Altered Platelet Function Research Articles

The effect of concurrent clopidogrel and omeprazole administration on clopidogrel metabolism and platelet function in healthy cats.

Some studies in humans show that the concurrent use of clopidogrel and omeprazole decreases plasma clopidogrel active metabolite (CAM) concentrations and clopidogrel antiplatelet effects. Whether this drug interaction occurs in cats is unknown. We hypothesized that administration of clopidogrel with omeprazole would decrease plasma CAM concentrations and decrease clopidogrel antiplatelet effects in healthy cats. Ten domestic cats. In this 2-sequence, 2-period, 2-treatment randomized crossover study, healthy cats were randomly assigned to receive clopidogrel only (18.75 mg PO q24h) or clopidogrel with omeprazole (1 mg/kg PO q12h) for 10 days, followed by a 2-week washout period, and then the opposite treatment for another 10 days. Blood was collected by jugular venipuncture on days 0, 5, and 10. Plasma CAM concentrations were measured using high-performance liquid chromatography-tandem mass spectrometry. Platelet function was evaluated using Plateletworks, Multiplate Analyzer, and Platelet Function Analyzer-100 (PFA-100). Multiplate Analyzer and PFA-100 detected no difference in platelet function between days or treatment groups. Plateletworks detected a significant difference (P < .001) in platelet function from day 0 to 5 and day 0 to 10 in both treatment groups but no difference between treatment groups. Plasma CAM concentrations were significantly lower on day 10 (P < .02) in cats receiving both medications versus clopidogrel only. Concurrent omeprazole and clopidogrel administration was associated with altered pharmacokinetics on day 10, but no difference in pharmacodynamics between the 2 treatment groups. The short-term use of clopidogrel and omeprazole does not seem to alter platelet function significantly.

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.

Biomedical application of TiO2NPs can cause arterial thrombotic risks through triggering procoagulant activity, activation and aggregation of platelets.

Titanium dioxide nanoparticles (TiO2NPs) are widely used in medical application. However, the relevant health risk has not been completely assessed, the potential of inducing arterial thrombosis (AT) in particular. Alterations in platelet function and susceptibility to arterial thrombosis induced by TiO2NPs were examined using peripheral blood samples from healthy adult males and an in vivo mouse model, respectively. Here, using human platelets (hPLTs) freshly isolated from health volunteers, we demonstrated TiO2NP treatment triggered the procoagulant activity of hPLTs through phosphatidylserine exposure and microvesicles generation. In addition, TiO2NP treatment increased the levels of glycoprotein IIb/IIIa and P-selectin leading to aggregation and activation of hPLTs, which were exacerbated by providing physiology-mimicking conditions, including introduction of thrombin, collagen, and high shear stress. Interestingly, intracellular calcium levels in hPLTs were increased upon TiO2NP treatment, which were crucial in TiO2NP-induced hPLT procoagulant activity, activation and aggregation. Moreover, using mice in vivo models, we further confirmed that TiO2NP treatment a reduction in mouse platelet (mPLT) counts, disrupted blood flow, and exacerbated carotid arterial thrombosis with enhanced deposition of mPLT. Together, our study provides evidence for an ignored health risk caused by TiO2NPs, specifically TiO2NP treatment augments procoagulant activity, activation and aggregation of PLTs via calcium-dependent mechanism and thus increases the risk of AT.

Trauma patients have reduced ex vivo flow-dependent platelet hemostatic capacity in a microfluidic model of vessel injury.

