Platelet Phosphatidylserine Research Articles

Unexpected bleeding with platelet phosphatidylserine exposure defect: new kindred motivates rethinking Scott syndrome

Unexpected bleeding with platelet phosphatidylserine exposure defect: new kindred motivates rethinking Scott syndrome

Platelet phosphatidylserine exposure and microparticle production as health bioindicators in marine mammals.

In human medicine, various pathologies, including decompression sickness, thrombocytopenia, and rheumatoid arthritis, have been linked to changes in cellular microparticles (MP) formation, particularly platelet microparticles (PMP). Similar disorders in marine mammals might be attributed to anthropogenic threats or illnesses, potentially impacting blood PMP levels. Thus, detecting platelet phosphatidylserine (PS) exposure and PMP formation could serve as a crucial diagnostic and monitoring approach for these conditions in marine mammals. Our group has developed a methodology to assess real-time PS exposure and PMP formation specifically tailored for marine mammals. This method, pioneered in species such as bottlenose dolphins, beluga whales, walruses, and California sea lions, represents a novel approach with significant implications for both clinical assessment and further research into platelet function in these animals. The adapted methodology for evaluating PS exposure and PMP formation in marine mammals has yielded promising results. By applying this approach, we have observed significant correlations between alterations in PMP levels and specific pathologies or environmental factors. These findings underscore the potential of platelet function assessment as a diagnostic and monitoring tool in marine mammal health. The successful adaptation and application of this methodology in marine mammals highlight its utility for understanding and managing health concerns in these animals.

Abstract 140: Exploring Protein Kinase C Theta (PKCθ) Signaling In The Generation Of Procoagulant Platelet: Insights From Phosphoproteomics And Systems Analysis

Background: Platelet activation plays a pivotal role in physiological hemostasis and thrombosis. Protein Kinase C theta (PKCθ) isoforms exert significant regulatory influence on platelet activation. While specific PKC family members have well-defined roles in platelet function, the roles of PKCθ remain less explored. Objectives: To determine mechanisms of PKCθ signaling that orchestrate procoagulant platelet generation. Methods: Platelets were prepared from healthy human donors for ex vivo experiments. Platelet signaling and function were studied following stimulation with GPVI agonist crosslinked collagen-related peptide (CRP-XL). To investigate PKCθ inhibition mechanisms in platelets, small molecule inhibitors targeting the PKCθ (CC-90005 and C20) were employed. Results: From phosphoproteomics, we noted prominent phosphorylation on the C-terminus PKCθ at Ser685. Pretreatment of platelets with inhibitors specific PKC isoforms revealed that PKCα/β, PKCθ, and PKCΔ activities determine PKCθ Ser685 phosphorylation. From the PKC Inhibitors Activity Curves, we noted a robust and selective inhibitory impact of CC-90005 on PKCθ, with specificity surpassing that of C20. Parallel platelet function analyses revealed that inhibiting PKCθ had minimal impact on platelet spreading and aggregation. However, PKCθ inhibition in the presence of Ca 2+ upregulated platelet phosphatidylserine (PS) exposure, as measured by flow cytometry. This was accompanied by an increase in platelet procoagulant activity, where PKCθ inhibition reduced the lag time for fibrin formation. Furthermore, fluorescence microscopy also found that PKCθ inhibition significantly augmented the proportion of procoagulant platelets and microparticles. Additionally, compared to the control group, the use of cyclosporin A (CsA) reduced the production of procoagulant platelets, but the addition of CC-90005 or C20 rescued this phenomenon, with signal intensity surpassing that of the control group. Conclusions: Our study proposes a model wherein PKCΔ and PKCα/β activities induce the phosphorylation of PKCθ at S685, influencing mitochondrial calcium signaling and supporting procoagulant platelet generation.

Comprehensive functional characterization of a novel ANO6 variant in a new patient with Scott syndrome

BackgroundScott syndrome is a mild platelet-type bleeding disorder, first described in 1979, with only 3 unrelated families identified through defective phosphatidylserine (PS) exposure and confirmed by sequencing. The syndrome is distinguished by impaired surface exposure of procoagulant PS on platelets after stimulation. To date, platelet function and thrombin generation in this condition have not been extensively characterized. ObjectivesGenetic and functional studies were undertaken in a consanguineous family with a history of excessive bleeding of unknown cause. MethodsA targeted gene panel of known bleeding and platelet genes was used to identify possible genetic variants. Platelet phenotyping, flow adhesion, flow cytometry, whole blood and platelet-rich plasma thrombin generation, and specialized extracellular vesicle measurements were performed. ResultsWe detected a novel homozygous frameshift variant, c.1943del (p.Arg648Hisfs∗23), in ANO6 encoding Anoctamin 6, in a patient with a bleeding history but interestingly with normal ANO6 expression. Phenotyping of the patient’s platelets confirmed the absence of PS expression and procoagulant activity but also revealed other defects including reduced platelet δ granules, reduced ristocetin-mediated aggregation and secretion, and reduced P-selectin expression after stimulation. PS was absent on spread platelets, and thrombi formed over collagen at 1500/s. Reduced thrombin generation was observed in platelet-rich plasma and confirmed in whole blood using a new thrombin generation assay. ConclusionWe present a comprehensive report of a patient with Scott syndrome with a novel frameshift variant in AN06, which is associated with no platelet PS exposure and markedly reduced thrombin generation in whole blood, explaining the significant bleeding phenotype observed.

