Platelet Function Research Articles

PAR for the liver course: Preserving platelet function mitigates outcomes in cirrhosis.

PAR for the liver course: Preserving platelet function mitigates outcomes in cirrhosis.

Platelet Function, Platelet Size and Content of Reticulated Platelets: Interactions in Patients Receiving Dual Antiplatelet Therapy.

Increased platelet activity is a risk factor of thrombotic events in cardiovascular patients. We studied the relationship between platelet function, platelet size, and the content of reticulated platelets (RP) in patients with coronary heart disease (CHD, n = 55) and acute coronary syndrome (ACS, n = 95) receiving acetylsalicylic acid + clopidogrel or ticagrelor, respectively. The control group consisted of patients with risk factors for CHD, but with no CHD/ACS and free of antiplatelet drugs (n = 66). Platelet function was evaluated by the exposure of activated glycoprotein (GP) IIb-IIIa and P-selectin. In the control group, platelets were activated by TRAP (Thrombin Receptor Activating Peptide) 10 µM, and ADP 20, 5, 2.5 µM, and in the CHD/ACS groups, by TRAP 10 µM, and ADP 20 5 µM (±epinephrine 20 µM). Platelet size was assessed by the mean volume, % large forms, and forward scattering. RP were stained by thiazole orange. In the control group, activated GP IIb-IIIa and P-selectin correlated with platelet size and RP content after platelet activation by all agonists. Despite the decrease in platelet activity by antiplatelet drugs, most correlations (primarily for activated GP IIb-IIIa) were preserved in the CHD/ACS patients. In conclusion, increased platelet size and RP content are associated with increased platelet activity and the reduced efficacy of antiplatelet therapy.

Effects of selective serotonin reuptake inhibitors on platelet functions: a literature review.

Many epidemiological studies have evidenced an increased bleeding risk associated with selective serotonin reuptake inhibitors (SSRIs), yet the underlying mechanisms remain unclear. This review summarizes data on SSRIs' effects on platelet functions assessed with assays used in clinical practice and highlights the areas that deserve further investigation. Conflicting results of SSRI effects on platelet aggregation were observed irrespectively of the agonist used, the antidepressant drug or the study type. Alike, discrepant results were reported with flow-cytometry-based assays assessing either platelet surface glycoprotein levels, integrin activation, agonist-induced secretion of intraplatelet granule content or membrane anionic phospholipid exposure. Other tests may have detected a platelet function defect in SSRIs samples, however, results were largely inconsistent. Critical literature examination unveils very low certainty of evidence on potential SSRI effect on platelet functions. Findings are often inconsistent even when similar methods are used, most likely because of differences in study design, included patients (age, comorbid conditions), SSRIs' type and dose, uncontrolled confounding factors, and statistical analysis power. Further studies are needed to disentangle any intrinsic antiplatelet effect of SSRIs and the multiple confounding factors, mainly the depression control itself and the degree of platelet SERT inhibition.

Outcome of tailored antiplatelet therapy in carotid stenting: a retrospective comparative study

BackgroundCarotid stenting requires dual antiplatelet therapy to effectively prevent thromboembolic complications. However, resistance to clopidogrel, a key component of this therapy, may lead to persistent risk of these complications. The aim of this study was to determine, if the implementation of routine platelet function testing and adjusting therapy was associated with lower incidence of thromboembolic complications and death.MethodsAll consecutive patients treated with carotid artery stenting in a single institution over 8 years were enlisted in a retrospective study. Platelet function testing was performed, and efficient antiplatelet therapy was set before the procedure. Incidence of procedure-related stroke or death within periprocedural period (0–30 days) was assessed. The results were evaluated in relation to the findings of six prominent randomized control trials.ResultsA total of 241 patients were treated for carotid stenosis, seven patients undergo CAS on both sides over time. There was 138 symptomatic (55,6%) and 110 asymptomatic stenoses (44,4%). Five thromboembolic complications (2,01%) occurred, four of them (1,61%) was procedure-related. Two patients died because of procedure-related stroke (0,82%). Incidence of procedure-related stroke or death was significant lower compared to the results of CREST study (2,01% vs. 4,81%, P = 0,0243) in the entire cohorts, and to the results of ICSS study in the symptomatic cohorts (2,86% vs. 7,37%, P = 0,0243), respectively.ConclusionsTailored antiplatelet therapy in carotid stenting is safe and seems to be related with lower incidence of procedure-related death or stroke rate. Larger prospective studies to assess whether platelet function testing-guided antiplatelet therapy is superior to standard dual antiplatelet should be considered.

