Platelet Integrin Activation Research Articles

Reduced platelet activation and thrombus formation in male transgenic model mice of Alzheimer's disease suggests early sex-specific differences in platelet pathophysiology

Alzheimer's disease (AD) is the most common form of dementia and characterized by extracellular amyloid-β (Aβ) plaques, intracellular neurofibrillary tau tangles and neurodegeneration. Over 80 % of AD patients also exhibit cerebral amyloid angiopathy (CAA). CAA is a cerebrovascular disease caused by deposition of Aβ in the walls of cerebral blood vessels leading to vessel damage and impairment of normal blood flow. To date, different studies suggest that platelet function, including activation, adhesion and aggregation, is altered in AD due to vascular Aβ deposition. For example, the transgenic AD model mice APP23 mice that exhibit CAA and parenchymal Aβ plaques, show pre-activated platelets in the blood circulation and increased platelet integrin activation leading to a pro-thrombotic phenotype in these mice late stages of AD. However, it is still an open question whether or not platelets exhibit changes in their activation profile before they are exposed to vascular Aβ deposits. Therefore, the present study examined platelets from middle-aged transgenic APP23 mice at the age of 8–10 months. At this age, APP23 mice show amyloid plaques in the brain parenchyma but not in the vasculature. Our analyses show that these APP23 mice have unaltered platelet numbers and size, and unaltered surface expression of glycoproteins. However, the number of dense granules in transgenic platelets was increased while the release was unaltered. Male, but not female APP23 mice, exhibited reduced platelet activation after stimulation of the thrombin receptor PAR4 and decreased thrombus stability on collagen under flow conditions ex vivo compared to control mice. In an arterial thrombosis model in vivo, male APP23 mice showed attenuated occlusion of the injured artery compared to controls. These findings provide clear evidence for early changes in platelet activation and thrombus formation in male mice before development of overt CAA. Furthermore, reduced platelet activation and thrombus formation suggest sex-specific differences in platelet physiology in AD that has to be considered in future studies of platelets and their role in AD.

Quantitative Label-Free Mass Spectrometry Identifies Molecular Content and Signaling Differences between Neonatal and Adult Platelets

Observational and randomized studies have associated platelet transfusions with increased morbidity and mortality in preterm neonates. Clinical practice changes have been hindered by a limited understanding of why adult donor-derived platelets might be inappropriate or harmful to infants. Neonatal and adult platelets differ in reactivity and function, but we lack an understanding of underlying molecular differences. As proteins and kinase-driven phosphorylation signaling dictate platelet biology, we aimed to characterize altered protein and phosphoprotein content between neonatal and adult platelets to help explain differences in platelet reactivity and function. We isolated resting platelets from full term cord blood (n=9) and adult peripheral blood (n=7) and used mass spectrometry-based platforms to ascertain protein and phosphoprotein content. We identified 4745 high-confidence proteins in adult and neonatal platelets, including 331 differentially abundant proteins (≥1.5-fold change, p<0.05). Neonatal platelets had increased metabolic and ribosomal proteins, consistent with transcriptional data. Adult platelets were enriched for inflammatory proteins, including complement components, consistent with pro-inflammatory function. We also identified 2115 phospho-proteins (17852 unique phosphopeptides) across all samples. These impacted actin cytoskeletal biology, cell adhesion, and GTPase signaling that regulate platelet functions. Kinase enrichment analysis identified FYN, SRC, ABL1, and AKT1 as kinases most responsible for platelet phosphorylation activities. Granule trafficking and degranulation proteins were among 1183 differentially abundant phosphoproteins in neonatal vs adult samples (p<0.05). For example, 31 different Reticulon 1 (RTN1) phosphopeptides were enriched in adult platelets (p<0.05). RTN1 regulates membrane trafficking and participates in SNARE-mediated exocytosis. Increased RTN1-directed membrane trafficking may promote increased activation and degranulation in adult vs neonatal platelets. Indeed, SNARE complex protein deficiency was previously suggested to mediate hyporeactive degranulation in neonatal platelets. We detected 2 phosphopeptides in adult samples that were absent from any neonatal sample. We reasoned that these could represent developmental stage-specific differences that exist only in adult platelets. One of the adult-specific phosphopeptides was in Rap1GAP2 (phospho-S588). Rap1GAP2 is a highly phosphorylated GTPase-activating protein that inhibits Rap1, a small guanine-nucleotide-binding protein that facilitates integrin activation in platelets. Rap1GAP2 binds synaptotagmin-like protein 1 (Slp1) to regulate dense granule secretion. These findings may link altered developmentally regulated Rap1GAP2 activities with platelet degranulation. A Platelet Cell Adhesion Molecule (PECAM) phosphopeptide (phospho-S726) was also found exclusively in adult platelets. PECAM facilitates inflammation and leukocyte transendothelial migration. PECAM phosphorylation in adult platelets may reflect an enhanced ability to participate in these processes. These findings reveal key differences in protein content and signaling that underlie differential neonatal vs adult platelet reactivity and function, including pro-inflammatory activities and enhanced activation and/or degranulation propensity in adult platelets. RTN1-mediated SNARE complex function and Rap1 signaling activities represent novel and potentially modifiable targets to manipulate platelet activation and/or degranulation. Our results also help provide a biological rationale for the higher morbidity and mortality observed in preterm infants transfused with adult platelets, including the inadvertent introduction of serum complement and other pro-inflammatory molecules within transfusions of donated adult platelets.