Circulating Platelets in Thrombotic Antiphospholipid Syndrome Display High Procoagulant Activity and Surface Complement Deposition: Correlation with Thrombotic Risk

Background Antiphospholipid syndrome (APS) is characterized by circulating antiphospholipid antibodies (aPL) accompanied by arterial and/or venous thrombosis, and/or recurrent pregnancy loss. It commonly affects young women in whom it dramatically increases their risk of myocardial infarction, ischemic stroke, deep vein thrombosis and pulmonary embolism. Although the thrombotic risk associated with aPL is established, the underlying mechanisms are still incompletely defined and there are no definitive predictive biomarkers. As a result the management of APS involves indefinite anticoagulation. Increased activation of vascular cells, including platelets is considered to underlie the pathogenesis of APS, and may be mediated in some cases by complement. There is a considerable crosstalk between platelets and complement at various levels during thrombosis. First, activated complement proteins stimulate platelets through their cognate receptors on the platelet surface, and genetic deficiency of complement proteins in mice leads to decreased platelet responsiveness. Second, platelets secrete complement proteins into the medium upon activation. Third, agonist-stimulated platelets contribute to activation of both the classical and alternative pathways of the complement cascade. Hence, we sought to investigate the association between the complement-platelet axis and the prothrombotic pathology observed in APS. Methods Venous blood was drawn from patients with APS and healthy controls, and washed human platelets were prepared by differential centrifugation. Platelets either remained unstimulated or were treated with thrombin (0.1, 0.25, 0.5 U/ml) for 5 min and then labelled with FITC-PAC1, PE-anti-CD62P antibody, Alexa Fluor 488-anti-C4d conjugate, PE-Annexin V, Mitotracker Red, MitoSOX Red, Fluo4-AM, Rhod2-AM, Cell Event Green Caspase 3/7 detection reagent and FITC-IETD-FMK for 30 min in the dark, and then analyzed by flow cytometry to measure integrin activation, P-selectin expression (α-granule secretion), complement C4d binding, phosphatidylserine (PS) exposure, mitochondrial membrane potential, mitochondrial ROS, cytosolic calcium, mitochondrial calcium, caspase 3/7 activity (apoptosis) and caspase 8 activity (extrinsic apoptosis pathway), respectively. Platelet markers were correlated with complement binding and clinical characteristics of APS patients including number of thrombotic events and circulating levels of aPL. Results Thrombin-induced platelet integrin activation and P-selectin expression were identical in APS patients and healthy controls. However, platelets isolated from APS patients had significantly higher procoagulant activity (PS exposure) as well as complement C4d binding in both unstimulated and thrombin-stimulated states compared to healthy controls. There was a strong positive correlation between platelet PS exposure and C4d binding in APS patients, but not healthy individuals, suggesting a role for complement activation in formation of procoagulant platelets in APS. A subset of APS patients with high platelet procoagulant activity (PS exposure > mean + 2SD observed in healthy individuals) had lower mitochondrial membrane potential and increased mitochondrial calcium transients compared to healthy individuals, suggesting mitochondrial permeability transition pore (mPTP)-driven PS exposure. Another subset of APS patients with PS exposing platelets showed caspase activation indicating apoptosis. Platelet PS exposure and C4d binding correlated strongly with the number of thrombotic events as well as levels of circulating aPL, which identifies complement-induced procoagulant platelets as predictors and possible underlying mechanisms for thrombosis in APS. Conclusion Our preliminary studies suggest that PS-exposing platelets are generated in APS by multiple mechanisms including agonist-induced mPTP formation and apoptosis-associated caspase activation. Platelet PS exposure in APS is positively correlated with C4d binding, suggesting a role for complement activation in driving platelet procoagulant activity. Complement activation and PS exposure on platelets are strong predictors of thrombotic risk in APS and could serve as promising targets for novel antithrombotic agents in APS management.

PB0342 Platelet Phosphatidylserine is the Critical Mediator of Thrombosis in Heparin-Induced Thrombocytopenia

PB0342 Platelet Phosphatidylserine is the Critical Mediator of Thrombosis in Heparin-Induced Thrombocytopenia

Abstract 123: Protein Kinase C Theta (PKCθ) Signaling Associated With The Generation Of Procoagulant Platelets Revealed By Phosphoproteomics And Systems Analysis