Pharmacological effects of small molecule BCR-ABL tyrosine kinase inhibitors on platelet function.

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL fusion protein, such as imatinib (Gleevec), have revolutionized targeted cancer therapies. However, drug resistance and side effects, particularly those affecting hemostasis, continue to pose significant challenges for TKI therapies. As tyrosine kinases serve pivotal roles in platelet hemostatic function, we investigated the potential impact of both established and emerging ABL TKIs on human platelet activities ex vivo Our study included standard-of-care agents (e.g., imatinib and nilotinib), and second-generation ABL inhibitors including ponatinib and bosutinib designed to mitigate drug resistance. Additionally, we explored the effects of allosteric inhibitors targeting the myristoyl pocket of ABL (e.g., asciminib and GNF-2), and novel agents in preclinical development, including ELVN-919, which uniquely exhibits high specificity for the ABL kinase active site. Our findings reveal that while ABL inhibitors such as ponatinib and bosutinib impede platelet activity, highly specific new-generation ABL inhibitors, including first-in-class therapeutics, do not impact platelet function ex vivo Overall, these new insights around the effects of ABL TKIs on platelet function could inform the development of targeted therapies with reduced hematologic toxicities. Significance Statement This study examines the effects of clinically relevant small molecule BCR-ABL tyrosine kinase inhibitors (TKIs) on platelet activity. This analysis includes first-time assessments of agents such as asciminib and ELVN-919 on human platelet function ex vivo, alongside established therapies (e.g., imatinib, ponatinib) with well-characterized effects on platelet function, to discern potential anti-platelet and other effects of BCR-ABL TKIs and inform clinical safety.

ACKR3 agonism induces heterodimerization with chemokine receptor CXCR4 and attenuates platelet function.

Platelet receptors ACKR3 and CXCR4 play a crucial role in a variety of cardiovascular diseases. Like most chemokine receptors, CXCR4 is a G protein coupled receptor that induces platelet activation. In contrast, the atypical chemokine receptor 3 (ACKR3) lacks the ability to activate heterotrimeric G proteins and its activation leads to platelet inhibition and attenuates thrombus formation. In nucleated cells, heterodimerization of ACKR3 with CXCR4 regulates CXCL12-dependent signalling. The aim of our study was to investigate the formation of ACKR3/CXCR4 heterodimers in platelets and the subsequent consequences for platelet function. Using a proximity ligation assay (PLA, Duolink®) to screen for CXCR4/ACKR3 heterodimerization inducing compounds, we found that ACKR3 agonism but not conventional platelet agonists or endogen ligands lead to heterodimer formation. To further characterize the formation of ACKR3/CXCR4 heterodimers, we studied the CXCL12-dependent platelet activation via CXCR4. Both, CXCL12-dependent platelet aggregation and collagen-dependent exvivo thrombus formation were significantly downregulated by ACKR3 agonism. Moreover, platelet intracellular calcium and Akt signalling were increased by CXCL12 and again suppressed by ACKR3-specific agonists. Previously, CXCL12 was shown to decrease platelet cAMP levels via CXCR4. Treatment with a specific ACKR3 agonist counteracted this CXCL12/CXCR4-dependent cAMP decrease. Our results reveal that the formation of platelet ACKR3/CXCR4 heterodimers is dependent on ACKR3 rather than CXCR4. Furthermore, ACKR3 agonism induced heterodimerization is associated with mitigating CXCL12/CXCR4-dependent platelet activation possibly by modulating CXCR4-dependent G protein signalling. Our results indicate possible ACKR3 agonist functions and reinforce the potential therapeutic applications of ACKR3 agonists.

Commonalities of platelet dysfunction in heart failure with preserved ejection fraction and underlying comorbidities.