Trauma is the leading cause of death in individuals up to 45 years of age. Alterations in platelet function are a critical component of trauma-induced coagulopathy (TIC), yet these changes and the potential resulting dysfunction is incompletely understood. The lack of clinical assays available to explore platelet function in this patient population has hindered detailed understanding of the role of platelets in TIC. The objective of this study was to assess trauma patient ex vivo flow-dependent platelet hemostatic capacity in a microfluidic model. We hypothesized that trauma patients would have flow-regime dependent alterations in platelet function. Blood was collected from trauma patients with level I activations (N = 34) within 60 min of hospital arrival, as well as healthy volunteer controls (N = 10). Samples were perfused through a microfluidic model of injury at venous and arterial shear rates, and a subset of experiments were performed after incubation with fluorescent anti-CD41 to quantify platelets. Complete blood counts were performed as well as plasma-based assays to quantify coagulation times, fibrinogen, and von Willebrand factor (VWF). Exploratory correlation analyses were employed to identify relationships with microfluidic hemostatic parameters. Trauma patients had increased microfluidic bleeding times compared to healthy controls. While trauma patient samples were able to deposit a substantial amount of clot in the model injury site, the platelet contribution to microfluidic hemostasis was attenuated. Trauma patients had largely normal hematology and plasma-based coagulation times, yet had elevated D-Dimer and VWF. Venous microfluidic bleeding time negatively correlated with VWF, D-Dimer, and mean platelet volume (MPV), while arterial microfluidic bleeding time positively correlated with oxygenation. Arterial clot growth rate negatively correlated with red cell count, and positively with mean corpuscular volume (MCV). We observed changes in clot composition in trauma patient samples reflected by significantly diminished platelet contribution, which resulted in reduced hemostatic function in a microfluidic model of vessel injury. We observed a reduction in platelet clot contribution under both venous and arterial flow ex vivo in trauma patient samples. While our population was heterogenous and had relatively mild injury severity, microfluidic hemostatic parameters correlated with different patient-specific data depending on the flow setting, indicating potentially differential mechanistic pathways contributing to platelet hemostatic capacity in the context of TIC. These data were generated with the goal of identifying key features of platelet dysfunction in bleeding trauma patients under conditions of flow and to determine if these features correlate with clinically available metrics, thus providing preliminary surrogate markers of physiological platelet dysfunction to be further studied across larger cohorts. Future studies will continue to explore those relationships and further define mechanisms of TIC and their relationship with patient outcomes.

Atherosclerosis associated with Chlamydia pneumoniae: Dissecting the etiology

Chlamydia pneumoniae related infections and atherosclerosis are both common entities. Today, the literature presents an enormous amount of data regarding the role of C. pneumoniae in the development and sustainment of atherosclerosis and allowing us to comprehend the molecular mechanisms behind better. The implications of C. pneumoniae in atherogenesis include altered platelet function, hypercoagulability, macrophage dysfunction, vascular smooth muscle proliferation, and increased neutrophilic migration. Therefore, it would not be wrong to implicate that, C. pneumoniae plays important roles in almost every stage of atherogenesis. Furthermore, various serological markers suggestive of active or past C. pneumoniae infection are known to be associated with multiple clinical presentations, such as abdominal aortic aneurysms, subclinical atherosclerosis in the young individuals, aggravated atherosclerosis in heterozygous familial hypercholesterolemia. This review, as a result, aims to provide detailed insights into the pathophysiological mechanisms of atherogenesis associated with C. pneumoniae and its clinical implications.

Deciphering Abnormal Platelet Subpopulations in COVID-19, Sepsis and Systemic Lupus Erythematosus through Machine Learning and Single-Cell Transcriptomics.

This study focuses on understanding the transcriptional heterogeneity of activated platelets and its impact on diseases such as sepsis, COVID-19, and systemic lupus erythematosus (SLE). Recognizing the limited knowledge in this area, our research aims to dissect the complex transcriptional profiles of activated platelets to aid in developing targeted therapies for abnormal and pathogenic platelet subtypes. We analyzed single-cell transcriptional profiles from 47,977 platelets derived from 413 samples of patients with these diseases, utilizing Deep Neural Network (DNN) and eXtreme Gradient Boosting (XGB) to distinguish transcriptomic signatures predictive of fatal or survival outcomes. Our approach included source data annotations and platelet markers, along with SingleR and Seurat for comprehensive profiling. Additionally, we employed Uniform Manifold Approximation and Projection (UMAP) for effective dimensionality reduction and visualization, aiding in the identification of various platelet subtypes and their relation to disease severity and patient outcomes. Our results highlighted distinct platelet subpopulations that correlate with disease severity, revealing that changes in platelet transcription patterns can intensify endotheliopathy, increasing the risk of coagulation in fatal cases. Moreover, these changes may impact lymphocyte function, indicating a more extensive role for platelets in inflammatory and immune responses. This study identifies crucial biomarkers of platelet heterogeneity in serious health conditions, paving the way for innovative therapeutic approaches targeting platelet activation, which could improve patient outcomes in diseases characterized by altered platelet function.