Protein kinase C (PKC) family members integrate platelet activation programs to support hemostasis, and, also drive inflammatory and vascular diseases. While some PKC isoforms have established roles in platelets, roles for PKCθ are less specified. Here, we aim to determine how PKCθ signaling orchestrates platelet function. We prepared washed platelets from human donors for biochemical, physiological and omics studies. Platelet signaling and physiology were studied following stimulation with agonists, including the glycoprotein receptor GPVI agonist crosslinked collagen-related peptide (CRP-XL), and protease activated receptor PAR1 and PAR4 agonists, TRAP6 and AYPGKF-NH 2 . Platelets were treated with inhibitors of PKC family members to study PKC activation mechanisms by Western blot using antisera against specific phosphorylation sites on PKC isoforms and PKC substrates. Phosphoproteomics studies of GPVI and PAR agonist-stimulated platelets quantified significant, site-specific changes in the phosphorylation of PKC isoforms as well as PKC substrates. In addition to established phosphorylation events (e.g., PKCδ Tyr311), we noted a novel, prominent phosphorylation on the C-terminus PKCθ at Ser685. After generating and characterizing antisera specifically reactive to PKCθ phospho-Ser685, Western blot analysis determined that PKCθ Ser685 phosphorylation occurred later following GPVI platelet activation (>5 min) relative to PKCδ Tyr311 phosphorylation and PKC substrate phosphorylation driven by calcium-dependent PKCs. Treatment of platelets with specific PKC inhibitors revealed that PKCα/β, PKCθ, and, to a lesser extent, PKCδ activities all determine PKCθ Ser685 phosphorylation. In parallel, platelet function studies found that inhibition of PKCθ had only minor effects on platelet secretion and aggregation; however, PKCθ inhibition significantly reduced platelet phosphatidylserine exposure - a marker of procoagulant platelet activity - in response to GPVI and PAR agonists. Together, our results suggest that PKCδ and PKCα/β activities drive the phosphorylation of PKCθ Ser685 to support procoagulant platelet generation in a manner that may be blunted by therapeutically relevant small molecule kinase inhibitors.

Platelet phosphatidylserine is the critical mediator of thrombosis in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a severe immune-mediated prothrombotic disorder caused by antibodies (Ab) reactive to complexes of platelet factor 4 and heparin. Platelets (PLT) and their interaction with different immune cells contribute to prothrombotic conditions in HIT. However, the exact mechanisms and the role of different PLT subpopulations in this prothrombotic environment remain poorly understood. In this study, we observed that HIT patient Ab induce a new PLT population that is characterized by increased P-selectin expression and phosphatidylserine (PS) externalization. Formation of this procoagulant PLT subpopulation was dependent on engagement of PLT Fc-γ-RIIA by HIT Ab and resulted in a significant increase of thrombin generation on the PLT surface. Using an ex vivo thrombosis model and multi-parameter assessment of thrombus formation, we observed that HIT Ab-induced procoagulant PLT propagated formation of large PLT aggregates, leukocyte recruitment and most importantly, fibrin network generation. These prothrombotic conditions were prevented via the upregulation of PLT intracellular cAMP with Iloprost, a clinically approved prostacyclin analogue. Additionally, the functional relevance of P-selectin and PS was dissected. While inhibition of P-selectin did not affect thrombus formation, the specific blockade of PS prevented HIT Ab-mediated thrombin generation and most importantly procoagulant PLT-mediated thrombus formation ex vivo. Taken together, our findings indicate that procoagulant PLT are critical mediators of prothrombotic conditions in HIT. Specific PS targeting could be a promising therapeutic approach to prevent thromboembolic events in HIT patients.

Platelet‑related parameters as potential biomarkers for the prognosis of sepsis.

Early diagnosis and accurate prognosis are key for reducing the fatality rate and medical expenses associated with sepsis. Platelets are involved in the delayed tissue injury that occurs during sepsis. Therefore, the aim of the present study was to investigate the usefulness of platelets and associated parameters as prognostic markers of sepsis. The present study collected patient samples based on The Third International Consensus Definitions for Sepsis and Septic Shock criteria. Platelet-associated parameters were detected by flow cytometry and their correlation with clinical scores and prognoses was analyzed. Considering the association between endothelial cells and platelet activation, levels of plasma tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and angiopoietin-2 (Ang-2) were analyzed by ELISA. The results showed significant differences in platelet P-selectin expression and phosphatidylserine exposure, mitochondrial membrane potential (Mmp)-Index values and plasma levels of TWEAK and Ang-2 between patients and healthy controls (P<0.05). Except for P-selectin and TWEAK levels, all parameters were correlated with clinical scores (acute physiology and chronic health evaluation II and sequential/sepsis-related organ failure assessment). Additionally, platelet Mmp-Index between admission and the end of therapy was only different in non-survivors (P<0.001) and platelet phosphatidylserine exposure was significantly lower in survivors (P=0.006). Therefore, of the parameters tested, the dynamic monitoring of phosphatidylserine exposure, platelet Mmp-Index values and plasma Ang-2 levels had the most potential for the assessment of disease severity and clinical outcomes.