Heart failure with preserved ejection fraction (HFpEF) is characterized by a lack of a specific targeted treatment and a complex, partially unexplored pathophysiology. Common comorbidities associated with HFpEF are hypertension, atrial fibrillation, obesity and diabetes. These comorbidities, combined with advanced age, play a crucial role in the initiation and development of the disease through the promotion of systemic inflammation and consequent changes in cardiac phenotype. In this context, we suggest platelets as important players due to their emerging role in vascular inflammation. This review provides an overview of the role of platelets in HFpEF and its associated comorbidities, including hypertension, atrial fibrillation, obesity and diabetes mellitus, as well as the impact of age and sex on platelet function. These major HFpEF-associated comorbidities present alterations in platelet behaviour and in features linked to platelet size, content and reactivity. The resulting dysfunctional platelets can contribute to further increase inflammation, oxidative stress and endothelial dysfunction, suggesting an active role of these cells in the initiation and progression of HFpEF. Recent evidence shows that reduced platelet count and elevated mean platelet volume are associated with worsening heart failure in HFpEF patients. However, the specific mechanisms by which platelets contribute to HFpEF development and progression are still largely unexplored, with only a few studies investigating platelet function in HFpEF. We discuss the limited yet significant body of research investigating platelet function in HFpEF, emphasizing the need for more comprehensive studies. Additionally, we explore the potential mechanisms through which platelets may influence HFpEF, such as their interactions with the vascular endothelium and the secretion of bioactive molecules like cytokines, chemokines and RNA molecules. These interactions and secretions may play a role in modulating vascular inflammation and contributing to the pathophysiological landscape of HFpEF. The review underscores the necessity for future research to elucidate the precise contributions of platelets to HFpEF, aiming to potentially identify novel therapeutic targets and improve patient outcomes. The evidence presented herein supports the hypothesis that platelets are not merely passive bystanders but active participants in the pathophysiology of HFpEF and its comorbidities.

Unbiased High-Throughput Screening of Drug-Repurposing Libraries Identifies Small-Molecule Inhibitors of Clot Retraction

Platelet clot retraction, the ultimate phase of platelet thrombus formation, is critical for clot stabilization. It requires functional αIIbβ3 receptors, fibrin, and the integrated actions of the actin-myosin contractile and cytoskeletal systems. Disturbances in clot retraction have been associated with both bleeding and thrombosis. We recently demonstrated that platelets treated with the αIIbβ3 antagonist peptide RGDW, which eliminates fibrinogen-mediated platelet aggregation, are still able to retract clots. We have exploited this observation to developed an unbiased, functional high-throughput assay to identify small molecule inhibitors of fibrin-mediated clot retraction adapted for a 384-well plate format. We tested 9,710 compounds from drug-repurposing libraries (DRL’s). These libraries contain compounds that are either FDA-approved or have undergone preclinical/clinical development. We identified 27 compounds from the LOPAC library as inhibitors of clot retraction of which 14 are known inhibitors of platelet function. From the DRL we identified 135 compounds (1.6% hit rate). After extensive curation, these compounds were categorized based on the activity of their reported target. Multiple kinase and phosphodiesterase inhibitors with known antiplatelet effects were identified, along with multiple deubiquitination and receptor inhibitors, as well as compounds that have not previously been reported to have antiplatelet activity. Studies of one of the deubiquitination inhibitors (degrasyn) suggests that its effects are downstream of thrombin-induced platelet-fibrinogen interactions and thus may permit the separation of platelet thrombin-induced aggregation-mediated events from clot retraction. Additional studies of the identified compounds may lead to novel mechanisms of inhibiting thrombosis.

