level of pro-thrombotic activity of platelets. Our work aimed to verify potential molecular abnormalities of the platelet P2Y12 receptor expression and functionality as a cause of an increased risk of thromboembolism observed in the course of MS. We have demonstrated an enhanced platelet reactivity in response to adenosine diphosphate (ADP) in SP MS relative to controls. We have also shown an increased mRNA expression for the P2RY12 gene in both platelets and megakaryocytes, as well as enhanced density of these receptors on the platelet surface. We postulate that one of the reasons for the elevated risk of ischemic events observed in MS may be a genetically or phenotypically reinforced expression of the platelet P2Y12 receptor. In order to analyze the effect of the PAR1 (protease activated receptor type 1) signaling pathway on the expression level of P2Y12, we also analyzed the correlation parameters between P2Y12 expression and the markers of platelet activation in MS induced by selective PAR1 agonist (thrombin receptor activating peptide-6, TRAP-6). Identifying the molecular base responsible for the enlarged pro-thrombotic activity of platelets in SP MS could contribute to the implementation of prevention and targeted treatment, reducing the development of cardiovascular disorders in the course of the disease.

Sex-Specific Relationship Between Parathyroid Hormone and Platelet Indices in Phenotypes of Heart Failure-Results From the MyoVasc Study.

Background: Heart failure (HF) is a multifactorial syndrome with pathophysiological complexities still not fully understood. Higher mean platelet volume (MPV), a potential marker of platelet activation, and high concentrations of parathyroid hormone (PTH) have been implicated in the pathogenesis of HF.Aim: This study aims to investigate sex-specifically the association between PTH concentrations and platelet indices in phenotypes of HF.Methods and Results: PTH and platelet indices (MPV and platelet count) were available in 1,896 participants from the MyoVasc study in Mainz, Germany. Multivariable linear regression models, adjusted for age, sex, season, vitamin D status, cardiovascular risk factors, comorbidities, estimated glomerular filtration rate, and medication, were used to assess the associations between platelet indices and PTH. The results showed distinct sex-specific associations between PTH and platelet indices. A positive association between PTH and MPV was found in females with symptomatic HF with reduced ejection fraction (HFrEF) only [β = 0.60 (0.19; 1.00)]. Platelet count was inversely associated with PTH in male HFrEF individuals [β = −7.6 (−15; −0.30)] and in both males and females with HF with preserved ejection fraction (HFpEF).Conclusion: This study reports differential, sex-specific relationships between PTH and platelet indices in HF individuals independent of vitamin D status and clinical profile. Particularly in phenotypes of symptomatic HF, distinct associations were observed, suggesting a sex-specific mechanism involved in the interaction between PTH and platelets.

Effects of whole blood storage in a polyolefin blood bag on platelets for acute normovolemic hemodilution

Acute normovolemic hemodilution (ANH) is a potential transfusion method for platelets, as well as for red blood cells. However, previous studies have shown that whole blood storage in ANH decreases platelet aggregability by 14.7–76.3% and that this decrease is not recovered by reinfusion. We investigated whether a new whole blood storage method for 6 h using a polyolefin bag, based on the platelet concentrates storage method, would maintain platelet function better than the conventional method using a polyvinyl chloride bag. We demonstrated that storage of whole blood in a polyolefin bag maintained ADP-induced aggregation rates at more than twofold higher than those in a polyvinyl chloride bag, and also significantly suppressed P-selectin expression, a platelet activation marker (ADP-induced aggregation rates: 24.6 ± 5.1% vs. 51.7 ± 11.5%, p = 0.002; P-selectin expression; 50.3 ± 8.4MFI vs. 31.6 ± 9.3MFI, p = 0.018). These results could be attributed to the high gas permeability of polyolefin, which lowered PCO2 and maintained a high pH with or without agitation. There were no significant changes in platelet count and red blood cell parameters due to the storage methods. Our results suggest that ANH using polyolefin bags is advantageous in improving hemostatic function compared to the conventional method.