SOD2 in platelets: with age comes responsibility

SOD2 in platelets: with age comes responsibility

Platelet Phosphatidylserine Is the Critical Mediator of Increased Thrombus Formation in Heparin-Induced Thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a severe adverse event that is caused by the interaction of platelets with ultra-large platelet factor 4 (PF4)/heparin IgG-immune complexes. Previous studies have shown that HIT Abs harbour the potential to interact with PLT Fc-gamma-RIIA leading to PLT activation and interplay with different actors of the innate and adaptive immune system. Despite this mechanistic insights, the role of different HIT Ab-induced PLT subpopulations that might contribute to an increased pro-thrombotic potential and severe patient outcome, remains poorly understood. In this study, we hypothesized that PF4/heparin HIT Abs induce a procoagulant PLT response that contribute to an increased pro-thrombotic environment. Using flow cytometry, we found that HIT patient Abs have the capability to induce a procoagulant (P-Selectin/phosphatidylserine double positive) PLT phenotype (p=0.0039) with increased thrombin generation potential (p<0.0001). Most importantly, the perfusion of whole blood samples that were spiked with HIT Ab-induced procoagulant PLTs resulted in significant increase of procoagulant (Annexin-V positive) PLT and fibrin deposition. Interestingly, Fc-gamma-RIIA inhibition via monoclonal antibody IV.3 resulted in a nearly complete abrogation of HIT Ab-induced thrombus formation, indicating that PLT Fc-gamma-RIIA signalling mechanisms are pivotal for Ab-induced pro-thrombotic PLT potential (p=0.0108, Figure 1A). Moreover, also the prostacyclin analogue Iloprost prevented procoagulant PLTs and increased thrombus formation despite the presence of HIT Abs and low dose (0.2 IUs/mL) heparin (p=0.0029). A finding that might be most probably due to increased intracellular concentrations of the second messenger cAMP due to increased PKA activity. To deeper investigate how increased CD62p expression and high PS externalization on the procoagulant PLT surface contribute to the prothrombotic potential of HIT Ab-induced procoagulant PLTs, we performed specific inhibition experiments. Although a marked reduction of neutrophil granulocyte deposition was observed, blocking of PLT CD62p did not reduced HIT Ab-induced increased thrombin generation or thrombus formation. In contrast, procoagulant PLTs that were treated with lactadherin, a PS binding and blocking protein resulted in a significant reduction of thrombin generation (p=0.0040) and thrombus formation compared to the vehicle control (p=0.0005, Figure 1B). Findings of our study indicate that HIT patient Abs harbour the ability to induce procoagulant PLTs via PLT FcγRIIA mediated signalling pathways. The observation that Iloprost reduced the formation of procoagulant PLTs and most importantly inhibits thrombus formation indicates a potential therapeutic use. Most importantly, based on our mechanistic studies we conclude that increased PS on the surface of procoagulant PLTs might be the causing factor for increased pro-thrombotic potential observed in HIT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Application of microfluidic chip technology to study the inhibitory effect of tetramethylpyrazine on platelet aggregation, activation, and phosphatidylserine exposure mediated by pathological high shear rate.

In order to study the antithrombotic effect and mechanism of tetramethylpyrazine (TMA). In this study, we developed a microfluidic chip model that can mimic normal arteries and stenotic arterial vessels, and studied the inhibitory effects of TMA on platelet aggregation, activation (P-selectin, GPIIb/IIIa, monocyte-platelet aggregates) and phosphatidyl serine (PS) exposure. In addition, we also investigated the effect of TMA on ADP and ristocetin-induced platelet aggregation by turbidimetry. The results showed that TMA significantly inhibited the platelet aggregation, activation and PS exposure induced by pathological high shear rate. Under static conditions, TMA can inhibit ADP and ristocetin-induced platelet aggregation. The results indicated that TMA mainly inhibited platelet aggregation, activation and PS exposure by inhibiting the binding of von Willebrand factor (vWF) to the GPIb/IX/V complex, and partially inhibited platelet aggregation through the platelet P2Y 12 -ADP receptor pathway.

Inhibition of platelet aggregation by activation of platelet intermediate conductance Ca2+‐activated potassium channels

BackgroundWithin the vasculature platelets and endothelial cells play crucial roles in hemostasis and thrombosis. Platelets, like endothelial cells, possess intermediate conductance Ca2+‐activated K+ (IKCa) channels and generate nitric oxide (NO). Although NO limits platelet aggregation, the role of IKCa channels in platelet function and NO generation has not yet been explored. ObjectivesWe investigated whether IKCa channel activation inhibits platelet aggregation, and per endothelial cells, enhances platelet NO production. MethodsPlatelets were isolated from human volunteers. Aggregometry, confocal microscopy, and a novel flow chamber model, the Quartz Crystal Microbalance (QCM) were used to assess platelet function. Flow cytometry was used to measure platelet NO production, calcium signaling, membrane potential, integrin αIIb/β3 activation, granule release, and procoagulant platelet formation. ResultsPlatelet IKCa channel activation with SKA‐31 inhibited aggregation in a concentration‐dependent manner, an effect reversed by the selective IKCa channel blocker TRAM‐34. The QCM model along with confocal microscopy demonstrated that SKA‐31 inhibited platelet aggregation under flow conditions. Surprisingly, IKCa activation by SKA‐31 inhibited platelet NO generation, but this could be explained by a concomitant reduction in platelet calcium signaling. IKCa activation by SKA‐31 also inhibited dense and alpha‐granule secretion and integrin αIIb/β3 activation, but maintained platelet phosphatidylserine surface exposure as a measure of procoagulant response. ConclusionsPlatelet IKCa channel activation inhibits aggregation by reducing calcium‐signaling and granule secretion, but not by enhancing platelet NO generation. IKCa channels may be novel targets for the development of antiplatelet drugs that limit atherothrombosis, but not coagulation.