6940 Thrombotic Events in Primary Hyperparathyroidism: Association or Coincidence?

Abstract Disclosure: I.A. Guatemala Funes: None. Z. Zavgorodneva: None. Y. EL-Soufi: None. A. Khan: None. T. Zahedi: None. F. Zhang: None. Introduction: The classical manifestations of primary hyperparathyroidism are nephrolithiasis, osteoporosis, and hypercalcemia-related symptoms, such as abdominal pain, constipation, polydipsia, and polyuria. Parathyroid crisis is a rare and life-threatening presentation of primary hyperparathyroidism characterized by calcium levels above 14-15 mg/dl and marked symptoms of hypercalcemia. Here, we describe a rare case of bilateral pulmonary embolism in a patient with severe hypercalcemia secondary to parathyroid adenoma. Case Report The patient is a 70-year-old female with a past medical history of primary hyperparathyroidism, who presented with profound fatigue, shortness of breath, polyuria and constipation. The patient denied any trauma, immobilization, or long-distance travel. On physical exam patient was confused, dehydrated, and nonhypoxic but with increased work of breathing. Initial labs showed severe hypercalcemia with calcium 19.2 mg/dL, significant elevated PTH 1113.9 pg/mL from her baseline PTH 380.8 pg/mL, normal 25 VitD, and acute kidney injury with creatinine increased from 0.9 mg/dL to 2.8 mg/dL. The calcium level in 24-hour urine collection was 323 mg/24 hours. CT chest angiogram revealed a bilateral lower lobe pulmonary embolism. The lower extremity duplex was negative for venous thrombosis. Thyroid ultrasound demonstrated suspected left parathyroid adenoma 2.1 x 2.1 x 1.4 cm. Renal ultrasound was negative for renal calculi. Following treatment with intravenous fluids, Calcitonin, and Denosumab, her calcium level improved to 10.1 mg/dL. The patient’s symptoms improved and underwent parathyroidectomy. PTH levels decreased significantly after parathyroidectomy. Pathology report confirmed parathyroid adenoma. Discussion The association between hypercalcemia and venous or arterial thrombosis has been rarely reported. Calcium participates directly in the platelet activation, platelet aggregation, and coagulation cascade. However, the true correlation between hypercalcemia and thrombosis is not well understood. It is believed that indirect factors such as vasoconstriction, dehydration, and cytotoxic effects due to hypercalcemia and the direct effects over platelet function and coagulation, may act together to induce thrombosis. Unfortunately, much of the evidence between hypercalcemia and hypercoagulability is based on case reports and our case could be one more evidence that this correlation exists. The patient’s age, absence of other risk factors of hypercoagulability, simultaneous manifestation of hypercalcemic crisis, and acute PE make hypercalcemia as a more likely pathogenetic origin of thrombus formation. Conclusion: Physicians should be cautious of the hypercalcemia-induced thrombotic events caused by dehydration and calcium-triggered vasoconstriction, clotting system activation, or platelet aggregation. Presentation: 6/3/2024

Comparative study of hemostatic profile between conventional methods and “Sonoclot Coagulation and Platelet Function Analyzer” in patients with septicemia

Abstract: BACKGROUND: Sonoclot Coagulation and Platelet Functional Analyzer (Sonoclot) operating on the principle of micro-viscometry is known to provide comprehensive information on the entire hemostatic process. We hypothesized that performing global hemostasis assessment in patients with sepsis would identify many more patients with altered hemostasis than the conventional tests. OBJECTIVES: We compared the hemostatic status determined by Sonoclot and that by conventional hemostatic tests. METHODS: It was a prospective analytical study conducted for 18 months in a tertiary care hospital. Sixty-one cases of septicemia admitted in the intensive care units were included in the study. RESULTS: Conventional hemostatic tests (platelet count, prothrombin time [PT], international normalized ratio, and activated partial thromboplastin time [APTT]) were compared with the Sonoclot parameters. Conventional hemostatic parameter PT was prolonged in 60.7% and APTT in 31.1%. Overall, 65.6% of the patients had prolonged coagulation on assessment by conventional hemostatic parameters. Sonoclot-derived parameter-activated clotting time was prolonged in 31.1%, indicating 31.1% of cases to have a hypocoagulable state. The clot rate was increased in 93.4% of subjects, indicating 93.4% to have a hypercoagulable state. Thrombocytopenia was seen in 34.4% and thrombocytosis in 10%. Defective platelet function was observed in 92% of the cases in Sonoclot, and all of them had normal platelet counts. This finding was statistically significant. CONCLUSION: Information about hemostatic status identified by Sonoclot is significantly higher and more complete than that identified by conventional coagulation tests in septicemic patients.