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

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

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

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

Effects of PCSK9 on thrombosis and haemostasis in a variety of metabolic states: Lipids and beyond (Review).

Proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitors are widely recognised as being able to induce a potent reduction in low‑density lipoprotein‑cholesterol. An increasing number of studies have suggested that PCSK9 also influences the haemostatic system by altering platelet function and the coagulation cascade. These findings have significant implications for anti‑PCSK9 therapy in patients with specific coagulation conditions, including expanded indications, dose adjustments and drug interactions. The present review summarises the changes in PCSK9 levels in individuals with liver diseases, chronic kidney diseases, diabetes mellitus, cancer and other disease states, and discusses their impact on thrombosis and haemostasis. Furthermore, the structure, effects and regulatory mechanisms of PCSK9 on platelets, coagulation factors, inflammatory cells and endothelial cells during coagulation and haemostasis are described.

Platelet Function in Mood Disorders: Interplay, Clinical Implications, and Future Perspectives – A Narrative Review

Abstract Mood disorders, encompassing major depressive disorder and bipolar disorder, present complex etiologies involving neuroanatomical, neurotransmitter, and inflammatory mechanisms. Recent research explores the role of platelets, traditionally associated with hemostasis, in the pathophysiology of mood disorders. We examine neuroanatomical insights, neurotransmitter dysregulation, and the link between inflammation and mood disorders, emphasizing the complex nature of mood disturbances. Emerging evidence of altered platelet function in mood disorders and the therapeutic potential of antiplatelet agents and serotonin reuptake inhibitors are discussed. While personalized medicine holds promise, further research is needed to fully comprehend this evolving field. This review offers comprehensive insights into mood disorder diagnosis, intervention, and pathophysiology. In this review, a comprehensive literature search was conducted using databases such as PubMed, Scopus, and Web of Science. The search focused on articles published from 2000 to 2023, using keywords like “platelet function,” “mood disorders,” and “neurotransmitter dysregulation.” We included studies that specifically investigated the role of platelets in mood disorders, encompassing various study designs and population demographics.

418 The antiplatelet effects of EPA, an omega-3 fatty acid, are mediated by its 12-lipoxygenase metabolite, 12-HEPE

OBJECTIVES/GOALS: To determine whether cardioprotective effects observed in individuals taking dietary supplementation with eicosapentaenoic acid (EPA), an ω-3 polyunsaturated fatty acid, are realized by altering platelet function, and if these effects are mediated through the 12-lipoxygenase derived metabolite, 12-hydroxyeicosapentaenoic acid (12-HEPE). METHODS/STUDY POPULATION: Washed platelets or platelet rich plasma from healthy human donors were treated with EPA and 12-HEPE to assess their ability to inhibit platelet activation. Platelets were stimulated with agonists targeting different steps of the hemostatic response to vascular injury. Platelet aggregation, dense granule secretion, surface expression of integrin αIIbβ3 and P-selectin, and clot retraction were analyzed. To assess signaling through Gαs-GPCRs and protein kinase A activity, phosphorylation of vasodilator-stimulated phosphoprotein (VASP) was examined via western blot following treatment with EPA or 12-HEPE. RESULTS/ANTICIPATED RESULTS: EPA and 12-HEPE dose-dependently inhibit both collagen and thrombin-induced platelet aggregation. Furthermore, 12-HEPE more potently attenuates dense granule secretion and surface expression of platelet activation markers, integrin αIIbβ3 and P-selectin, in comparison to EPA. Plasma treated with EPA delayed thrombin-induced clot retraction, while 12-HEPE had no effect. Additionally, treatment with 12-HEPE increases phosphorylation of VASP, suggesting it could signal through the activation of the eicosanoid Gαs-GPCRs. DISCUSSION/SIGNIFICANCE: Here, we show for the first time that EPA directly inhibits platelet activation through its 12-LOX metabolite, 12-HEPE. These findings provide further insight into the mechanisms underlying the cardioprotective effects of EPA. A better understanding of current PUFA supplementations can inform treatment and prevention of cardiovascular diseases.