Dual preparation of plasma and platelet concentrates in platelet additive solution from platelet concentrates in plasma using a novel filtration system.

Platelet concentrates suspended in a platelet additive solution (PAS-PC) are associated with a reduction in allergic response and are suitable for preparing pathogen-inactivated PC. We aimed to develop an efficient platform for the dual preparation of PAS-PC and platelet-poor plasma. PAS-PC was prepared in six steps by using a hollow-fibre system based on cross-flow filtration: priming, loading PC, loading PAS, collection of filtered liquid (flow-through) and collection of platelets by washing with PAS followed by washing with air. In this study, the efficacy of platelet and plasma protein recovery and characteristics of recovered PAS-PC and flow-through plasma were analysed in detail. Recoveries of platelet in PAS-PC and plasma protein in the flow-through were 95.4% ± 3.7% and 61.6% ± 5.0%, respectively. The residual plasma protein in PAS-PC was 34.1% ± 2.8%. Although the expression level of CD62P, a platelet activation marker, in recovered platelets was approximately 1.2-fold of that in original platelets, swirling patterns were well retained, and aggregation in PAS-PC was not visible. Agonist-induced aggregabilities, platelet morphology and hypotonic shock recovery were conserved. The patterns of plasma protein and lipoprotein in the flow-through were comparable with those in the original PCs. The multimeric pattern analysis of VWF remained unaltered. We propose a highly efficient preparation system that enables the simultaneous production of PAS-PC and platelet-poor plasma. It also achieves a high recovery of functionally well-retained platelets with very low activation.

The Vasculopathy of Juvenile Dermatomyositis: Endothelial Injury, Hypercoagulability, and Increased Arterial Stiffness.

Vasculopathy is considered central to the pathogenesis of juvenile dermatomyositis (DM) and is associated with severe extramuscular manifestations. We undertook this study to investigate the hypothesis that the vasculopathy of juvenile DM can be noninvasively tracked by examining biomarkers of endothelial injury, subclinical inflammation, hypercoagulability, and vascular arterial stiffness. The study population was a UK cohort of children with juvenile DM. Circulating endothelial cells (CECs) and microparticles (MPs) were identified using immunomagnetic bead extraction and flow cytometry, respectively. Plasma thrombin generation was determined using a fluorogenic assay. Cytokine and chemokine levels were measured by electrochemiluminescence. Arterial stiffness was assessed using pulse wave velocity (PWV). Results were expressed as the median and interquartile range (IQR), and statistical significance was assessed using nonparametric analyses. Ninety patients with juvenile DM and 79 healthy control subjects were included. The median age of the patients was 10.21 years (IQR 6.68-13.40), and the median disease duration was 1.63 years (IQR 0.28-4.66). CEC counts were higher in all patients with juvenile DM compared to controls (median 96 cells/ml [IQR (40-192] and 12 cells/ml [IQR 8-24], respectively; P < 0.0001). Circulating MP numbers were also significantly higher in patients with active juvenile DM compared to controls (median 204.7 × 103 /ml [IQR 87.9-412.6] and 44.3 × 103 /ml [IQR 15.0-249.1], respectively; P < 0.0001). MPs were predominantly of platelet and endothelial origin. Enhanced plasma thrombin generation was demonstrated in patients with active juvenile DM compared to those with inactive disease (P = 0.0003) and controls (P < 0.0001). Carotid-radial PWV adjusted for age was increased in patients with juvenile DM compared to controls (P = 0.003). We observed increased endothelial injury and increased levels of proinflammatory cytokines in patients with active juvenile DM. MP profiles reflected distinct disease activity status in juvenile DM and are markers of vascular pathology, platelet activation, and thrombotic propensity. Ongoing long-term vascular injury may result in increased arterial stiffness in patients with juvenile DM.

Lessons learnt from COVID-19 coagulopathy.

