Surface Expression Of P-selectin Research Articles

Platelet bioenergetic profiling uncovers a metabolic pattern of high dependency on mitochondrial fatty acid oxidation in type 2 diabetic patients who developed in-stent restenosis

Although platelet bioenergetic dysfunction is evident early in the pathogenesis of diabetic macrovascular complications, the bioenergetic characteristics in type 2 diabetic patients who developed coronary in-stent restenosis (ISR) and their effects on platelet function remain unclear. Here, we performed platelet bioenergetic profiling to characterize the bioenergetic alterations in 28 type 2 diabetic patients with ISR compared with 28 type 2 diabetic patients without ISR (non-ISR) and 28 healthy individuals. Generally, platelets from type 2 diabetic patients with ISR exhibited a specific bioenergetic alteration characterized by high dependency on fatty acid (FA) oxidation, which subsequently induced complex III deficiency, causing decreased mitochondrial respiration, increased mitochondrial oxidant production, and low efficiency of mitochondrial ATP generation. This pattern of bioenergetic dysfunction showed close relationships with both α-granule and dense granule secretion as measured by surface P-selectin expression, ATP release, and profiles of granule cargo proteins in platelet releasates. Importantly, ex vivo reproduction of high dependency on FA oxidation by exposing non-ISR platelets to its agonist mimicked the bioenergetic dysfunction observed in ISR platelets and enhanced platelet secretion, whereas pharmaceutical inhibition of FA oxidation normalized the respiratory and redox states of ISR platelets and diminished platelet secretion. Further, causal mediation analyses identified a strong association between high dependency on FA oxidation and increased angiographical severity of ISR, which was significantly mediated by the status of platelet secretion. Our findings, for the first time, uncover a pattern of bioenergetic dysfunction in ISR and enhance current understanding of the mechanistic link of high dependency on FA oxidation to platelet abnormalities in the context of diabetes.

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

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

Inhibition of mitochondrial calcium transporters alters adp-induced platelet responses.

ADP-stimulated elevation of cytosolic Ca2+ is an important effector mechanism for platelet activation. The rapidly elevating cytosolic Ca2+ is also transported to mitochondrial matrix via Mitochondrial Ca2+ Uniporter (MCU) and extruded via Na+/Ca2+/Li+ Exchanger (NCLX). However, the exact contribution of MCU and NCLX in ADP-mediated platelet responses remains incompletely understood. The present study aimed to elucidate the role of mitochondrial Ca2+ transport in ADP-stimulated platelet responses by inhibition of MCU and NCLX with mitoxantrone (MTX) and CGP37157 (CGP), respectively. As these inhibitory strategies are reported to cause distinct effects on matrix Ca2+ concentration, we hypothesized to observe opposite impact of MTX and CGP on ADP-induced platelet responses. Platelet aggregation profiling was performed by microplate-based spectrophotometery while p-selectin externalization and integrin αIIbβ3 activation were analyzed by fluorescent immunolabeling using flow cytometery. Our results confirmed the expression of both MCU and NCLX mRNAs with relatively low abundance of NCLX in human platelets. In line with our hypothesis, MTX caused a dose-dependent inhibition of ADP-induced platelet aggregation without displaying any cytotoxicity. Likewise, ADP-induced p-selectin externalization and integrin αIIbβ3 activation was also significantly attenuated in MTX-treated platelets. Concordantly, inhibition of NCLX with CGP yielded an accelerated ADP-stimulated platelet aggregation which was associated with an elevation of p-selectin surface expression and αIIbβ3 activation. Together, these findings uncover a vital and hitherto poorly characterized role of mitochondrial Ca2+ transporters in ADP-induced platelet activation.

Developmental Differences between Neonatal and Adult Platelets in Regard to Immune Protein Content and Regulation of Monocyte Inflammatory Responses