Role of Genetic Polymorphisms in the Development of Complications in Patients with Implanted Left Ventricular Assist Devices: HeartWare, HeartMate II, and HeartMate 3

Left ventricular assist device (LVAD) implantation is one of the mechanical circulatory support (MCS) treatments for advanced heart failure (HF) patients. MCS has emerged as a lifesaving therapy that improves patients’ quality of life. However, MCS remains limited by a paradoxical coagulopathy accompanied by thrombosis and bleeding. The mechanisms of MCS thrombosis are increasingly being defined, but MCS-related bleeding, which is related to shear-mediated alteration of platelet function, remains poorly understood. Complications might develop due to the high non-physiological shear stress in the device and as a consequence of individual variability in response to the antithrombotic therapy. Thromboelastography (TEG) and genotyping of gene polymorphisms that are involved in the coagulation cascade and in the metabolism of the antithrombotic therapy might be valuable sources of information for the reduction of complication development. The aim of the study was to identify genetic factors related to the development of device complications according to the implanted LVAD type. We compared the clinical and genetic data of HF patients (n = 98) with/without complications with three types of implanted devices: HeartWare HVAD (HW), HeartMate II (HMII), and HeartMate 3 (HM3). rs9923231 in VKORC1 (95%CI −6.28–0.22, p = 0.04) and rs5918 in ITGB3 genes (95%CI 0.003–4.36, p = 0.05) showed significant association with the TEG coagulation index parameter, which identified hyper- and hypo-coagulation states. The wild genotype of rs5918 in the ITGB3 gene prevailed in patients implanted with HM3 devices, which developed fewer complications than with HMII (p = 0.04). Individual genetic information could be useful in the management of patients with HF and the implantation of MCS to reduce the development of complications.

Superiority of CS585 as a selective prostacyclin receptor agonist in prevention of thrombosis

Abstract Background A major challenge with development of new targets for antithrombotic treatment is the sustained effect on the platelet and the target selectivity. While increasing cAMP in the platelet through activation of the prostacyclin (IP) receptor represents a viable antiplatelet target for prevention of thrombosis, current IP receptor agonists lack stability and selectivity. Development of a selective IP receptor agonist that is stable in the blood and selective for the IP receptor would represent a new approach for prevention of platelet activation and thrombosis in the blood and expand the utility of IP receptor agonists which are currently used for the treatment of pulmonary arterial hypertension (PAH). Purpose Develop an IV and oral IP receptor agonist, CS585, with sustained activity in vivo in the blood and eliminating the off-target effects observed with other IP agonists not having selective activation of the IP receptor. These characteristics would make CS585 an attractive new therapeutic approach for limiting platelet activation and thrombosis. Methods We assessed platelet activation and thrombosis in human blood ex vivo and mouse models in vivo. For mouse models, platelets and fibrin were labelled and accumulation at the site of injury was measured using intravital microscopy. Thrombosis in the vessel was assessed in the laser-induced cremaster thrombosis assay. Selectivity was assessed in human blood pharmacologically using prostaglandin receptor inhibitors and in mouse with an IP receptor knockout mouse. Flow cytometry, aggregometry, and western blot techniques were used to assess selectivity. Results IV and oral administration of CS585 resulted in sustained inhibited clot formation and fibrin formation at the site of injury in the laser-induced cremaster arteriole thrombosis assay in wild-type mice but was unable to alter platelet function in IP knockout mice. CS585 inhibition of platelet activation was fully prevented in platelets from IP knockout mice as assessed by flow cytometry (integrin activation and granule secretion) and by VASP phosphorylation. Inhibition of human platelet activation by CS585 was inhibited by pharmacological inhibition of the IP receptor, but not by inhibition of the DP1, EP2, or EP4 receptors as assessed by aggregation, flow cytometry, and VASP phosphorylation. Conclusions We have shown for the first time that CS585, a novel IP receptor agonist, not only inhibits platelet activation and clot formation, but gives sustained inhibition through multiple routes of administration in an in vivo mouse model of thrombosis. Importantly, the challenge in selectivity observed with other IP receptor agonists is not observed with CS585 supporting its development for a number of diseases in the cardiovascular system including thrombosis, VTE, secondary prevention after MI and stroke, and PAH.CS585 inhibition of thrombosis in vivoCS585 selectively signals through IP