Evaluation of the effects and plasma concentration of the platelet inhibitor ticagrelor, after crushed and non-crushed intake, after cardiac arrest and after semi-urgent coronary artery bypass surgery

Background Ticagrelor, used in acute coronary syndrome (ACS), can be administered via nasogastric tube when oral intake is impossible. We investigated platelet inhibition and pharmacokinetics in resuscitated ACS patients and those undergoing semi-urgent coronary artery bypass graft (CABG) surgery. Our study aimed to assess platelet inhibition with use of the Platelet Function Analyser (PFA) and measured plasma concentrations of ticagrelor and its active metabolite in these ACS patients. Methods We included resuscitated cardiac arrest patients (STEMI/NSTEMI) and semi-urgent CABG patients. Crushed ticagrelor tablets were administered using a nasogastric tube. PFA closure time (CT) was determined with CT longer than 113 s as reference range. Plasma concentrations of ticagrelor and its active metabolite were measured after protein precipitation, by using liquid chromatography with mass spectrometry detection. Results In 20 resuscitated patients, 89% showed platelet inhibition at 24 h and 92% at day 4. For semi-urgent CABG patients, 85% exhibited platelet inhibition at 24 h and 84% at day 4. For ticagrelor in resuscitated patients, the median time to peak plasma concentration (Tmax) was 100 h [8; 100] for a median maximal concentration (Cmax) of 615.5 ng/mL [217.5; 1385.0]. For AR-C124910XX median Tmax was 100 h [8; 100] for a Cmax of 131.0 ng/mL [52.1; 177.7]. Among 20 patients undergoing semi-urgent CABG, Tmax for ticagrelor was 100 h [100; 100] for a median Cmax of 857.0 ng/ml [496.8; 1157.5]. For AR-C124910XX, median Tmax was 100 h [43; 100] for a Cmax of 251.0 ng/ml [173.0; 396.5]. Conclusion Crushed ticagrelor via nasogastric tube achieved targeted platelet inhibition. Pharmacokinetics aligned with previous studies. EudraCT number: 2013-004191-35; Study protocol code: AGO/2013/011; EC/2014/1061; ClinicalTrial.gov identifier: NCT02341729

A gain of function variant in RGS18 candidate for a familial mild bleeding syndrome.

Inherited platelet diseases (IPDs) are bleeding disorders characterized by either defect in platelet count or in platelet function, the latter being less common and very heterogeneous. Numerous gene variants associated with abnormal receptors, granules, and signaling pathways have been reported. Despite significant advancements in our understanding, many patients still lack a precise diagnosis. To identify the genetic basis of a novel mild bleeding syndrome in a family exhibiting a selective defect of platelet aggregation. Our study included 6 family members across three generations, who displayed reduced platelet aggregation in response to ADP, PAR1-AP, arachidonic acid, and epinephrine, but not collagen. Platelet morphology, granule content and expression of major surface glycoproteins were all found to be normal. Whole exome sequencing was performed for affected and non-affected family members. We identified RGS18, which encodes the regulator of G protein signaling (RGS) 18, as a candidate gene for the platelet function defect observed in this family. The RGS18 protein serves as a crucial negative regulator of G protein-coupled receptor (GPCR) signaling and coordinates the signaling pathways of natural platelet inhibitors. The heterozygous RGS18 c.643C>T, p.Arg215* variant was found to co-segregate among all six affected subjects. Truncation at Arg215 removes the S216 and S218 phosphorylation sites, which are crucial regulatory domains for RGS18 activation. The impaired platelet function is thought to arise from excessive platelet downregulation due to constitutive activation of RGS18, resulting from a loss of association of the truncated form with the 14-3-3 protein.