The coronavirus disease 2019 (COVID‐19) pandemic has already left an indelible mark in human lives. Despite the havoc it created, this pandemic also saw significant advances in the management of an infectious disease wherein worldwide collaborative efforts from health care professionals have been unprecedented. One of the commonest complications recognised early in the pandemic is the development of coagulopathy. In this review, the lessons learnt from COVID‐19 coagulopathy are summarised with some perspectives on future clinical and research strategies. These include how local versus systemic coagulopathy can matter, how we can put D‐dimers to effective use, exhort more input into identifying a simple platelet activation marker, rethink the role of fibrinogen, look differently at lupus anticoagulant and heparin‐induced thrombocytopenia, bring back disseminated intravascular coagulation into our differential diagnosis slate and most importantly channel more funding into haemostasis and thrombosis research.

Platelet bound complement split product (PC4d) is a marker of platelet activation and arterial vascular events in Systemic Lupus Erythematosus

Platelet-bound complement activation products (PC4d) are associated with thrombosis in Systemic Lupus Erythematosus (SLE). This study investigated the effect of PC4d on platelet function, as a mechanistic link to arterial thrombosis. In a cohort of 150 SLE patients, 13 events had occurred within five years of enrollment. Patients with arterial events had higher PC4d levels (13.6 [4.4–24.0] vs. 4.0 [2.5–8.3] net MFI), with PC4d 10 being the optimal cutoff for event detection. The association of arterial events with PC4d remained significant after adjusting for antiphospholipid status, smoking, and prednisone use (p = 0.045). PC4d levels correlated with lower platelet counts (r = −0.26, p = 0.002), larger platelet volumes (r = 0.22, p = 0.009) and increased platelet aggregation: the adenosine diphosphate (ADP) concentration to achieve 50% maximal aggregation (EC50) was lower in patients with PC4d 10 compared with PC4d < 10 (1.6 vs. 3.7, p = 0.038, respectively). These results suggest that PC4d may be a mechanistic marker for vascular disease in SLE.

English

Thalassemia, derived from Thalassa [Greek: Sea] refers to a cluster of hereditary haemoglobinopathies initially reported in areas of Sub-Saharan Africa, the Indian subcontinent, Southeast Asia, and the Mediterranean where malaria was (or is) endemic. Currently, it is the most frequently recognized haemoglobinopathy, and has been correlated with a plethora of immune system changes such as neutropenia, natural killer cell dysfunctions, increased activity of CD8 suppressor cells, along with disturbances in macrophage function, chemotaxis, phagocytosis, and interferons INFγ production.1 The reduction in the alpha or beta chain that leads to the production of haemoglobin is the key pathophysiology behind thalassemia. Therefore, weakened red blood cells can contribute to inadequate erythropoiesis and haemolytic anaemia. Thalassemia diseases are known as transfusion-dependent or non-dependent according to either their beta-globin chain genotype or their clinical path. The most serious type of thalassemia is beta-thalassemia major, and it is transfusiondependent, presenting in early infancy.2 A diagnosis of β-thalassemia major (BTM) means lifelong, regular transfusions, supplemented with adequate iron chelation therapy for the patient.3 This also means that unless adequate blood screening and hygiene procedures are in place, patients with BTM are at a risk of transfusion transmitted infections (TTI).4 Many countries have been prompted by the spread of Covid-19 infection to shutter routine outpatient coronary health care units before the pandemic is over, which also extends to people with thalassemia, who require regular follow-ups. While Covid-19 infection rates remain high, patients with thalassemia with planned cardiovascular examinations are advised to delay their medical appointments for a minimum of 3 months.1 In addition, according to the statement published by the Thalassemia International Foundation on the 13th of July, 2020, patients with hemoglobinopathies are at a greater risk in acquiring the Covid-19 infection, posing an additional challenge to the patients, families, and medical staff managing these disorders.5 Thalassaemic patients have reduced levels of protein S and protein C; elevated aggregation of platelets; and recruitment of monocytes, granulocytes, and endothelial cells. Patients of thalassemia also show elevated markers of platelet and coagulation activation, even in the absence of major thromboembolic events. An increase in D-Dimer levels is also seen in most Covid-19 patients who are hospitalised, attributable to the underlying inflammatory process.1 A variety of questions have been raised during the ongoing Covid-19 pandemic with respect to diagnostic and clinical approaches towards this particular population. The precise path of infection with Covid-19 in those patients has yet to be elucidated.2 The progression of Covid-19 infection in patients with hemoglobinopathy in general and thalassemia in particular is not fully known and is still an area of discussion and under review. We report a case of a 30-year-old male patient, a known case of beta thalassemia major on maintenance transfusion and iron chelation therapy who acquired Covid-19 infection.