Nonsteroidal Anti-inflammatory Medications in Cranial Neurosurgery: Balancing Opioid-Sparing Analgesia with Bleeding Risk

Post-craniotomy pain is a common problem frequently encountered by neurosurgeons. This is typically managed with opioids; however, opioids have been shown to increase intracranial pressure by way of hypercapnia and straining from the associated constipation. Additionally, opioids can confound and mask the neurologic examination of post-craniotomy patients as well as be the nidus for a potential opioid addiction. Thus, alternative solutions for opioids have been a major topic of investigation within the neurosurgical community. Non-steroidal anti-inflammatory drugs (NSAIDs), present as a potential solution due to their non-addictive and analgesic properties, but utilization of NSAIDs in neurosurgical patients has been controversial given that NSAIDs alter platelet function. Whether the degree to which NSAIDs alter platelet function and bleeding time to clinically relevant manner has remained controversial; although, there have been several well-designed studies concluding that the utilization of NSAIDs in post-craniotomy patients does not increase the risk of postoperative bleeding. Herein, we review the pharmacology, efficacy, and safety of NSAIDs with a particular emphasis on NSAID use for post intracranial neurosurgical procedure pain management.

Monitoring platelet function in marine mammals: Intracellular Ca2+ mobilization as a biomarker of platelet activation

Platelet functionality plays a crucial role in marine mammals. Alterations in platelet function can result from stress, pathologies, or exposure to xenobiotics, among others. The early detection of platelet function abnormalities is essential in these species to prevent advanced pathology and mitigate potential risks. Our main objective was to establish a range of physiological values of platelet function in bottlenose dolphins (Tursiops truncatus), beluga whales (Delphinapterus leucas), sea lions (Otaria flavescens) and walruses (Odobenus rosmarus). Intraplatelet Ca2+ mobilization using adenosine diphosphate (ADP) as a platelet agonist was used as a platelet function biomarker, adapting the methodology previously described by us in dolphins (Felipo-Benavent et al., 2022) to the rest of the species. The assay was also adapted to a seal (Phoca vitulina). Numerical indicators of intraplatelet Ca2+ mobilization kinetics were established, and statistical analyses were performed to compare the effects of species, sex, age, aquarium and species. Significant differences were observed between species, being the platelets of the sea lions the more reactive to the agonist. This work demonstrates the usefulness of this assay in the diagnosis or monitoring of animals with hemostatic diseases, showing two clinical cases in which intraplatelet calcium mobilization values were altered in marine mammals suffering haemorrhages. This assay may also serve as a means to monitor environmental changes and their potential impact on the health of marine mammal populations.

No impact of polymorphism in the phosphodiesterase 5A gene in Cavalier King Charles Spaniels on pimobendan-induced inhibition of platelet aggregation response.