Ayurvedic preparations of Raudra Rasa inhibit agonist-mediated platelet activation and restrict thrombogenicity without affecting cell viability.

Ayurveda is considered to be one of the most ancient forms of medicine still practiced. The Ayurvedic preparation Raudra Rasa and its derivatives have been widely employed against cancer since the 12th century, but the effect of these traditional formulations on platelet function and signaling has not previously been examined. Here we demonstrate that Raudra Rasa and its derivatives significantly reduce thrombin-induced integrin activation and granule secretion in platelets, as observed by reduced PAC-1 binding and P-selectin externalization, respectively. These formulations also inhibited thrombin-stimulated phosphatidylserine exposure, mitochondrial reactive oxygen species generation, and mitochondrial transmembrane potential in platelets. Consistent with the above, Raudra Rasa significantly reduced thrombin-induced tyrosine phosphorylation of the platelet proteins, as well as phosphorylation of the enzymes AKT and GSK-3β. In summary, Raudra Rasa inhibits agonist-mediated platelet activation without affecting cell viability, suggesting it may have therapeutic potential as an anti-platelet/anti-thrombotic agent.

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.

Platelet functional abnormalities in pediatric patients with kaposiform hemangioendothelioma/Kasabach-Merritt phenomenon.

Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy that is commonly associated with a life-threatening thrombocytopenic condition, Kasabach-Merritt phenomenon (KMP). Platelet CLEC-2, tumor podoplanin interaction is considered the key mechanism of platelet clearance in these patients. Here, we aimed to assess platelet functionality in such patients. Three groups of 6 to 9 children were enrolled: group A with KHE/KMP without hematologic response (HR) to therapy; group B with KHE/KMP with HR; and group C with healthy children. Platelet functionality was assessed by continuous and end point flow cytometry, low-angle light scattering analysis (LaSca), fluorescent microscopy of blood smears, and exvivo thrombi formation. Platelet integrin activation in response to a combination of CRP (GPVI agonist) and TRAP-6 (PAR1 agonist), as well as calcium mobilization and integrin activation in response to CRP or rhodocytin (CLEC-2 agonist) alone, were significantly diminished in groups A and B. At the same time, platelet responses to ADP with or without TRAP-6 were unaltered. Thrombi formation from collagen in parallel plate flow chambers was also noticeably decreased in groups A and B. In silico analysis of these results predicted diminished amounts of CLEC-2 on the platelet surface of patients, which was further confirmed by immunofluorescence microscopy and flow cytometry. In addition, we also noted a decrease in GPVI levels on platelets from group A. In KHE/KMP, platelet responses induced by CLEC-2 or GPVI activation are impaired because of the diminished number of receptors on the platelet surface. This impairment correlates with the severity of the disease and resolves as the patient recovers.

X-rays Stimulate Granular Secretions and Activate Protein Kinase C Signaling in Human Platelets.

X-rays can induce morphological as well as functional changes in cells. Platelets are anuclear cellular fragments originating from megakaryocytes and are the major regulators in hemostasis and thrombosis. Platelet products are irradiated to avoid medical complications associated with platelet transfusion. So far, gamma, UV, and laser radiation have been used for this purpose. However, scientists are divided about the effects of radiation on platelet quality. The present study was designed to explore the possible effects of X-rays in washed human platelets and understand the molecular mechanism behind them. In the present study, we exposed washed human platelets to 10 or 30 Gy X-rays at 0.25 Gy/min. Flow cytometry, aggregometry, and western blot were performed to investigate the effect of X-rays on platelet degranulation, integrin activation, platelet aggregation, and apoptosis. It was found that X-rays immediately induced granular secretions with no effect on GP IIb/IIIa activation. Not surprisingly, due to granule secretions in irradiated platelets, platelet aggregation was significantly reduced. In contrast to granular secretions and platelet aggregation, X-rays induced mitochondrial transmembrane potential depolarization in a time-dependent manner to induce apoptosis and activated protein kinase C (PKC) signaling. This study revealed and explained the molecular mechanism activated by X-rays in washed human platelets. Here we also introduced Gö 6983, a PKC inhibitor, as an agent that counteracts X-ray-induced changes and maintains the integrity of platelets.

Uncoupling of platelet granule release and integrin activation suggests GPIIb/IIIa as a therapeutic target in COVID-19.

Thromboembolic events are frequent and life-threating complications of COVID-19, but are also observed in patients with sepsis. Disseminated thrombosis can occur despite anticoagulation, suggesting that platelets play a direct, but yet incompletely understood role. Several studies demonstrated altered platelet function in COVID-19 with in part controversial findings, while underlying disease-specific mechanisms remain ill-defined. We performed a comprehensive cohort study with 111 patients, comprising 37 with COVID-19, 46 with sepsis, and 28 with infection, compared to controls. Platelet phenotype and function were assessed under static and flow conditions, revealing unexpected disease-specific differences. From hospital admission on, platelets in COVID-19 failed to activate integrin GPIIb/IIIa in response to multiple agonists. Dense granule release was markedly impaired due to virtually missing granules, also demonstrated by whole mount electron microscopy. In contrast, alpha-granule marker CD62P exposure was only mildly affected, revealing a subpopulation of PAC-1-/CD62P+ platelets, independently confirmed by automated clustering. This uncoupling of alpha-granule release was not observed in sepsis patients, despite a similar disease severity. We found overall unaltered thrombus formation in COVID-19 and sepsis samples under venous shear rates, which was dependent on the presence of tissue factor. Unexpectedly, under arterial shear rates thrombus formation was virtually abrogated in sepsis, while we detected overall normal-sized and stable thrombi in blood from COVID-19 patients. These thrombi were susceptible to subthreshold levels of GPIIb/IIIa blockers eptifibatide or tirofiban that had only a minor effect in control blood. We provide evidence that low dose GPIIb/IIIa blockade could be a therapeutic approach in COVID-19.