In Vitro Effect of Mitochondria-Targeted Triphenylphosphonium-Based Compounds (Honokiol, Lonidamine, and Atovaquone) on the Platelet Function and Cytotoxic Activity.

Introduction: Obtaining triphenylphosphonium salts derived from anticancer compounds to inhibit mitochondrial metabolism is of major interest due to their pivotal role in reactive oxygen species (ROS) production, calcium homeostasis, apoptosis, and cell proliferation. However, the use of this type of antitumor compound presents a risk of bleeding since the platelet activation is especially dependent on the mitochondrial function. In this study, we evaluated the in vitro effect of three triphenylphosphonium-based compounds, honokiol (HNK), lonidamine (LDN), and atovaquone (ATO), on the platelet function linked to the triphenylphosphonium cation by a lineal 10-carbon alkyl chain and also the decyltriphenylphosphonium salt (decylphos). Methods: Platelets obtained by phlebotomy from healthy donors were exposed in vitro to different concentrations (0.1–10 μM) of the three compounds; cellular viability, exposure of phosphatidylserine, the mitochondrial membrane potential (∆Ψm), intracellular calcium release, and intracellular ROS generation were measured. Platelet activation and aggregation were induced by agonists (adenosine diphosphate, thrombin receptor-activating peptide-6, convulxin, or phorbol-12-myristate-13-acetate) and were evaluated by flow cytometry and light transmission, respectively. Results: The three compounds showed a slight cytotoxic effect from 1 μM, and this was concomitant with a decrease in ∆Ψm and intracellular calcium increase. Only ATO produced a modest but significant increase in intra-platelet ROS. Also, the three compounds increased the exposure to phosphatidylserine in platelets expressed in platelets positive for annexin V. None of the compounds had an inhibitory effect on the aggregation or activation markers of platelets stimulated with three different agonists. Similar results were obtained with decylphos. Conclusion: Triphenylphosphonium derivatives showed slight platelet toxicity below 1 μM, probably associated with their effect on ∆Ψm and exposure to phosphatidylserine, but no significant effect on platelet activation and aggregation, making them an antitumoral alternative with a low risk of bleeding. However, future assays on animal models and human trials are required to evaluate if their effects with a low risk for hemostasis are replicated in vivo.

Maintaining flippase activity in procoagulant platelets is a novel approach to reducing thrombin generation

BackgroundDuring thrombosis, procoagulant platelets expose phosphatidylserine (PS), which enhances local thrombin generation. Reducing platelet PS exposure could be a novel anti‐thrombotic approach. PS is confined to the inner leaflet of the plasma membrane in unstimulated platelets by ATP‐dependent “flippase” activity. Ca2+ ionophores trigger all platelets to expose a high level of PS by activating a scramblase protein and inactivating the flippase. Although R5421 was previously shown to reduce Ca2+ ionophore‐induced PS exposure, its mechanism of action is unknown. ObjectivesTo determine the mechanism by which R5421 reduces platelet PS exposure. MethodsWashed human platelets were stimulated with the Ca2+ ionophore, A23187, to induce procoagulant platelet formation while bypassing proximal receptor signalling. Platelets PS exposure was detected using annexin V or lactadherin in flow cytometry. NBD (7‐nitro‐2‐1,3‐benzoxadiazol‐4‐yl)‐PS was used to assess scramblase and flippase activity. Thrombin generation was monitored using a fluorogenic substrate. Results and conclusionsR5421 reduced the extent of A23187‐stimulated platelet PS exposure, as demonstrated with annexin V or lactadherin binding. R5421 also maintained flippase activity in procoagulant platelets. Although R5421 appeared to inhibit scramblase activity in procoagulant platelets, it did not once the flippase had been inhibited, demonstrating that scramblase activity is not directly inhibited. Furthermore, R5421 inhibited the contribution of A23187‐stimulated platelets to thrombin generation. Together these data demonstrate that R5421 reduces the extent of PS exposure in procoagulant platelets by maintaining flippase activity. Maintaining flippase activity in procoagulant platelets is a novel and effective approach to reducing thrombin generation.