Preterm neonates frequently receive platelet transfusions (PTx), owing to their high incidence of thrombocytopenia and bleeding. However, a recent large randomized trial found a significantly higher incidence of death or major bleeding among more liberally transfused preterm neonates. The mechanisms underlying these deleterious effects are unknown, but it has been suggested that they are related to the developmental differences between adult and neonatal platelets, and to a potential “developmental mismatch” occurring when adult platelets (comparatively hyperreactive) are transfused into neonates. While most studies so far have focused on the hemostatic effects of PTx, it is now clear that platelets are key modulators of immune and inflammatory responses. We hypothesized that adult platelets would be more pro-inflammatory than neonatal platelets. To test this hypothesis, we first compared the proteome of resting cord blood (CB, n=9) and adult (n=7) platelets. Using data-independent acquisition mass spectrometry, we identified 4173 high-confidence proteins present in both adult and neonatal platelets. In a principal component analysis, neonatal and adult platelets clustered separately, and 359 proteins were present at significantly different relative abundances between the two groups (≥1.5-fold change, p<0.05). Pathway analysis of the 169 proteins significantly more abundant in the adult platelet proteome revealed enrichment for immune and inflammatory pathways, while neonatal platelets were enriched for pathways involved in metabolic processes. Next, we compared the effects of neonatal and adult platelets on the inflammatory response of human neonatal monocytes. Platelets and monocytes were isolated from healthy adult blood or full-term CB (n=6/group). Platelet P-selectin surface expression was determined by flow cytometry, and key platelet-derived immune regulators (RANTES, β-2 Microglobulin [β2M], and TGF-β) were quantified in the supernatant of resting and activated platelets by ELISA. After washing, neonatal and adult platelets had similarly low P-selectin surface expression levels. Following thrombin activation, P-selectin surface expression was significantly higher in adult than in neonatal platelets (MFI 4010±1645 vs. 1987±816, p=0.04). Thrombin activation increased RANTES and TGF-β release from adult and neonatal platelets to similar levels. In contrast, β2M levels were significantly higher in the supernatant from activated adult compared to neonatal platelets (37.9±10.2 vs 17.9±5.0 ng/mL, p=0.0004). Next, we co-cultured activated adult or neonatal platelets with neonatal monocytes and quantified the pro-inflammatory cytokines IL-8 and MCP-1 (CCL2) in the supernatants after 18 hours. The presence of activated platelets significantly increased IL-8 production from neonatal monocytes compared to monocytes alone, 2.9±1.2-fold when neonatal platelets were added and 4.2±1.8-fold with addition of adult platelets (p=0.01 and p=0.001, respectively). MCP-1 increased to similar levels with the addition of either neonatal or adult platelets. In a regression model, both P-selectin expression and platelet-released β2M levels were significantly associated with monocyte-produced IL-8 levels (p=0.001 and p=0.04, respectively). In summary, we found that proteins related to inflammatory and immune responses are more abundant in adult compared to neonatal platelets, and that activated adult platelets express more P-selectin and release more β2M than neonatal platelets, resulting in higher IL8 production by neonatal monocytes. These findings support the hypothesis that the pro-inflammatory effects of adult platelets might contribute to the increased morbidity and mortality associated with neonatal PTx.

Exposure of hiPSC-Derived Mature Megakaryocytes to Vincristine Boosts Platelet Biogenesis in Vitro

Millions of healthy donor platelet (PLT) units are needed each year for therapeutic transfusions of patients with thrombocytopenia and bleeding disorders, but shortages are common. The pandemic worsened donor shortages in the US and Japan. To ensure the predictability of supply, and to mitigate clinical complications associated with conventional donor PLTs, we have developed human induced pluripotent stem cell (hiPSC)-derived megakaryocyte (MK) cell lines, imMKCLs, as a source of in vitro PLT production (iPSC-PLTs)(Nakamura et al., Cell Stem Cell, 2014; Sone et al., Stem Cell Rep, 2021), and manufactured platelets are now in clinical trials using a previously published protocol (Sugimoto et al., Blood, 2022; Sugimoto et al., Blood Adv, 2022). To optimize and improve the in vitro manufacturing protocol of iPSC-PLTs from imMKCLs, we conducted a comprehensive imaging-based compound screening which identified microtubule (MT) destabilizing agents, including the vinca alkaloid vincristine (VCR), that appear to boost proplatelet formation. Combined use of VCR at 10 μM and turbulent hydrodynamic culture conditions (Ito et al., Cell, 2018) allowed production of 23.0 PLTs per imMKCL, 2.5 fold higher than without VCR. This successful result was observed only when VCR was administered at day 3 of 6-days of the maturation phase, when imMKCLs have become polyploid, while exposure at earlier timepoints induced apoptosis. However, PLTs produced in this manner showed poor agonist-induced surface expression of P-selectin or the PAC1 epitope. Therefore, we next examined dose-dependent action of VCR on both yield of iPSC-PLTs and their function. At nanomolar concentrations (10 nM), VCR still exhibited significant PLT biogenesis-boosting activity, but without apparent impairment of PLT in vitro functionality. Interestingly, this low concentration of VCR still resulted in reduced marginal MT band staining in imMKCLs, suggesting a potential role of weakened membrane structure/status in enhanced iPSC-PLT generation. Currently, we are attempting to address if PLTs made from low-dose VCR-exposed imMKCLs have reduced marginal MT bands and whether they restore hemostasis when transfused into immunodeficient thrombocytopenic mice. Our study unveils the potential of VCR as a potent enhancer of PLT biogenesis in the imMKCL system, particularly when combined with optimized turbulent flow production systems. Our results provide a means to make production of iPSC-PLTs more cost-efficient and suggest a mechanism for the previously unexplained observation that vinca alkaloids raise PLT counts in vivo.