Effect of antiplatelet agents and tyrosine kinase inhibitors on oxLDL-mediated procoagulant platelet activity.

Low-density lipoprotein (LDL) contributes to atherogenesis and cardiovascular disease through interactions with peripheral blood cells, especially platelets. However, mechanisms by which LDL affects platelet activation and atherothrombosis, and how to best therapeutically target and safely prevent such responses remain unclear. Here, we investigate how oxidized low-density lipoprotein (oxLDL) enhances glycoprotein VI (GPVI)-mediated platelet hemostatic and procoagulant responses, and how traditional and emerging antiplatelet therapies affect oxLDL-enhanced platelet procoagulant activity exvivo. Human platelets were treated with oxLDL and the GPVI-specific agonist, crosslinked collagen-related peptide, and assayed for hemostatic and procoagulant responses in the presence of inhibitors of purinergic receptors (P2YR), cyclooxygenase (COX), and tyrosine kinases. Exvivo, oxLDL enhanced GPVI-mediated platelet dense granule secretion, α-granule secretion, integrin activation, thromboxane generation and aggregation, as well as procoagulant phosphatidylserine exposure and fibrin generation. Studies of washed human platelets, as well as platelets from mouse and nonhuman primate models of hyperlipidemia, further determined that P2YR antagonists (eg, ticagrelor) and Bruton tyrosine kinase inhibitors (eg, ibrutinib) reduced oxLDL-mediated platelet responses and procoagulant activity, whereas COX inhibitors (eg, aspirin) had no significant effect. Together, our results demonstrate that oxLDL enhances GPVI-mediated platelet procoagulant activity in a manner that may be more effectively reduced by P2YR antagonists and tyrosine kinase inhibitors compared with COX inhibitors.

Actin Bundling Protein L-Plastin Regulates Megakaryocyte Membrane Rigidity and Platelet Spreading

An adequate number of normally functioning platelets is required for normal hemostasis, while thrombocytopenia and platelet dysfunction are common causes of pathologic bleeding. Platelets are released into the blood from proplatelets (PPs) that extend from megakaryocyte (MK) intracellular invaginated membrane system (IMS) that is continuous with the plasma membrane. Tubulin provides the motor for PP extension, and actin reorganization is believed to be critical for MK migration and PP branching. However, the role for actin in PP formation (PPF) is less well understood. The initiation of PPF requires podosomes - actin-based structures with an F-actin core surrounded by a ring of adhesion molecules (e.g., integrins) and many actin-binding proteins. We have been intrigued by how podosomes form and how the IMS is able to penetrate the dense cortical actin cytoskeleton to form PPs. We recently showed that human MKs and platelets express a novel actin bundling protein, L-plastin. L-plastin levels were inversely correlated with platelet number, and its expression was down-regulated during MK maturation. L-plastin knockdown in cultured human CD34+ derived MKs caused increased PP, IMS, and podosome formation. These data indicate L-plastin is a negative regulator of MK platelet production. To assess the in vivo effects of L-plastin, we have investigated platelet production and function in vivo using L-plastin knockout (KO) mice (Lcp1-/-). Compared to wildtype (WT) littermate controls, Lcp1 KO mice showed both significantly increased platelet counts and increased PPF from cultured murine bone marrow MKs. Elegant work in both HUVECs and epithelial cells has shown high membrane proximal F-actin (MPA) prohibits membrane protrusions, and lowering MPA density is required to sensitize areas for membrane protrusion (Bisaria, Science 2020). We hypothesize that normal MK levels of L-plastin inhibits PPF in less mature MKs by F-actin bundling, which raises the MPA. Conversely, lower L-plastin levels result in lower MPA and enabling PP protrusion. Enhanced L-plastin-mediated F-actin bundling is expected to increase membrane stiffness, so we measured MK membrane stiffness at single cell level using a state-of-the-art Biomembrane Force Probe (BFP). MKs from KO mice showed 68% reduction in membrane stiffness as compared to WT controls (p<0.0001) (Fig 1). Ultrastructure of MKs also revealed increased IMS in Lcp1-/- MKs. Preliminary data suggests that L-plastin deficiency also associated with increased MK podosome formation in vitro, which is consistent with our findings in human CD34+ derived MKs. Although L-plastin levels decrease during terminal MK differentiation, they persist at moderate levels in platelets. Platelet integrin activation, granule release and clot retraction also depend on actin reorganization, but L-plastin function in platelets has not been studied. We observed no difference between WT and Lcp1 KO platelets for measures of inside-out integrin signaling (activation-induced JON/A binding or P-selectin expression). However, when plated on fibrinogen and stimulated with thrombin, platelets from KO mice showed significantly increased spreading compared to WT mice (Fig 2). A similar trend was observed for platelet clot retraction. Taken together these results show that L-plastin regulates MK membrane stiffness, which likely inhibits PPF. L-plastin also appears to participate in platelet outside-in, but not inside-out, integrin signaling. On-going studies are determining whether integrin beta3 is the molecular link between these two findings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Asebogenin suppresses thrombus formation via inhibition of Syk phosphorylation.