Characterizing the Metabolic Determinants of Thromboinflammation in Myeloproliferative Neoplasms

BACKGROUNDMyeloproliferative neoplasms (MPNs) are a group of clonal hematopoietic disorders characterized by an overproduction of terminally differentiated myeloid elements. Venous and arterial thrombotic events represent the leading causes of morbidity and mortality in patients with JAK2 positive MPNs (essential thrombocythemia [ET] and polycythemia vera [PV]) in chronic phase. Platelet hyperreactivity is a hallmark finding of MPNs but the biological mechanisms remain to be elucidated. We have previously identified that platelets from patients with PV and ET exhibit dysmorphic mitochondria, however, the detailed bioenergetic and metabolomic implications of such alterations in mitochondrial mass and morphology remain to be investigated. Here in, we aim to identify the metabolic determinants that underpin thromboinflammation in patients with JAK2 positive MPNs in chronic phase. METHODSPlatelets from sex and age-matched healthy control subjects and individuals with either JAK2 positive PV or ET were isolated and washed following standard protocols. Platelet activation by flow cytometry was determined at baseline conditions. Platelet activated fibrinogen binding site (αIIbβIII), P-selectin, and phosphatidylserine (PS) surface marker expression (as measured by mean fluorescence intensity [MFI]) was determined with PAC-1 and P-selectin antibodies and lactadherin, respectively. The bioenergetic profile of washed platelets was determined by the 24-well format Seahorse extracellular flux analyzer. Metabolomic profile was developed with the Vanquish UHPLC system coupled online to a Q Exactive mass spectrometer. Statistical analyses were performed using the unpaired 2-tailed Student t test (GraphPad Software v9.1.2). Data expressed as mean plus or minus standard error of the mean (SEM). Significance was determined at p < 0.05. RESULTSPlatelets from patients with MPNs had higher surface marker expression of PS under resting conditions. A trend of higher surface marker expression of αIIbβIII and P-selectin was seen (Figure 1). The platelet bioenergetic profile in individuals with MPN demonstrated decreased basal respiration (p = 0.0008), ATP-linked respiration (p = 0.001), and basal extracellular acidification rate (ECAR) (p = 0.09) (Figure 2). Liquid chromatography-mass spectrometry-based metabolomics revealed elevated pentose phosphate pathway metabolites (pentose phosphates and 6-Phospho-D-gluconate), and diminished adenosine diphosphate (ADP) and adenosine triphosphate (ATP) pools (Figure 3). CONCLUSIONThe findings from this study suggest that platelets from patients with JAK2 positive PV and ET have a procoagulant phenotype given the increased expression of PS, an anionic phospholipid known to facilitate the generation of thrombin. The decreased basal respiration, ATP-linked respiration, basal ECAR (a surrogate marker of glycolysis), and energy pools (ADP and ATP) in platelets from MPN patients suggest a hypometabolic phenotype. Additionally, it appears there is preferential shunting of glucose into the pentose phosphate pathway. Such metabolic switch in conjunction with the hypometabolic state of platelets in MPN, could potentially represent a compensatory mechanism of platelets against oxidative stress.The bioenergetic dysregulation of platelets in MPNs may precipitate a cascade of events that leads to the loss of plasma membrane integrity (increased PS exposure) therefore increasing its procoagulant potential. Higher amounts of platelet PS could underly the elevated incidence of thrombosis observed in patients with JAK2 positive ET and PV. Further studies are underway to identify the inciting factor(s) that lead to the platelet bioenergetic failure and elevated PS exposure seen in patients with MPNs. These investigations will provide significant mechanistic insight for identifying therapies aimed at preventing thrombotic events in patients with MPNs. [Display omitted] DisclosuresNemkov: Omix Thecnologies: Other: Co-founder. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Brothers in arms: platelets and neutrophils in ischemic stroke.

In this review, we will describe how the combined ability of platelets and neutrophils to interact with each other drives ischemic stroke brain injury. Neutrophils are one of the first cells to respond during ischemic stroke. Although animals stroke models have indicated targeting neutrophils improves outcomes, clinical trials have failed to yield successful strategies. Platelets play a critical role in recruiting neutrophils to sites of injury by acting as a bridge to the injured endothelium. After initial platelet adhesion, neutrophils can rapidly bind platelets through P-selectin and glycoprotein Ibα. In addition, recent data implicated platelet phosphatidylserine as a novel key regulator of platelet-neutrophil interactions in the setting of ischemic stroke. Inhibition of procoagulant platelets decreases circulating platelet-neutrophil aggregates and thereby reduces infarct size. Platelet binding alters neutrophil function, which contributes to the injury associated with ischemic stroke. This includes inducing the release of neutrophil extracellular traps, which are neurotoxic and pro-thrombotic, leading to impaired stroke outcomes. Platelet-neutrophil interactions significantly contribute to the pathophysiology of ischemic stroke brain injury. Better understanding the mechanisms behind their formation and the downstream consequences of their interactions will lead to improved therapies for stroke patients.