Impact of electric cardioversion on platelet activation.

IntroductionAtrial fibrillation (AF) comes along with high risk of stroke. This risk continues even after re-establishing sinus rhythm with cardioversion. Aim of this study is to evaluate the contribution of electric cardioversion (EC) to platelet activation and procoagulatory tendency.MethodsExtent of platelet activation before and after electric cardioversion was quantified using flow cytometry, impedance aggregation measurements with Multiplate®, and quantification of serum levels of platelet factor 4 (PF4) and ß-thromboglobulin (ß-TG) in patients with AF (N = 10).ResultsNo significant differences were observed in any of the measured parameters comparing the values from before and after cardioversion. Geometric means of P-selectin expression and integrin αIIbβ3 activation were 0.27 (+/- 0.07) and 2.30 (+/- 2.61) before EC and 0.28 (+/- 0.17) and 1.67 (+/- 1.82) after EC. Levels of ß-TG were 110.11 ng/ml (+/- 3.78) before and 110.51 ng/ml (+/- 2.56) after EC, levels of PF4 were 35.64 ng/ml (+/- 12.94) before and 32.40 ng/ml (+/- 4.95) after EC. Platelet aggregation triggered with adenosine diphosphate (ADP), arachidonic acid, collagen, Ristocetin, or thrombin receptor activating peptide (TRAP) revealed results within the normally expected ranges without significant changes before and after EC.DiscussionElectric cardioversion has no influence on platelet activation markers which is in agreement with other studies reporting electrical cardioversion to be safe.

The Importance of Platelet Glycoside Residues in the Haemostasis of Patients with Immune Thrombocytopaenia.

Loss of sialic acid from the carbohydrate side chains of platelet glycoproteins can affect platelet clearance, a proposed mechanism involved in the etiopathogenesis of immune thrombocytopaenia (ITP). We aimed to assess whether changes in platelet glycosylation in patients with ITP affected platelet counts, function, and apoptosis. This observational, prospective, and transversal study included 82 patients with chronic primary ITP and 115 healthy controls. We measured platelet activation markers and assayed platelet glycosylation and caspase activity, analysing samples using flow cytometry. Platelets from patients with ITP with a platelet count <30 × 103/µL presented less sialic acid. Levels of α1,6-fucose (a glycan residue that can directly regulate antibody-dependent cellular cytotoxicity) and α-mannose (which can be recognised by mannose-binding-lectin and activate the complement pathway) were increased in the platelets from these patients. Platelet surface exposure of other glycoside residues due to sialic acid loss inversely correlated with platelet count and the ability to be activated. Moreover, loss of sialic acid induced the ingestion of platelets by human hepatome HepG2 cells. Changes in glycoside composition of glycoproteins on the platelets’ surface impaired their functional capacity and increased their apoptosis. These changes in platelet glycoside residues appeared to be related to ITP severity.

Impact of platelet population behaviours in patients undergoing the Glenn procedure.