Thrombosis is a major cause of morbidity and mortality worldwide. Platelet activation by exposed collagen through glycoprotein VI (GPVI) and formation of neutrophil extracellular traps (NETs) are critical pathogenic factors for arterial and venous thrombosis. Both events are regulated by spleen tyrosine kinase (Syk)-mediated signalling events. Asebogenin is a dihydrochalcone whose pharmacological effects remain largely unknown. This study aims to investigate the antithrombotic effects of asebogenin and the underlying molecular mechanisms. Platelet aggregation was assessed using an aggregometer. Platelet P-selectin exposure, integrin activation and calcium mobilization were determined by flow cytometry. NETs formation was assessed by SYTOX Green staining and immunohistochemistry. Quantitative phosphoproteomics, microscale thermophoresis, in vitro kinase assay and molecular docking combined with dynamics simulation were performed to characterize the targets of asebogenin. The in vivo effects of asebogenin on arterial thrombosis were investigated using FeCl3 -induced and laser-induced injury models, whereas those of venous thrombosis were induced by stenosis of the inferior vena cava. Asebogenin inhibited a series of GPVI-induced platelet responses and suppressed NETs formation induced by proinflammatory stimuli. Mechanistically, asebogenin directly interfered with the phosphorylation of Syk at Tyr525/526, which is important for its activation. Further, asebogenin suppressed arterial thrombosis demonstrated by decreased platelet accumulation and fibrin generation and attenuated venous thrombosis determined by reduced neutrophil accumulation and NETs formation, without increasing bleeding risk. Asebogenin exhibits potent antithrombotic effects by targeting Syk and is a potential lead compound for the development of efficient and safe antithrombotic agents.

The Connection Between Rap1 and Talin1 in the Activation of Integrins in Blood Cells.

Integrins regulate the adhesion and migration of blood cells to ensure the proper positioning of these cells in the environment. Integrins detect physical and chemical stimuli in the extracellular matrix and regulate signaling pathways in blood cells that mediate their functions. Integrins are usually in a resting state in blood cells until agonist stimulation results in a high-affinity conformation (“integrin activation”), which is central to integrins’ contribution to blood cells’ trafficking and functions. In this review, we summarize the mechanisms of integrin activation in blood cells with a focus on recent advances understanding of mechanisms whereby Rap1 regulates talin1-integrin interaction to trigger integrin activation in lymphocytes, platelets, and neutrophils.

Calcium-Induced Conformational Changes in EF hands and Autoinhibitory Linker Region are Critical for Catalytic Activity of CalDAG-GEFI Towards the Small GTPase Rap1

IntroductionSignaling by the small GTPase Rap1B is critical for the rapid inside-out activation of platelet integrins, a prerequisite for platelet aggregation and hemostatic plug formation at sites of vascular injury. The guanine nucleotide exchange factor (GEF), CalDAG-GEFI (CDGI), is the predominant Rap-GEF in platelets. Cell biological studies demonstrated that agonist receptor-mediated increases in cytosolic calcium are necessary for CDGI activity, but it is currently not known if and how calcium affects CDGI activity.MethodsRecombinant CDGI (1-551) and Rap1B (1-181) proteins were purified to test nucleotide exchange activity in a fluorescence-based functional assay. Hydrogen-deuterium exchange mass spectrometry was utilized to measure dynamic differences between the active and inactive forms of CDGI.ResultsNucleotide exchange activity of CDGI was strongly diminished by substitution of calcium-binding residues E450A or E479A within EF hand 1 and EF hand 2, respectively. Addition of exogenous calcium restored catalytic activity in CDGI (E479A) but not CDGI (E450A) or CDGI (E450A+E479A). Calcium-bound active CDGI exhibited faster deuterium exchange compared to the calcium-free inactive form, in particular for residues within the EF hands and a potential autoinhibitory linker region connecting the EF hand domains and the Cdc25 catalytic domain.Consistent with regulation by an autoinhibitory linker, catalytic activity was fully restored in CDGI (E450A+E479A) by a gain-of-function substitution in valine 406.ConclusionsThese findings propose a model where calcium-induced conformational changes within the EF hands and autoinhibitory linker region are necessary for Rap1B to gain access to the catalytic binding pocket in the Cdc25 domain of CDGI. These findings will be important in our understanding of how mutations in patients impair CDGI function and in efforts to design inhibitors of CDGI signaling. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Aging-Associated Inflammation Due to Elevated Levels of TNF-α Increases Activation of the Integrin αIIbβ3 and the Procoagulant Potential of Platelets