A Portable System for Processing Donated Whole Blood into High Quality Components without Centrifugation

Introduction: Centrifugation is the cornerstone of blood processing because it has been the only technology capable of separating large volumes of whole blood (WB) into components for storage under optimal conditions in a reasonable amount of time. However, the logistical complications and potential cellular damage associated with centrifugation-based manufacturing of blood products are well documented. In this study, we present a new dual-stage, passive separation system for processing WB into components, which does not require costly or complex machinery, and can be used on-site by minimally-trained personnel immediately following blood donation. This approach consists of two passive blood-processing modules, neither of which requires electricity: (i) a blood-bag compression apparatus designed to maximize the speed and efficiency of passive RBC sedimentation for separating RBCs and platelet-rich plasma (PRP) from WB at normal gravity (i.e. 1×g), and (ii) a flow-through microfluidic concentrator for continuous high-throughput enrichment of platelets from the PRP fraction, to produce platelet concentrate (PC) and platelet-poor plasma (PPP). The ability of this technology to produce high-quality blood components quickly without the use of a centrifuge was demonstrated.Materials and Methods: Freshly-donated units (~500mL) of whole blood (WB) were randomly selected (under an IRB approved protocol) from blood donations by healthy donors who met AABB/FDA eligibility criteria. The WB units (n = 6) were divided into two equal parts, with one half processed by the conventional centrifugation technique (‘PRP method,‘ control arm), and the other half with our new portable blood separation system (test arm). Both separation procedures were performed concurrently, within 4 hours of donation. The initial quality of blood products (RBCs, platelets and plasma) generated by the two approaches were compared using an extensive panel of hematological parameters, including hematocrit, mean corpuscular volume, platelet count, mean platelet volume, white blood cell count, RBC and platelet phosphatidylserine (PS) exposure, platelet P-selectin expression, total free protein, free hemoglobin (Hb), % hemolysis, RBC deformability, pH, K+, Na+, glucose, lactate, ATP, 2,3-diphosphoglyceric acid concentration (2,3-DPG), platelet aggregability (agonists: thrombin receptor agonist peptide, adenosine diphosphate, collagen), factors VIII and XI, soluble CD40-ligand (sCD40L), and thromboxane B2 (TxB2).Results and Discussion: To perform separation using our new portable separation system, 250mL (‘half-unit‘) of WB was placed in a 1L sedimentation bag between two plates of a custom-made compression apparatus at a slight incline (~10°) to allow natural sedimentation of WB into PRP and RBCs at unit gravity. After 150 minutes of sedimentation, the supernatant PRP was expressed from the sedimentation bag through the high-throughput microfluidic platelet concentrator at ~3.2 mL/min to process PRP into PC and PPP. Following PRP expression, the sedimented RBCs were mixed with AS-3 and leukoreduced in the same manner as RBCs produced via centrifugation. The entire separation process for a half-unit of WB was completed within ~3 hours. Comparison of nearly all RBC parameters showed no significant differences between the two separation approaches, although the portable system generated RBC units with a slight, statistically significant improvement in 2,3-DPG (portable system: 15.8 ± 1.2 µmol/g of Hb; centrifugation: 15.3 ± 1.2 µmol/g of Hb; p < 0.05). Importantly, key markers of platelet damage were significantly and meaningfully reduced in PC units generated using the portable, centrifuge-free system: platelet activation (assessed via P-selectin expression) was less than half of that for PC units produced via centrifugation (portable system: 5.2 ± 2.1%; centrifugation: 12.2 ± 3.6%; p < 0.01), and the release of pro-inflammatory mediators (sCD40L, TxB2) was also significantly lower in PC units produced with the portable system than via centrifugation (p < 0.01).Conclusion: This study demonstrated that a simple, portable, passive system for separating donated blood into components could be a viable alternative to centrifugation - particularly for applications in remote or resource-limited settings, or for patients requiring highly functional and undamaged platelets. [Display omitted] DisclosuresGifford:Halcyon Biomedical Incorporated: Employment, Equity Ownership. Shevkoplyas:New Health Sciences, Inc.: Consultancy, Research Funding; Halcyon Biomedical Incorporated: Employment, Equity Ownership, Patents & Royalties: U.S. Patent Appl. 61/929,357, Research Funding.

Avatrombopag, a Novel Thrombopoietin Receptor Agonist, Increases Platelet Counts without Increasing Platelet Activation in Patients with Thrombocytopenia Due to Chronic Liver Disease