Pulmonary pressure is one of the most important parameters in the postoperative follow-up of patients who have undergone the Glenn procedure. Platelet activation markers, which are inexpensive and easily accessible blood count parameters, have been shown to be associated with the aetiology and pathogenesis of primary pulmonary artery hypertension. We examined the relationship between platelet activation markers and pulmonary pressures in the early postoperative period of patients who underwent the Glenn procedure.Eighty-five patients who underwent the Glenn procedure in our clinic between January 2011 and March 2020 were included in the study retrospectively. Fifty-one patients were male and 34 were female, and age varied from 4 to 416 months, with a mean of 28.64±51 months.Patients with increased pulmonary blood flow on palliation before Glenn surgery had higher mean platelet volume values. However, no correlation was found between pulmonary pressures and platelet activation markers in the early postoperative period.There was not similar study evaluating platelet activation markers in the paediatric age group before and after postoperative Glenn surgery in the literature. Therefore, even if platelet activation markers provide information about the pulmonary bed, they may be misleading due to other reasons that trigger bleeding and inflammatory processes in the early postoperative period.

131 Burn-Induced Coagulopathy: Burn Patient Plasma Causes Platelet Dysfunction

Abstract Introduction Burn-induced coagulopathy (BIC) greatly increases the risk of thrombosis, leading to organ dysfunction and death. Although BIC is attributed to a multitude of factors including hemodilution, SIRS, and excess coagulation, the exact molecular mechanisms are elusive. Determination of these mechanisms is essential to develop new strategies aimed at reducing the morbidity and mortality of BIC as commonly used anticoagulants are ineffective at preventing the consequences of BIC. Platelet dysfunction, which has been associated with bleeding, thrombosis, poor wound repair, and susceptibility to infection, occurs in BIC, but the etiology of this phenomenon is unknown. Burn injuries provoke a dramatic increase in plasma levels of inflammatory, coagulation, and fibrinolytic factors, potentially altering the physiology of blood cells and platelets. Therefore, we hypothesized that burn patient plasma pathologically alters normal platelet function. Methods In this prospective study, plasma samples were collected from 32 adult patients with burns &amp;gt;10% TBSA at a regional burn center. To assess the effects of burn plasma on platelet function, platelets isolated from healthy individuals were incubated with heparinized plasma from burn patients or control plasmas (N=15) for 2 hours. Following incubation, platelets were stimulated with various agonists, and markers of platelet activation (GPIIb/IIIa activation and P-selectin expression) were measured using flow cytometry. Results Platelets incubated with burn plasmas exhibited a reduction in response to stimulation compared with those incubated with control plasma. Both GPIIb/IIIa activation and P-selectin expression were affected, suggesting a defect in platelet signaling. This dysfunction did not strongly correlate with TBSA affected or modified Baux score but was significant when compared to healthy controls (P&amp;lt; 0.05). However, measurement of markers of inflammation, coagulation, and fibrinolysis in burn plasmas revealed a significant correlation of both plasma D-dimer (P&amp;lt; 0.02) and soluble P-selectin (P&amp;lt; 0.05) with induced platelet dysfunction. This suggests circulating factors indicative of coagulation, fibrinolysis, and endothelial dysfunction may play a role in platelet dysfunction observed in BIC. Conclusions This study demonstrates an in vitro effect of burn patient plasma on platelet function, suggesting a possible benefit for resuscitation with FFP. BIC is a principal source of morbidity and mortality in burn patients, and current thromboprophylaxis for burn patients may not address critical elements of BIC. Future studies are required to optimize resuscitation and minimize the consequences of BIC.

Decreased platelet surface phosphatidylserine predicts increased bleeding in patients with severe factor VIII deficiency