Background: Aging is an independent risk factor for thrombosis and the molecular mechanisms underpinning this higher thrombosis risk in aging remains largely unknown. Chronic inflammation due to elevated levels of TNF-α contributes to the pathobiology of aging. Nevertheless, whether increased TNF-α levels during aging alter platelet function to promote thrombosis remains unexamined.Aims: To investigate the mechanism(s) by which aging-associated inflammation by TNF-α may promote platelet hyperreactivity in mice and humans.Methods: We analyzed megakaryocyte maturation, platelet number and activation indices by FACS, light transmission aggregometry (LTA) and a microfluidic assay in the following murine models of aging: 1) aged C57BL/6J mice (>18 months old, where circulating TNF-α levels are high); 2) young mice (~8 weeks old) with constitutive overexpression of TNF-α (TNFΔARE) ; and 3) young C57BL/6J mice injected in vivo with TNF-α or IL1b (for up to 20 days). In select experiments, we also neutralized TNF-α in vivo in aged mice. To determine if murine findings were recapitulated in humans, we examined platelet integrin activation in younger and older adults. Finally, to establish whether inflammation alters the molecular signature of megakaryocytes (MKs-the platelet parent cell) during aging, we performed next-generation RNA-sequencing (both bulk and single-cell) in bone marrow MKs from young and aged mice.Results: Aged mice exhibit thrombocytosis and accelerated megakaryocyte maturation as determined by ploidy analysis. In addition, platelets from aged mice, TNFΔARE mice, and from young mice injected with TNF-α exposed significantly higher amounts of membrane phosphatidylserine and activated integrin αIIbβ3 faster after stimulation with thrombin and convulxin. Treating mice in vivo with IL1b did not recapitulate these effects. Moreover, platelets from old mice adhered faster to collagen and formed larger clots under flow when assessed in a microfluidic assay using reconstituted whole blood (pooled plasma and erythrocytes in addition to platelets from young or old mice). Similarly, integrin αIIbβ3 activation was significantly greater in activated platelets from older adults (mean age 78.0 ± 8.4) compared to younger adults (mean age 35.0 ± 10.1). Interestingly, platelets from old mice injected with an anti-TNF-α antibody had a markedly decreased activation profile. Bulk RNA-seq analysis of native megakaryocytes from aged mice and from young mice treated with TNF-⍺ both had a robust TNF-⍺ transcriptional signature. Analyses of single cell RNA-seq of bone marrow MKs demonstrated dysregulation of the mTOR and mitochondrial dependent pathways in aged mice. These transcriptional findings indicate there is MK reprogramming in aging, perhaps due to chronic inflammation, and serve to identify potential candidates regulating aging-associated platelet hyperactivity.Conclusions: TNF-associated inflammation of aging is an important factor in the development of platelet hyperreactivity during aging and may contribute to the increased incidence of age-associated thrombosis. DisclosuresNo relevant conflicts of interest to declare.

CD36 Promotes Platelet Phosphatidylserine Exposure through MAP Kinase ERK5

Dyslipidemic conditions can promote atherosclerotic plaque instability and rupture, leading to thrombosis that can cause life threatening complications. Thrombosis in this setting is augmented by circulating oxidized lipids on oxidized low density lipoprotein particles (oxLDL) that are recognized by key scavenger receptors. CD36 is a major platelet scavenger receptor that recognizes oxLDL and consequently promotes a prothrombotic platelet phenotype through multiple signaling pathways. We previously reported that recognition of oxLDL by CD36 generates specific reactive oxygen species through Src family kinases and NADPH oxidase. These species modulate redox sensitive signaling, such as the activation of the oxidant sensor MAP kinase ERK5, leading to platelet integrin activation, aggregation, and thrombus formation. Although ERK5 was a key effector for CD36 to promote thrombosis, the downstream signaling mechanisms are unclear. We hypothesize that CD36 promotes a procoagulant phenotype that requires ERK5. Platelets isolated and washed from healthy human donors were stimulated with buffer, native low-density lipoprotein (LDL), or oxLDL, and were stained with fluorophore-conjugated Annexin V or Lactadherin to detect exposure of procoagulant phosphatidylserine (PS). OxLDL-stimulated platelets showed dose-dependent 13.4 ± 2.8% and 18.5 ± 3.5% increases in Lactadherin binding at 50 and 100μg/mL oxLDL compared to control LDL (p=0.026; p<0.01), and a rapid binding within 5 min (11.0±0.35%, p<0.001). Furthermore, pre-treatment with oxLDL followed by addition of convulxin, a snake venom that activates platelets through the collagen signaling receptor GPVI, showed a 58.1 ± 4.62% increase in Lactadherin binding compared to oxLDL and convulxin alone (p<0.0001). Addition of ADP or thrombin, agonists that activates specific G-protein coupled receptors, following pre-treatment with oxLDL did not enhance Lactadherin binding. Platelets treated with FA6, a monoclonal CD36 antibody which prevents oxLDL binding, or BIX02188, an ERK5 inhibitor, showed an 80-90% decrease in Annexin V binding (p=0.039; p=0.02) after oxLDL exposure compared to a control non-immunizing IgG or DMSO. To link known pathways for PS exposure with oxLDL-induced PS exposure, platelets were stimulated with oxLDL followed by treatment with Z-VAD-FMK, a “pan” caspase inhibitor, or cyclosporin A, a cyclophilin D/mitochondrial permeability transition pore inhibitor. Z-VAD-FMK treated platelets showed 68% decrease in Lactadherin binding compared to control DMSO (p=0.0196), whereas cyclosporin A did not inhibit Lactadherin binding. Relevance of CD36/ERK5-mediated PS exposure was demonstrated using an in vivo arterial thrombosis model that relies on autologous transplantation of the epigastric artery, which has an adventitial surface that serves as a collagen-rich thrombogenic substrate for thrombosis, into the carotid artery. This model of thrombosis was chosen due to the potential impact of free radical generation in the standard ferric chloride or Rose Bengal-mediated arterial thrombosis models. Bone marrows from platelet ERK5-deficient mice (ERK5 flox/Pf4-cre+) or control mice (ERK5 flox/flox) were transplanted into irradiated atherogenic apoE null mice fed a high fat or control diet. Fibrin deposition, detected by a fluorophore-conjugated antibody to fibrin, was enhanced in hyperlipidemic conditions for mice with platelet ERK5. High fat diet-fed chimeric mice without ERK5 or animals on control diet do not show enhanced fibrin deposition. In conclusion, oxLDL promoted PS exposure through a CD36-Src family kinases-ERK5-caspase-dependent mechanism. PS exposure by this pathway potentially enhances fibrin deposition in an atherogenic arterial in vivo thrombosis model, which suggests a role for CD36 and ERK5 in thrombosis and coagulation in dyslipidemic conditions. DisclosuresNo relevant conflicts of interest to declare.