NLRP3 Inflammasome-Mediated Platelet Activation and Thrombus Formation in Sickle Cell Mice Can be Targeted By the BTK Inhibitor Ibrutinib

Introduction: Platelets are abnormally activated in sickle cell disease (SCD) and are involved in promoting vascular occlusions and thrombosis. The NOD like receptor protein 3 (NLRP3) inflammasome is a critical inflammatory mediator that has recently been found to be upregulated in platelets in SCD. Our recent discovery of Bruton Tyrosine Kinase (BTK) as a regulator of the NLRP3 inflammasome has provided an opportunity to suppress NLRP3 inflammasome activation in platelets via the BTK inhibitor ibrutinib. The role of the NLRP3 inflammasome in regulating platelet activation in SCD and its potential targetability through BTK inhibition is poorly understood. Methods: Townes SCD mice, which express human sickle hemoglobin (HbSS), and control mice (HbAA) at age 16 to 28 weeks were used for this study. Where indicated, mice were injected with the NLRP3 inhibitor MCC950, the BTK inhibitor ibrutinib or vehicle control 4 hours prior to blood collection. NLRP3 inflammasome activation was assessed in isolated platelets with fluorescent labeled inhibitor of caspase-1 (FLICA) assay and immunofluorescence staining of intraplatelet NLRP3 and the adaptor apoptosis-associated speck-like protein containing a CARD (ASC) with confocal microscopy. We measured platelet surface expression of P-selectin (CD62P), platelet ATP secretion, platelet spreading, platelet aggregation, and in vitro thrombus formation using flow cytometry, luminescence, scanning electron microscopy, whole blood impedance aggregometry, and a flow chamber with blood perfused at shear rates of 1,700 s -1, respectively. Isolated platelets or blood samples were treated with the NLRP3 activator nigericin where indicated. Collagen was used as platelet agonist. Data are shown as mean ± SD. Results: We found increased platelet P-selectin expression (CD62P%: 74.57 ± 3.03 vs 35.18 ± 0.73, p<0.01), platelet ATP secretion (AUC: 16.24 ± 2.17 vs 3.16 ± 0.94, p<0.01), platelet spreading (spreading area, µm 2: 5.47 ± 1.97 vs 2.35 ± 0.23, p<0.01), platelet aggregation (AUC: 68.18 ± 6.84 vs 36.28 ± 3.29, p<0.01), and thrombus formation (thrombus area %: 64.24 ± 14.64 vs 27.44 ± 5.99, p<0.05) in blood from SCD mice as compared to control mice. NLRP3:ASC speck formation (Pearson coefficient: 0.62 ± 0.03 vs 0.38 ± 0.02, p<0.001) and caspase-1 activation levels (RFUs: 476.6 ± 54.84 vs 237.8 ± 50.09, p<0.001) were elevated in platelets from SCD mice as compared to control mice, indicating platelet NLRP3 inflammasome activation in SCD mice. Next, we investigated the role of the NLRP3 inflammasome in regulating platelet function in SCD mice. Treatment of mice with the NLRP3 inhibitor MCC950 resulted in a reduction of upregulated platelet P-selectin expression (CD62P%: 33.55 ± 17.21 vs 74.23 ± 9.95, p<0.001), platelet ATP secretion (AUC: 5.98 ± 1.2 vs 13 ± 2.91, p<0.001), platelet spreading (spreading area, µm 2: 2.42 ± 0.75 vs 5.47 ± 1.97, p<0.001), platelet aggregation (AUC: 39.85 ± 2.5 vs 64.65 ± 6.5, p<0.001), and thrombus formation (thrombus area %: 41.63 ± 7.78 vs 70.22 ± 5.5, p<0.001) (MCC950 vs vehicle) in SCD mice but had no effect in control mice. We next investigated the effect of the BTK inhibitor ibrutinib. Injection of ibrutinib interfered with elevated platelet NLRP3 inflammasome activation in SCD mice, as shown by quantification of intraplatelet NLRP3:ASC specks (Pearson coefficient: 0.56 ± 0.04 vs 0.72 ± 0.03, p<0.001) and platelet caspase-1 activity (RFUs: 193 ± 55.24 vs 386.8 ± 88.08, p<0.001) (ibrutinib vs vehicle) but had no effect in control mice. Ibrutinib treatment decreased upregulated platelet P-selectin (CD62P%: 37.81 ± 10.65 vs 64.74 ± 17.73, p<0.01), platelet ATP secretion (AUC: 6.78 ± 0.24 vs 13.9 ± 1.12, p<0.001), platelet spreading (spreading area, µm 2: 2.32 ± 0.79 vs 6.45 ± 0.99, p<0.001), platelet aggregation (AUC: 40.08 ± 3.56 vs 62.13 ± 3.99, p<0.001), and thrombus formation (thrombus area, %: 44.42 ± 8.24 vs 66.97 ± 11.83, p<0.001) (ibrutinib vs vehicle) in SCD mice. The NLRP3 activator nigericin reversed these inhibitory effects of ibrutinib in SCD mice, indicating that ibrutinib interferes with platelet activation and thrombus formation in SCD mice through the NLRP3 inflammasome. Conclusion: The NLRP3 inflammasome is a critical mediator of platelet activation and thrombus formation in SCD mice, which is targetable by the BTK inhibitor ibrutinib, making it a potentially useful therapeutic approach for managing the prothrombotic state in SCD.