A variant in the canine phosphodiesterase (PDE) 5A gene (PDE5A:E90K) is associated with decreased concentrations of circulating cyclic guanosine monophosphate (cGMP) and response to PDE5 inhibitor treatment. Pimobendan is a PDE inhibitor recommended for medical treatment of certain stages of myxomatous mitral valve disease (MMVD) in dogs. PDE5A:E90K polymorphism attenuates the inhibitory effect of pimobendan on in vitro platelet aggregation and increases basal platelet aggregation in Cavalier King Charles Spaniels (CKCS). Selected clinical variables (MMVD severity, sex, age, hematocrit, platelet count in platelet-rich plasma [PRP], and echocardiographic left ventricular fractional shortening [LV FS]) will not show an association with results. Fifty-two privately owned CKCS with no or preclinical MMVD. Using blood samples, we prospectively assessed PDE5A genotype using Sanger sequencing and adenosine diphosphate-induced platelet aggregation response (area under the curve [AUC], maximal aggregation [MaxA], and velocity [Vel]) with and without pimobendan using light transmission aggregometry. Dogs also underwent echocardiography. Pimobendan inhibited platelet function as measured by AUC, MaxA, and Vel at a concentration of 10 μM (P < .0001) and Vel at 0.03 μM (P < .001). PDE5A:E90K polymorphism did not influence the inhibitory effect of pimobendan or basal platelet aggregation response. The PDE5A:E90K polymorphism did not influence in vitro basal platelet aggregation response or the inhibitory effect of pimobendan on platelet aggregation in CKCS. Dogs with the PDE5A:E90K polymorphism did not appear to have altered platelet function or response to pimobendan treatment.

High-throughput microfluidic blood testing to phenotype genetically linked platelet disorders: an aid to diagnosis

AbstractLinking the genetic background of patients with bleeding diathesis and altered platelet function remains challenging. We aimed to assess how a multiparameter microspot-based measurement of thrombus formation under flow can help identify patients with a platelet bleeding disorder. For this purpose, we studied 16 patients presenting with bleeding and/or albinism and suspected platelet dysfunction and 15 relatives. Genotyping of patients revealed a novel biallelic pathogenic variant in RASGRP2 (splice site c.240-1G>A), abrogating CalDAG-GEFI expression, compound heterozygosity (c.537del, c.571A>T) in P2RY12, affecting P2Y12 signaling, and heterozygous variants of unknown significance in the P2RY12 and HPS3 genes. Other patients were confirmed to have Hermansky-Pudlak syndrome type 1 or 3. In 5 patients, no genetic variant was found. Platelet functions were assessed via routine laboratory measurements. Blood samples from all subjects and day controls were screened for blood cell counts and microfluidic outcomes on 6 surfaces (48 parameters) in comparison with those of a reference cohort of healthy subjects. Differential analysis of the microfluidic data showed that the key parameters of thrombus formation were compromised in the 16 index patients. Principal component analysis revealed separate clusters of patients vs heterozygous family members and control subjects. Clusters were further segregated based on inclusion of hematologic values and laboratory measurements. Subject ranking indicated an overall impairment in thrombus formation in patients carrying a (likely) pathogenic variant of the genes but not in asymptomatic relatives. Taken together, our results indicate the advantages of testing for multiparametric thrombus formation in this patient population.

CARD29: Sodium Bicarbonate as an Adjunctive Local Anti-Thrombotic Agent for Cardiovascular Therapeutic Devices