Janus kinase inhibitors ruxolitinib and baricitinib impair glycoprotein-VI mediated platelet function

Several Janus kinase (JAK) inhibitors (jakinibs) have recently been approved to treat inflammatory, autoimmune and hematological conditions. Despite emerging roles for JAKs and downstream signal transducer and activator of transcription (STAT) proteins in platelets, it remains unknown whether jakinibs affect platelet function. Here, we profile platelet biochemical and physiological responses in vitro in the presence of five different clinically relevant jakinibs, including ruxolitinib, upadacitinib, oclacitinib, baricitinib and tofacitinib. Flow cytometry, microscopy and other assays found that potent JAK1/2 inhibitors baricitinib and ruxolitinib reduced platelet adhesion to collagen, as well as platelet aggregation, secretion and integrin αIIbβ3 activation in response to the glycoprotein VI (GPVI) agonist collagen-related peptide (CRP-XL). Western blot analysis demonstrated that jakinibs reduced Akt phosphorylation and activation following GPVI activation, where ruxolitinib and baricitinib prevented DAPP1 phosphorylation. In contrast, jakinibs had no effects on platelet responses to thrombin. Inhibitors of GPVI and JAK signaling also abrogated platelet STAT5 phosphorylation following CRP-XL stimulation. Additional pharmacologic experiments supported roles for STAT5 in platelet secretion, integrin activation and cytoskeletal responses. Together, our results demonstrate that ruxolitinib and baricitinib have inhibitory effects on platelet function in vitro and support roles for JAK/STAT5 pathways in GPVI/ITAM mediated platelet function.

Loss of the exocyst complex component EXOC3 promotes hemostasis and accelerates arterial thrombosis

AbstractThe exocyst is an octameric complex comprising 8 distinct protein subunits, exocyst complex components (EXOC) 1 to 8. It has an established role in tethering secretory vesicles to the plasma membrane, but its relevance to platelet granule secretion and function remains to be determined. Here, EXOC3 conditional knockout (KO) mice in the megakaryocyte/platelet lineage were generated to assess exocyst function in platelets. Significant defects in platelet aggregation, integrin activation, α-granule (P-selectin and platelet factor 4), dense granule, and lysosomal granule secretion were detected in EXOC3 KO platelets after treatment with a glycoprotein VI (GPVI)-selective agonist, collagen-related peptide (CRP). Except for P-selectin exposure, these defects were completely recovered by maximal CRP concentrations. GPVI surface levels were also significantly decreased by 14.5% in KO platelets, whereas defects in proximal GPVI signaling responses, Syk and LAT phosphorylation, and calcium mobilization were also detected, implying an indirect mechanism for these recoverable defects due to decreased surface GPVI. Paradoxically, dense granule secretion, integrin activation, and changes in surface expression of integrin αIIb (CD41) were significantly increased in KO platelets after protease-activated receptor 4 activation, but calcium responses were unaltered. Elevated integrin activation responses were completely suppressed with a P2Y12 receptor antagonist, suggesting enhanced dense granule secretion of adenosine 5′-diphosphate as a critical mediator of these responses. Finally, arterial thrombosis was significantly accelerated in KO mice, which also displayed improved hemostasis determined by reduced tail bleeding times. These findings reveal a regulatory role for the exocyst in controlling critical aspects of platelet function pertinent to thrombosis and hemostasis.