Background: The thrombopoietin (TPO) receptor c-mpl is expressed on the surface of megakaryocytes and platelets. In addition to its effect on increasing megakaryocyte growth and maturation, TPO also lowers the threshold for platelet activation in vivo and in vitro . TPO receptor agonists (TPO-RA) may, perhaps to differing extents, also lead to platelet activation. Indeed, in a recent study of patients with chronic liver disease (CLD), the TPO-RA eltrombopag reduced the need for platelet transfusions, but was associated with an increased incidence of thrombosis. Therefore, the goal of this study was to evaluate the effect of avatrombopag, a novel TPO-RA, on in vivo platelet activation and in vitro platelet reactivity in response to agonists in patients with CLD.Methods: CLD patients participating in the Phase 3 E5501-G000-310 and -311 studies (Randomized, Global, Double-blind, Placebo-controlled, Parallel-group Studies to Evaluate the Efficacy and Safety of Once-daily Oral Avatrombopag for the Treatment of Adults with Thrombocytopenia Associated with Liver Disease Prior to an Elective Procedure) could participate in a substudy to evaluate the effects of avatrombopag on platelet function. IRB-approved informed consent was obtained prior to sample collection. Patients (n = 30) were randomized to treatment on Days 1 - 5 with placebo or avatrombopag: 60 mg daily in patients with platelet counts <40 x 109/L, and 40 mg daily in patients with platelet counts 40 to ≤50 x 109/L. Samples were collected at Baseline (Day 1, pre-drug) and after treatment (Day 4 and Day 10). No patient received a platelet transfusion within the 10 days prior to these blood draws.Platelet activation was evaluated by whole blood flow cytometry (which, unlike other methods, is accurate in samples with low platelet counts) with and without in vitro stimulation with adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP). Endpoints included platelet surface activated glycoprotein (GP) IIb-IIIa (as reported by the activation-dependent monoclonal antibody PAC1), platelet surface P-selectin, platelet surface GPIb, platelet phosphatidylserine expression and generation of platelet-derived microparticles. Citrate-anticoagulated whole blood was incubated (15 min, room temperature) with an antibody cocktail containing fluorescein isothiocyanate (FITC)-conjugated PAC1, phycoerythrin (PE)-conjugated anti-P-selectin, and PE-Cy5-conjugated anti-CD42b (GPIb) with or without ADP 0.5 µM or 20 µM or TRAP 1.5 µM or 20 µM. Samples were fixed with 1% formaldehyde, then shipped cold together with the remaining blood sample (for analysis of phosphatidylserine expression and microparticles) to the Center for Platelet Research Studies for analysis. Phosphatidylserine expression on platelets was determined by staining with FITC-conjugated annexin V. Platelet-derived microparticles were identified by light scattering properties and positive staining for CD41 and CD42b. Changes from Baseline for drug-treated (60 mg and 40 mg avatrombopag cohorts combined, n=20) vs. placebo-treated patients (n=10) were compared using a mixed effects model with p<0.05 being considered significant.Results: Avatrombopag, 60 mg and 40 mg treatment, but not placebo treatment, resulted in significant increases in platelet counts at Day 10 (Table). Circulating activated platelets were not increased in avatrombopag-treated patients compared with placebo-treated patients (Table, “no agonist” data), as measured by platelet surface activated GPIIb-IIIa and platelet surface P-selectin. Platelet reactivity, as measured by platelet surface activated GPIIb-IIIa and platelet surface P-selectin after low and high concentrations of ADP and TRAP, was also not increased in avatrombopag-treated patients compared with placebo-treated patients (Table). Similarly, there was no evidence of increased platelet activation in avatrombopag-treated patients compared with placebo-treated patients as assessed by platelet surface GPIb, platelet phosphatidylserine expression and generation of platelet-derived microparticles.Conclusions: In this randomized, double-blind, placebo-controlled, parallel-group study of patients with thrombocytopenia due to CLD, treatment with avatrombopag resulted in increased platelet counts, but did not increase either platelet activation in vivo or platelet reactivity in vitro . [Display omitted] DisclosuresFrelinger:Ionis: Research Funding; Sysmex: Research Funding; Pfizer: Research Funding; Ironwood: Research Funding; GLSynthesis: Research Funding; Baxalta: Research Funding. Michelson:Elsevier: Patents & Royalties; Eisai: Research Funding; GLSynthesis: Research Funding; Instrumentation Laboratory: Consultancy; Baxalta: Research Funding; Ionis: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Sysmex: Research Funding; Pfizer: Research Funding; Ironwood: Research Funding.

Tyrosine Kinase Inhibitor Sunitinib Delays Platelet-Induced Coagulation: Additive Effects of Aspirin.

Sunitinib is a multitarget tyrosine kinase inhibitor (TKI) used for cancer treatment. In platelets, sunitinib affects collagen-induced activation under noncoagulating conditions. We investigated (1) the effects of sunitinib on thrombus formation induced by other TK-dependent receptors, and (2) the effects under coagulating conditions. Cardiovascular disease is a comorbidity in cancer patients, resulting in possible aspirin treatment. Sunitinib and aspirin are associated with increased bleeding risk, and therefore we also investigated (3) the synergistic effects of these compounds on thrombus and fibrin formation. Blood or isolated platelets from healthy volunteers or cancer patients were incubated with sunitinib and/or aspirin or vehicle. Platelet activation was determined by TK phosphorylation, flow cytometry, changes in [Ca2+]i, aggregometry, and whole blood perfusion over multiple surfaces, including collagen with(out) tissue factor (TF) was performed. Sunitinib reduced thrombus formation and phosphatidylserine (PS) exposure under flow on collagen type I and III. Also, sunitinib inhibited glycoprotein VI-induced TK phosphorylation and Ca2+ elevation. Upon TF-triggered coagulation, sunitinib decreased PS exposure and fibrin formation. In blood from cancer patients more pronounced effects of sunitinib were observed in lung and pancreatic as compared to neuroglioblastoma and other cancer types. Compared to sunitinib alone, sunitinib plus aspirin further reduced platelet aggregation, thrombus formation, and PS exposure on collagen under flow with(out) coagulation. Sunitinib suppresses collagen-induced procoagulant activity and delays fibrin formation, which was aggravated by aspirin. Therefore, we urge for awareness of the combined antiplatelet effects of TKIs with aspirin, as this may result in increased risk of bleeding.