Purpose: Despite clinical efficacy, mechanical circulatory support (MCS) systems remain challenged in maintaining physiologic hemostatic balance. The Impella heart pump is a micro-axial percutaneous MCS device that utilizes a purge flow for maintaining bearing lubrication, purge patency, and anti-thrombosis purposes, forming a purge gap micro-environment wherein purge fluid and blood may mix. During operation, a continuous 5% dextrose in water (D5W) + heparin purge solution flows through the Impella catheter to inhibit protein adsorption and thrombosis in internal purge gaps. This exogenous heparin can increase bleeding risk and complicate systemic heparin and anticoagulation management. Recent FDA approval has made available sodium bicarbonate (SB) as an alternative to purge heparin. Despite promising early clinical results, the influence of SB on blood platelets, central mediators of thrombosis and bleeding, remains unclear. Here, we investigated the effect of SB on human platelet function (adhesion, aggregation, activation) in vitro following incubation with relevant concentrations of heparin or SB. Methods: Human gel-filtered platelets (20,000/µL) or platelet rich plasma (100,000/µL) were obtained from ACD-A anticoagulated whole blood. Platelets were incubated (6:4 platelets: purge) at 37°C for 15 min with purge solutions (D5W; 50U/mL heparin + D5W; 25mEq/L SB + D5W; 50mEq/L SB + D5W; 100mEq/L SB + D5W). Platelet activation was measured as thrombin generation rate utilizing a previously-described modified prothrombinase assay. Platelet adhesion was measured via imaging (Nikon DS-Qi2; 100X) of fluorescent-labelled platelets (BioTracker490) adhered to glass. Platelet aggregation was quantified from agonist-mediated aggregation using a light-transmission aggregometer (PAP8E). All experiments were performed in duplicate with N≥3 donors. Results: SB altered overall platelet function in a concentration-dependent manner. 100mEq/L SB led to a significant decrease in platelet adhesion (0.11% decrease; Fig 1A), whereas only 25mEq/L SB led to a significant reduction in thrombin generation rate (14.0% decrease; Fig 1B). Collagen-mediated aggregation was significantly reduced after addition of 100mEq/L SB (42.2% decrease; Fig 1C). Traditional D5W+heparin solution significantly reduced platelet function, in all categories, to a greater degree than 25mEq/L SB, but had a similar effect to 50 or 100mEq/L SB when comparing platelet adhesion and activation metrics. Conclusion: SB in the concentrations tested led to altered platelet function, modulating surface adhesion, activation, and collagen-mediated aggregation –useful effects to limit device fouling and purge gap thrombosis. Use of SB as a localized anti-thrombotic agent, where its concentration and effect are not rapidly diluted, is promising for MCS such as the Impella system. SB use offers the potential for locally limiting thrombogenicity at the purge gap, while avoiding the need for exogenous heparin.

P74: Plasma Osmolarity as a Determinant of Platelet Morphology and Function in Heart Failure

Purpose: Heart failure (HF) is associated with increased sodium and water retention, though disproportionately, leading to hypervolemia and reduced plasma osmolality (<275 mOsm/kg). HF is also associated with thrombosis/thromboembolic adverse advents, either in the native heart, or in mechanical circulatory support (MCS) devices implanted to restore hemodynamics. Platelets, critical cells in thrombosis, are mechanosensitive, responding to altered shear flow associated with HF or MCS devices. Excess plasma water and hypo-osmolarity can induce a gradient via which cells become swollen and stiff as water is forced into cells. It is recognized in cell biology that volume regulation and swelling can impact cell mechanics and function. However, it is not well understood how these processes are regulated or affect platelet function. Here, we investigate the affect of altered tonicity on platelet morphology/geometry and corresponding affect on platelet activation and aggregation. Methods: Human gel-filtered platelets (GFP; 20,000/µL) or platelet rich plasma (PRP; 100,000/µL) were obtained from fresh ACD-A anticoagulated whole blood. Modified Tyrode’s buffer osmolality was measured (Advanced Instruments; Osmo1), mixed with platelets and adjusted to a final concentration of either 250mOsm/kg (“Hypotonic”), 285mOsm/kg (“Isotonic”), or 325mOsm/kg (“Hypertonic”). Scanning ion conductance microscopy (SICM; Park Systems NX12) was utilized to quantify platelet morphology and volume. Platelet activation was measured as thrombin generation rate utilizing a previously-described modified prothrombinase assay. Platelet aggregation was quantified from agonist-mediated light-transmission aggregometry (BIO/DATA Corp; PAP-8E). All experiments were performed in duplicate with N ≥ 3 donors. Results: Overall, platelet geometry and function were altered in an osmolarity-dependent fashion. Hypotonic conditions led to an average 2.1µm3 (17.7%) increase in platelet volume, compared to isotonic conditions (Fig 1A). Despite this large increase in volume, there was a non-significant increase in thrombin generation rate in hypotonic conditions; however, a hypertonic environment led to a significant increase (0.14 min-1 average increase; Fig 1B). Collagen-mediated aggregation had a negative correlation to osmolarity, with significantly higher aggregation in hypotonic conditions (average 11.7% increase; Fig 1C). Conclusions: Hypotonicity leads to increased platelet volume and increased aggregability to collagen; however, platelet thrombin generation remains unaffected. The results of our study provide insight into operative mechanisms of thrombogenicity and altered platelet function under heart failure hypo-osmotic conditions. Further, these studies are hypothesis-generating as to the potential role of water, ion, and membrane mechanisms, providing the foundation for defining the impact of platelet volume regulation on shear-mediated platelet dysfunction in future studies.