Impact of Dipyrone Administration on Postoperative Analgesia and Aspirin Effect in Patients Undergoing Coronary Artery Bypass Grafting: The Prospective Randomized DipASA Study

ObjectiveThe aim of the study was to investigate the impact of dipyrone administration on postoperative analgesia and acetylsalicylic acid (ASA) effect in patients undergoing coronary artery bypass grafting (CABG). DesignA prospective randomized study. SettingSingle-university hospital setting. ParticipantsNinety-eight patients who underwent CABG between April 2022 and May 2023. InterventionsASA effect were measured at six time points with impedance aggregometry (Multiplate) and thromboelastography (TEG6sPlateletMapping). Patients were randomized to one of three groups: intravenous ASA and dipyrone at the same time (group 1), intravenous ASA and dipyrone with a 2-hour delay (group 2), and intravenous ASA alone (group 3). Postoperative analgesic effects (numeric rating scale, NRS) and the prevalence of potential ASA non-response (defined as ASPI>40U and TEG-ASA-inhibition<50%) were recorded. Measurements and Main Results80 of 90 analyzed patients took ASA preoperatively. All patients received intravenous ASA 100mg from postoperative day 1. The effect of ASA did not significantly differ between the study groups at any time for either platelet function test. NRS values did not differ between the study groups at any time (p=0.469). Patients in group 3 received significantly more additional co-analgesics than patients who received dipyrone (p=0.005). ASA non-response was detected in 38.9% and 67.8% on 7th postoperative day, respectively. ConclusionsDipyrone given after CABG seems to be safe and did not show any significant effect on platelet inhibition after ASA administration. Patients taking dipyrone postoperatively need significantly less additional co-analgesics. The ASA effect on platelet function should be checked at least once after surgery.

Management of Bleeding Diathesis in Elective and Orthopaedic Trauma: A Review.

There is a general need among orthopaedic surgeons for practical advice on managing patients with bleeding disorders. Appropriate diagnosis and management of these disorders is paramount once discovered before, during, or after the patient's surgical course. Bleeding disorders disrupt the body's ability to control bleeding, commonly through platelet function and blood clotting. Normally, the vessel contracts and retracts once disruption of blood vessels occurs, limiting blood loss. Blood platelets adhere to exposed collagen, aggregate at the site, and obstruct blood loss. Because platelet aggregates are temporary, blood clotting is needed to back up the platelet plug and provide a milieu for the healing process that completes the hemostatic events. Disorders that interfere with any of these events can result in hemorrhage, drainage, or rebleeding. Bleeding disorders are a group of conditions, either hereditary or acquired, marked by abnormal or excessive bleeding and/or bruising. The most effective methods for assessing coagulation disorders include a detailed history and a series of blood tests. Clinical examination findings are notable but may be less specific. If a surgical patient has a bleeding disorder discovered preoperatively, postoperatively, or intraoperatively, treatments exist with medications, surgical management, interventional radiology procedures, and replacement therapy.

Bleeding and quality of life in people with Glanzmann thrombasthenia—insights from the Glanzmann’s 360 study

BackgroundGlanzmann thrombasthenia (GT) is a rare platelet function disorder that results in severe bleeding. We assessed clinical symptoms and psychological parameters to identify the unmet needs associated with GT. ObjectivesGlanzmann’s 360 is a mixed-methods study designed to give a contemporary snapshot of the impact of living with GT. MethodsThe study comprised a self-completion online survey complemented by interviews conducted with affected individuals and carers recruited via social media and hemophilia treatment centers. ResultsThe survey was completed by 88 people with GT and 29 carers of children/young people with GT aged <16 years. The population ranged in age from <2 years to >70 years; 56% were female. Although 47% had been diagnosed with GT under the age of 2 years, 12% were diagnosed after 20 years of age. For 82%, a bleeding phenotype was apparent by the age of 5 years. Most respondents (88%) had experienced at least one bleed in the past week. Bleeding disproportionally affected women. Bleeds resulted in frequent hospital contact and considerable psychological distress: 26% of the population had scores suggestive of low self-esteem, while 30% met criteria suggestive of symptomatic depression. Exploratory analyses suggest that bleed experiences are associated with impaired health-related quality of life. ConclusionThe Glanzmann’s 360 study reveals the significant physical, psychosocial, and quality-of-life impairments that are likely to be linked to the frequent bleeds experienced by those with GT. Clinicians treating people with GT should promote access to multidisciplinary comprehensive care, including psychosocial support.