Platelet Function Decreases with Increasing Severity of Liver Cirrhosis and Portal Hypertension-A Prospective Study.

Cirrhotic patients display an increased risk for both bleeding and thrombosis. We investigated platelet activation across Child-Pugh stages (CPSs) and portal hypertension (PH) severity. A total of 110 cirrhotic patients were prospectively included. CPS and hepatic venous pressure gradient (HVPG) were determined. Platelet surface expression of P-selectin and activated glycoprotein (GP) IIb/IIIa were measured by flow cytometry before/after stimulation with protease-activated receptor (PAR)-1 (thrombin receptor activating peptide, TRAP) and PAR-4 (AYPGKF) agonists, epinephrine, and lipopolysaccharide (LPS). Platelet count was similar across CPS but lower with increasing PH severity. Expression of P-selectin and activated GPIIb/IIIa in response to TRAP and AYPGKF was significantly reduced in platelets of CPS-B/C versus CPS-A patients (all p < 0.05). Platelet P-selectin expression upon epinephrine and LPS stimulation was reduced in CPS-C patients, while activated GPIIb/IIIa in response to these agonists was lower in CPS-B/C (all p < 0.05). Regarding PH severity, P-selectin and activated GPIIb/IIIa in response to AYPGKF were lower in HVPG ≥20 mmHg patients (both p < 0.001 vs. HVPG < 10 mmHg). Similarly, activated GPIIb/IIIa was lower in HVPG ≥20 mmHg patients after TRAP stimulation (p < 0.01 vs. HVPG < 10 mmHg). The lower platelet surface expression of P-selectin and activated GPIIb/IIIa upon stimulation of thrombin receptors (PAR-1/PAR-4) in CPS-B/C and HVPG ≥20 mmHg patients was paralleled by reduced antithrombin-III levels in those patients (all p < 0.05). Overall, PAR-1- and PAR-4-mediated platelet activation correlated with antithrombin-III levels (p < 0.001). Platelet responsiveness decreases with increasing severity of liver cirrhosis and PH but is potentially counterbalanced by lower antithrombin-III levels.

Binding of respiratory syncytial virus particles to platelets does not result in their degranulation in vitro.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory infection in infants and the elderly. The mechanisms behind severe RSV disease are incompletely understood, but a dysregulated immune response probably plays an important role. Platelets are increasingly being recognized as immune cells and are involved in the pathology of several viruses. The release of chemokines from platelets upon activation may attract, for example, neutrophils to the site of infection, which is a hallmark of RSV pathology. In addition, since RSV infections are sometimes associated with cardiovascular events and platelets express several known RSV receptors, we investigated the effect of RSV exposure on platelet degranulation. Washed human platelets were incubated with sucrose-purified RSV particles. P-selectin and CD63 surface expression and CCL5 secretion were measured to assess platelet degranulation. We found that platelets bind and internalize RSV particles, but this does not result in degranulation. Our results suggest that platelets do not play a direct role in RSV pathology by releasing chemokines to attract inflammatory cells.