P82: Platelet Exogenous Lipid Uptake: A Step Toward Lipid Membrane Mechanoceuticals

Background: Mechanical circulatory support (MCS) remains limited by bleeding and clotting adverse events driven by altered platelet function related to supraphysiologic shear exposure. As the primary target of platelet shear exposure is the lipid-rich plasma membrane, in prior work we demonstrated that organic solvents altering membrane properties limit SMPA while preserving platelet function to avoid bleeding. These chemical agents limiting mechanical (shear) activation have been termed “mechanoceuticals.” Natural lipids exhibit membrane altering properties similar to solvents, yet avoid the risk of toxicity, are safe for human use and offer potential therapeutic translation. As a first step here we investigated whether lipids could be taken up and incorporated into the platelet membrane. Methods: Two distinct methods were utilized - fluorescence microscopy and flow cytometry. Gel-filtered platelets (GFP) were prepared from aspirin/NSAID-free human volunteers. Platelet uptake of three differing lipids were tested: 1. cholesterol, 2. Palmitic acid (16:0 – a saturated medium chain fatty acid) and 3. Oleic acid (18:1 – partially unsaturated fatty acid). GFP were stained with Nile Red (to allow platelet visualization, fluoresces red) and aliquots incubated with one of the three lipids (30 minutes at 37.) pre-tagged with nitrobenzoxadiazole (NBD – fluoresces green). GFP was imaged via fluorescence microscopy (1000X) using red and green filters successively to visualized platelets (red), platelets with lipid uptake (green), and to confirm co-registration (green and red overlap). GFP were also incubated with NBD-tagged lipids, followed by CD-41a antibody for platelet detection and analyzed via flow cytometry. Results: Platelets were readily stained and detected via Nile Red fluorescent imaging. All lipids tested appeared to be taken up by platelets with clear incorporation in their membrane. Overlap of imaging revealed clear registration of “platelet-lipid” images (green) with platelets alone (red), with evident co-registration confirming uptake and ruling out micelle artifact. Lipids alone did not reveal detectable particle imaging (see Figure). Flow Cytometry similarly demonstrated overlap of fluorescent lipid signals with those of anti-platelet antibody (CD41-a) confirming lipid uptake by platelets. Conclusion: Platelets appear to readily uptake lipids. Both saturated and unsaturated fatty acids, as well as cholesterol are incorporated into the platelet membrane with reasonable time exposure. As these differing lipids have a range of membrane-modulating properties, such as altering fluidity and stiffness, they offer promise as potential mechanoceutical agents. As next steps, studies are now ongoing to examine the cell mechanobiological effects of these lipids and their ability to modulate shear-mediated alteration of platelet function.Figure 1. Fluorescence microscopy imaging of 100000u/uL platelets stained with Nile Red upon incubation with NBD- Cholesterol, NBD-16:0 (Palmitic acid), and NBD-18:1 (Oleic acid) lipids each at 4uM concentrations for 30 minutes at 37ο C. Magnification ×1000. Scale bar =4 μm.