Platelet Activation through GPVI Receptor: Variability of the Response

Platelets are nuclear-free cell fragments responsible for preventing blood loss in case of the vascular wall injury. After a contact with collagen exposed when the vessel is damaged, platelets become activated through the receptor glycoprotein GPVI. This leads to platelet shape change, degranulation, aggregation, and procoagulant responses. The aim of this study was to observe changes in the concentration of Ca2+ in the cytosol and functional responses of platelets upon stimulation through the GPVI receptor. The study involved healthy adult volunteers and house mice Mus musculus of the C57Bl6 line (wild type). Calcium signaling was monitored by Fura-Red fluorophore fluorescence using BD FACS Canto II flow cytometer. Functional responses were observed on a flow cytometer by binding of human fibrinogen conjugated to a fluorescent label or by Biola optical aggregometry. When tested on a cytometer, platelets were activated by collagen-related peptide CRP and when tested by aggregometry, platelets were activated using collagen. As a result of the study, the following phenomenon was revealed: despite a significant variability in the human platelet cytosolic Ca2+ levels in response to stimulation, the variability in activation of platelet integrins, shape change, and the aggregation response was significantly lower. No variability in the platelet cytosolic Ca2+ levels in response to stimulation was observed in mice. The observed variability of the calcium response of platelets could be caused by differences in the GPVI expression or polymorphisms of the GPVI receptor gene in the studied donors. Similarities in the functional responses of platelets with different signaling suggest a variable contribution of the phosphoinositide branch of intracellular signaling in platelets in response to activation of GPVI.

Novel Splice-Site Mutation in RASGRP2 Leading to Caldag-GEFI Deficiency

Introduction: CalDAG-GEFI (Calcium and diacyglycerol-regulated guanine exchange factor I) deficiency or platelet-type bleeding disorder-18 (BDPLT18) is a relatively new, rare hemorrhagic disorder with autosomal recessive inheritance (OMIM #615888). CalDAG-GEFI activates Rap1 and is essential for αIIbβ3 (GP IIb/IIIa) integrin activation in platelets, a key step in platelet aggregation. The deficiency is due to mutations of the encoding gene, RASGRP2 (HGNC ID 9879), which is located on chromosome 11q13.1. To date, 23 pathogenic variants in the RASGRP2 gene are listed in the Human Gene Mutation Database (HGMD®). Two other RASGRP2 splice-site mutations (c.74-1G&amp;gt;C and 372-3C&amp;gt;G) are described in the literature in patients suffering from CalDAG-GEFI loss of function. Patients and Methods: We investigated a 3 yr-old index patient who was referred to our outpatient clinic because of Glanzmann thrombasthenia (GT)-like symptoms and GT-typical pattern in platelet aggregometry analyses. The boy presented with hematomas and recurrent infections, postnatally, he had shown petechiae. After a tongue bite at the age of 1 yr, he needed tranexamic acid to stop bleeding. So far, no surgery has been performed. His family members including mother, father, elder sisters and brother had no history of bleeding. Informed consent for our study of biochemical and molecular genetic characterization for inherited platelet disorders using NGS was given by all family members. Platelet functions were assessed by light transmission aggregometry using citrated PRP. Membrane glycoprotein quantification (CD41, CD42a and CD42b), fibrinogen binding, von Willebrand factor (VWF) binding and the investigation of alpha- and delta- granule secretion were performed by flow cytometry. For the index patient and his parents we performed NGS panel sequencing (Nextera Rapid Custom Enrichment, Illumina). 80 genes known to be associated with platelet disorders were screened. The variants detected were classified based on the 2015 American College of Medical Genetics (ACMG) guidelines. ClinVar, Human Gene Mutation Database, and in silico pathogenicity prediction were used to assess the detected variants. The segregation of the putative mutation was confirmed by Sanger sequencing of three unaffected siblings. Results: The index patient presented with normal values for platelet count, aPTT, INR, FXIII and VWF parameters. ADP (4, 10 and 20µM/L), epinephrine (8, 16 and 40µM/L) and low concentration collagen (2µM/mL) and TRAP6 (5µM/mL) induced aggregation was markedly reduced. However, normal values after stimulation with 10µM/mL collagen and TRAP6 respectively were achieved. Agglutination after stimulation with ristocetin (1,2mg/mL) was normal. Flow cytometry revealed severely decreased fibrinogen binding after stimulation with ADP (0.25-5.0 μmol/l) compared to control platelets. For CD41 (GP IIb/IIIa-complex), CD42a/b (GP Ib/IX-complex), ristocetin-induced VWF-binding, CD62- and CD63-expression, normal values were measured. Molecular genetic analysis revealed a novel homozygous splice-site mutation of the RASGRP2 gene in the index patient. The mutation was heterozygous in the non- consanguineous parents (Tab.1). Conclusion: We have identified a novel recessive mutation of the RASGRP2 gene in a boy affected by a Glanzmann thrombasthenia-like disorder. Platelet aggregometry analysis showed typical failure of aggregation with low dose collagen and TRAP6, whereas aggregation after stimulation with higher concentration reached normal values. Flow cytometric measured severely reduced fibrinogen binding indicated a functional defect of the αIIbβ3 fibrinogen receptor with normal expression. Comprehensive platelet function analysis and panel sequencing allowed early and precise diagnosis and therapy of this ultra-rare platelet signaling defect. Disclosures Zieger: Biotest: Research Funding; DFG: Research Funding; CSL Behring: Research Funding; Takeda: Research Funding.