Functional characterization of a nanobody-based glycoprotein VI-specific platelet agonist

BackgroundGlycoprotein (GP)VI is a platelet-specific collagen receptor required for platelet activation during hemostasis. Platelet reactivity toward collagen is routinely assessed during diagnostic workup of platelet disorders. GPVI can be activated by inducing receptor clustering with suspensions of fibrillar collagen or synthetic cross-linked collagen-related peptide (CRP-XL). However, these suspensions are poorly standardized or difficult to produce. Nanobodies are small recombinant camelid-derived heavy-chain antibody variable regions. They are highly stable, specific, and ideal candidates for developing a stable GPVI agonist for diagnostic assays. ObjectivesDevelop a stable nanobody-based GPVI agonist. MethodsNanobody D2 (NbD2) was produced as dimers and purified. Tetramers were generated via C-terminal fusion of dimers with click chemistry. Nanobody constructs were functionally characterized with light transmission aggregometry (LTA) in platelet-rich plasma and whole blood flow cytometry. Diagnostic performance was assessed in patients with inherited platelet function disorders with LTA and flow cytometry. ResultsNbD2 was specific for human platelet GPVI. Dimers did not result in platelet activation in LTA or flow cytometry settings and fully inhibited CRP-XL-induced P-selectin expression and fibrinogen binding in whole blood and attenuated collagen-induced platelet aggregation in platelet-rich plasma. However, NbD2 tetramers caused full platelet aggregation, as well as P-selectin expression and fibrinogen binding. NbD2 tetramers were able to discriminate between inherited platelet function disorder patients and healthy controls based on fibrinogen binding, similar to CRP-XL. ConclusionNanobody tetramers to GPVI induce platelet activation and can be used to assess the GPVI pathway in diagnostic assays.

Beyond basic characterization and omics: Immunomodulatory roles of platelet-derived extracellular vesicles unveiled by functional testing.

Renowned for their role in haemostasis and thrombosis, platelets are also increasingly recognized for their contribution in innate immunity, immunothrombosis and inflammatory diseases. Platelets express a wide range of receptors, which allows them to reach a variety of activation endpoints and grants them immunomodulatory functions. Activated platelets release extracellular vesicles (PEVs), whose formation and molecular cargo has been shown to depend on receptor-mediated activation and environmental cues. This study compared the immunomodulatory profiles of PEVs generated via activation of platelets by different receptors, glycoprotein VI, C-type lectin-like receptor 2 and combining all thrombin-collagen receptors. Functional assays in vivo in zebrafish and in vitro in human macrophages highlighted distinct homing and secretory responses triggered by the PEVs. In contrast, omics analyses of protein and miRNA cargo combined with physicochemical particle characterization found only subtle differences between the activated PEV types, which were insufficient to predict their different immunomodulatory functions. In contrast, constitutively released PEVs, formed in the absence of an exogenous activator, displayed a distinct immunomodulatory profile from the receptor-induced PEVs. Our findings underscore that PEVs are tunable through receptor-mediated activation. To truly comprehend their role(s) in mediating platelet functions among immune cells, conducting functional assays is imperative.

Suppressed ORAI1-STIM1-dependent Ca2+ entry by protein kinase C isoforms regulating platelet procoagulant activity

Agonist-induced rises in cytosolic Ca2+ control most platelet responses in thrombosis and hemostasis. In human platelets we earlier demonstrated that the ORAI1-STIM1 pathway is a major component of extracellular Ca2+ entry, in particular when induced via the ITAM-linked collagen receptor, glycoprotein VI (GPVI). In the present paper, using functionally defective platelets from patients with a loss-of-function mutation in ORAI1 or STIM1, we show that Ca2+ entry induced by the endoplasmic reticulum ATPase inhibitor, thapsigargin, fully relies on this pathway. We demonstrate that both the GPVI-induced and thapsigargin-induced Ca2+ entry is strongly suppressed by protein kinase C (PKC) activation, while leaving intracellular Ca2+ mobilization unchanged. Comparing effects of a PKC inhibitory panel pointed to redundant roles of beta and theta PKC isoforms in Ca2+-entry suppression. In contrast, tyrosine kinases positively regulated GPVI-induced Ca2+ entry and mobilization. Label-free and stable isotope phosphoproteome analysis of GPVI-stimulated platelets suggested a regulatory role of bridging integrator-2 (BIN2), known as important mediator of the ORAI1-STIM1 pathway in mouse platelets. Identified were 25-45 regulated phospho- sites in BIN2 and 16-18 in STIM1. Five of these were characterized as direct substrates of the expressed PKC isoforms alpha, beta delta and theta. Functional platelet testing indicated that the downregulation of Ca2+ entry by PKC resulted in suppressed phosphatidylserine exposure and plasmatic thrombin generation. Conclusively, our results indicate that in platelets multiple PKC isoforms constrain the store-regulated Ca2+ entry via ORAI1-BIN2-STIM1, and hence downregulate platelet-dependent coagulation.