BackgroundBleeding phenotypes among individuals with severe factor VIII (FVIII) deficiency are variable, even with routine prophylactic FVIII administration. Activated platelets, in addition to their role in primary hemostasis, play a major role in coagulation by providing a phospholipid surface to which coagulation factors bind. ObjectivesThe aim of this study was to determine whether platelet function is associated with past and/or future bleeding in patients with severe FVIII deficiency on prophylaxis. Patients/MethodsPlatelet function quantified by platelet surface expression of phosphatidylserine, platelet surface glycoprotein (GP) VI, platelet surface activated GPIIb‐IIIa, platelet surface P‐selectin, the percentage of coated platelets, and the percentage of platelet‐derived microparticles in the presence or absence of in vitro activation by various agonists was assessed in 34 patients. ResultsDecreased platelet surface phosphatidylserine expression (identified by annexin V binding), both in the presence and absence of ADP/thrombin receptor activating peptide, demonstrated a significant association with both prior and subsequent bleeding in any location and specifically with hemarthrosis. No significant difference between patients with and without bleeding was observed in any of the other platelet activity markers. ConclusionsDecreased platelet surface phosphatidylserine expression measured by annexin V binding predicts increased bleeding in severe FVIII deficient patients on prophylaxis.

Platelet activation and aggregation in different centrifugal-flow left ventricular assist devices

Left-ventricular assist devices (LVADs) improve outcomes in end-stage heart failure patients. Two centrifugal-flow LVAD systems are currently approved, HeartMate 3 (HM3) and Medtronic/Heartware HVAD (HVAD). Clinical findings suggest differences in thrombogenicity between both systems. We compared markers of platelet activation and aggregation between HM3 and HVAD. We prospectively included 59 LVAD patients (40 HM3, 19 HVAD). Platelet P-selectin expression, activated glycoprotein (GP) IIb/IIIa and monocyte-platelet aggregates (MPA) were assessed by flow-cytometry. Platelet aggregation was measured by light-transmission aggregometry (LTA) and multiple-electrode aggregometry (MEA). Von-Willebrand factor (VWF) antigen (VWF:Ag), VWF activity (VWF:Ac), and VWF multimer pattern analysis were determined. Soluble P-selectin (sP-selectin) was measured with an enzyme-linked immunoassay. P-selectin, GPIIb/IIIa and MPA levels in vivo and in response to arachidonic acid, adenosine diphosphate, and thrombin receptor activating peptide were similar between HM3 and HVAD (all p > .05). Likewise, agonist-inducible platelet aggregation by LTA and MEA did not differ between HM3 and HVAD (all p > .05). VWF:Ag levels and FVIII:C were similar between both systems (both p > .05), but patients with HVAD had significantly lower VWF:Ac (p = .011) and reduced large VWF multimers (p = .013). Finally, sP-selectin levels were similar in patients with HVAD and HM3 (p = .845). In conclusion, on-treatment platelet activation and aggregation are similar in HM3 and HVAD patients. Potential clinical implications of observed differences in VWF profiles between both LVAD systems need to be addressed in future clinical trials.

Identification of Statin’s Action in a Small Cohort of Patients with Major Depression

Statins are widely used as an effective therapy for ischemic vascular disorders and employed for primary and secondary prevention in cardiac and cerebrovascular diseases. Their hemostatic mechanism has also been shown to induce changes in cerebral blood flow that may result in neurocognitive improvement in subjects with Major Depressive Disorder. Behavioral data, various blood tests, and resting-state brain perfusion data were obtained at the start of this study and three months post-therapy from a small cohort of participants diagnosed with Major Depressive Disorder. Subjects received either rosuvastatin (10 mg) or placebo with their standard selective serotonin reuptake inhibitors therapy. At the end of the study, patients using rosuvastatin reported more positive mood changes than placebo users. However, standard statistical tests revealed no significant differences in any non-behavioral variables before and after the study. In contrast, feature selection techniques allowed identifying a small set of variables that may be affected by statin use and contribute to mood improvement. Classification models built to assess the distinguishability between the two groups showed an accuracy higher than 85% using only five selected features: two peripheral platelet activation markers, perfusion abnormality in the left inferior temporal gyrus, Attention Switching Task Reaction latency, and serum phosphorus levels. Thus, using machine learning tools, we could identify factors that may be causing self-reported mood improvement in patients due to statin use, possibly suggesting a regulatory role of statins in the pathogenesis of clinical depression.