Differential Use of Rap1 Effectors for Integrin Activation in Platelets and Lymphocytes

Rap1 is a major convergence point of the platelet and lymphocyte signaling pathways that result in talin1 binding to the integrin β-cytoplasmic domain and consequent integrin activation, effective hemostasis and immune functions. The nature of the connection between Rap1 and talin1 in integrin activation is an important remaining gap in our understanding of this process. Deletion of RIAM in T cells partially suppressed αLβ2 and α4β7 integrin activation but did not block activation of α4β1. In sharp contrast, deletion of both Rap1a and Rap1b isoforms or deletion of talin1 profoundly suppressed activation of α4β1 in addition to the other two integrins. The loss of homing of T cells to peripheral lymphoid organs was correlated with the loss of integrin activation in these mutant T cells. We recently identified two Rap1 binding sites in talin1 F0 and F1 domains and generated mice bearing point mutations, which block Rap1 binding to both talin1 F0 and F1 domains (R35E,R118E). In platelets, which lack endogenous RIAM, a talin1 (R35E,R118E) mutation suppresses activation of integrin αIIbβ3 and platelet aggregation to a similar extent to that observed in Rap1a,b null platelets. In contrast, talin1 (R35E,R118E)-expressing T cells manifest only partial loss of integrin activation, whereas RIAM null T cells expressing talin1 (R35E,R118E) exhibit a much more profound defect. Importantly, overexpression of RIAM can completely rescue the integrin activation defect in talin1 (R35E,R118E)-expressing T cells indicating that Rap1 binding to RIAM and talin1 are partially redundant in T cells. These findings reveal that although both Rap1 and talin1 are essential for integrin action in blood cells, the connections between them vary in different blood cells and that the final steps in integrin activation and blood cell function can be manipulated in a cell-type specific manner. Disclosures No relevant conflicts of interest to declare.

Platelet Dysfunction and Persistent Fibrinolysis in Pediatric Scoliosis Surgery Patients Receiving Tranexamic Acid

High blood loss requiring transfusion is a common complication in pediatric scoliosis surgery. Bleeding in patients undergoing spinal surgery is exacerbated by the development of coagulopathy in response to extensive activation of the acute phase response (APR). Coagulopathy is characterized by consumption of coagulation factors, inflammation, and hyperfibrinolysis. Although the administration of antifibrinolytics (e.g. tranexamic acid, TXA) has significantly reduced transfusions, patients still experience high rates of blood loss, suggesting either that TXA dosing needs optimization or that plasmin-independent aspects of coagulopathy could be driving bleeding. Major orthopedic surgeries are characterized by a reduction in platelet counts indicating the coagulopathic conditions of surgery exert a tropism on the circulating platelet population. Indeed, platelet dysfunction concomitant with trauma-induced coagulopathy has been extensively described in literature. Platelet dysfunction during adult orthopedic surgery has been observed but has not been correlated with bleeding, and platelet function in pediatric spine surgery has yet to be assessed. To determine if pediatric patients also experience platelet dysfunction, we measured platelet activation by whole blood flow cytometry during the course of scoliosis surgery in 8 pediatric patients. In parallel, circulating markers of the APR (i.e. IL-6, IL-10, IL-8), products of fibrinolysis (D-dimer), and indicators of consumptive coagulopathy (Protein C) were measured by multiplex ELISA. Samples were collected preoperatively (Preop), intraoperatively (every 2 hours, Intraop 1 and Intraop 2), immediately after closure (Postop) and the following morning (POD1). Samples were stimulated with submaximal concentrations of thrombin, convulxin (GPVI agonist, collagen receptor), and ADP. Platelet integrin activation was measured by PAC1 binding (GPIIbIIIa activation) and platelet secretion was measured by CD62p binding (P-selectin surface expression). We observed a progressive and statistically significant reduction in the platelet response (GPIIbIIIa activation and P-selectin expression) to both thrombin and convulxin stimulation during the procedure but with distinct temporal patterns. The reduction in response to thrombin became significant at Postop, remaining down even at POD1 (PAC1 max reduction, Postop 1: 32.5%, p=0.0403; P-selectin max reduction, POD1: 33.9%, p=0.016). The reduction in response to convulxin, on the other hand developed almost immediately after the initiation of surgery, starting with Intraop1 and resolving by Postop (PAC1 max reduction, Intraop 1: 25.1%, p=0.0087; P-selectin max reduction, Intraop 1: 18.3%, p=0.0369). Intra-operative responses to thrombin (both PAC1 and CD62p binding) significantly correlated with estimates of blood loss based on red blood cell mass (Estimated Red Blood Cell Mass, ERCM def) and hemoglobin mass (Hg mass loss) (see Table I below). Although we observed a consistent and statistically significant reduction in platelet number it correlated with blood loss at POD1 only, suggesting platelet function is a better predictor of blood loss than platelet count. Despite all patients receiving a bolus of TXA with continuous low dose infusion, multiplex results indicated a progressive increase in circulating D-dimer, becoming statistically significant at Postop and persisting through POD 1. Elevations in D-dimer, the byproduct of plasmin degradation of fibrin, suggest elusive fibrinolysis despite administration of TXA. Significant intraoperative elevations in IL-6, IL-10, and IL-8 and consumption of Protein C were also noted that persisted through POD1, indicating the development of coagulopathy. In conclusion, pediatric scoliosis surgery is characterized by persistent fibrinolysis platelet dysfunction, activation of the APR, and coagulopathy despite administration of the anti-fibrinolytic TXA. Future studies will focus on altering the dose of TXA to determine if an optimal dosing can be achieved that effectively inhibits fibrinolysis and answer important questions about the dependence of the APR and platelet hypofunction on the persistent fibrinolysis seen in these patients. Figure Disclosures No relevant conflicts of interest to declare.