Prevalence and management of depressive symptoms in coronary heart disease patients and relationship with cardiovascular prognosis: a prospective cohort study

AimsDepressive symptoms are comorbid with coronary heart disease (CHD). There is a controversial debate about whether screening and intervention for depressive symptoms could improve cardiovascular prognosis. This study characterizes the prevalence, characteristics, cardiovascular prognosis and management need of depressive symptoms among CHD patients.MethodsCHD patients were recruited between November 18, 2020 and November 26, 2021. Depressive symptoms were evaluated with the Patient Health Questionnaire (PHQ-9). During the 12-month follow-up, cardiovascular disease (CVD) was the endpoint. Time-to-event data were estimated by Kaplan-Meier curves and Cox models.ResultsOf 582 patients (25% women), 21.0% had mild depressive symptoms, and 7.5% had moderate-to-severe depressive symptoms during hospitalization. Mild and moderate-to-severe depressive symptoms were risk factor-adjusted predictors of the primary composite endpoints (adjusted HR = 2.20; 95%CI 1.19–4.03, and adjusted HR = 2.70; 95%CI 1.23–5.59, respectively). Platelet count and low-density lipoprotein were higher in mild depressive symptoms compared to no depressive symptoms.ConclusionDepressive symptoms are prevalent in CHD patients. Mild and moderate-to-severe depressive symptoms are associated with higher risk of further CVD in CHD patients. Platelet function and behavioral mechanisms may contribute to this association.Trial registrationThis research was registered at https://www.chictr.org.cn. Full data of first registration is 11/09/2020. The registration number is ChiCTR2000038139.

The differential effects of eicosapentaenoic acid and docosahexaenoic acid on cardiovascular risk factors: an updated systematic review of randomized controlled trials.

Cardiovascular disease remains a major global health concern. The combination of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) has been shown to beneficially modify a range of cardiovascular risk factors. However, whether EPA and DHA have differential effects or potencies is currently unclear. A systematic review of randomized controlled trials (RCTs) that compared ≥2 g/day of near pure EPA and DHA was conducted. A total of 24 publications from nine unique RCTs were included. EPA and DHA both lower triglyceride levels, with DHA most likely having a slightly greater effect. Furthermore, both EPA and DHA increase high density lipoprotein (HDL) 2 cholesterol, which is cardioprotective, with the increase being greater with DHA. DHA appears to increase low density lipoprotein (LDL) cholesterol; however, DHA also increases LDL particle size, which would render LDL less atherogenic. DHA seems more effective than EPA in decreasing heart rate and blood pressure. Both EPA and DHA alter platelet function decreasing thrombogenicity, although they may have different actions on platelets. Both EPA and DHA decrease F2-isoprostanes, interpreted as a reduction in oxidative stress. They both decrease inflammatory gene expression and promote an anti-inflammatory oxylipin profile. These are all favorable effects with regard to cardiovascular disease risk. Effects of EPA and DHA on blood glucose are inconsistent. This review is constrained by the small number of high quality RCTs that directly compare EPA to DHA and report on outcomes other than blood lipids. There is a need for additional high-quality research to assess the independent effects of EPA and DHA on cardiovascular risk factors (e.g., inflammation, blood pressure, vascular function, platelet function) in larger and more diverse study populations.