Abstract 263: Prothrombotic State Exists In Long-COVID Even After Mild Acute Infection

Long-term consequences of COVID-19 (long-COVID) are becoming a significant burden to health care. Many people remain symptomatic for months or years after recovery from COVID-19, including some who had only mild symptoms during acute infection. Sustained increases in pro-thrombotic markers have been reported in patients who were initially hospitalized with acute COVID-19. However, whether patients with less severe acute infection develop a prothrombotic-state during long-COVID is unclear. Our objective was to measure prothrombotic factors in long-COVID patients with mild disease (non-hospitalization) during acute infection. We recruited 47 consecutive adult patients with long-COVID symptoms seen in the Post-COVID clinic. All patients had a confirmed COVID-19 diagnosis by RT-PCR or antibody test during acute infection and had at least one of the symptoms of long-COVID per WHO criteria. Twenty asymptomatic healthy subjects not previously diagnosed with COVID-19 were recruited as controls. Markers of platelet activation and platelet-neutrophil aggregates (PNA) were quantified using whole blood flow cytometry. Serum levels of cell-free DNA (cfDNA) and citrullinated histones (H3Cit) and thrombin generation potential in plasma was measured. At the time of recruitment (6 weeks to 2 years after acute infection), the most common post-COVID symptoms included fatigue, chest pain, shortness of breath, headache, and dizziness. In whole blood, activation with TRAP caused increased surface expression of P-selectin without inducing activation of integrin αII b β 3 on platelets (P&lt;0.001 and P=0.5 respectively vs control). Concurrently, there was increased PNA in the long-COVID cohort (P&lt;0.01 vs. control). H3Cit and cfDNA were elevated in long-COVID (P&lt;0.01 and P&lt;0.05 respectively vs. control) and thrombin generation potential was increased, reflected by shorter lag-time, higher peak thrombin and increased velocity index (P&lt;0.01, P&lt;0.05 and P&lt;0.0001 respectively vs control). Within the long-COVID cohort, these markers were not different for age, sex, BMI or time since infection. We conclude that a prothrombotic state persists for months to years in long-COVID patients who exhibited mild disease at the time of initial infection.

Abstract 131: Identification Of A Molecule To Modulate A Platelet Olfactory Receptor And Limit Thrombosis

Background and objective: Olfactory Receptors (ORs) are G protein-coupled receptors (GPCR) we recently discovered in human platelets. OR2L13 activation by an olfactory ligand Levo-Carvone (L-Carvone) limits platelet activation. Our goal was to better characterize the pharmacology of OR2L13 ligands through structural modifications of L-Carvone and by using an 8K bioactive non-olfactory ligand library and deciphering their role as anti-thrombotic. Methods and Results: With an IC 50 of 58 μM, 2-tert-Butyl-5-Methylphenol was the best odorant for inhibiting ADP-mediated platelet activation (Fig. 1). 169 molecular hits &gt;25% threshold in an OR2L13-expressiong reporter cell line for cyclic AMP (cAMP) production were found. By counter screen (cell line without OR2L13 receptor), 12 ligands were identified for OR2L13 and 6 (CCF0054500, CCF0054432, CCF0053070, CCF0052249, CCF0051970, CCF0058399) made endogenous cAMP. Healthy platelet rich plasma (PRP) (n=7) was incubated with OR2L13 ligand for 30 minutes, then subjected to Light Transmission Aggregometry (LTA). 3/6 ligands (CCF0054500, CCF0052249 and CCF0058399) inhibited platelet aggregation through the P2Y 12 receptor and so were further validated by FACS using washed healthy platelets (n=8, attenuation of agonist-activated platelets surface P-selectin expression) for the following receptors: ADP, TRAP-6, U46619, and CRP. CCF0054500 suppressed platelet activation and aggregation through P2Y 12, PAR-1, Thromboxane and GPVI receptors. Whole blood (n=5) traversing collagen-coated capillaries reduced thrombosis when compared to vehicle under arterial sheer stress with CCF0054500. Conclusions: Like neurons, platelets have a fully functional OR signal transduction cascade. Platelet OR2L13 operates as a negative regulator of platelet reactivity and thrombosis in response to odorant and non-odorant surface agonists (biochemical activation) and under shear stress (biomechanical activation).

Supplemental fibrinogen restores thrombus formation in cardiopulmonary bypass-induced platelet dysfunction ex vivo

BackgroundMajor cardiac surgery related blood loss is associated with increased postoperative morbidity and mortality. Platelet dysfunction is believed to contribute to post-cardiopulmonary bypass (CPB)-induced microvascular bleeding. We hypothesised that moderately hypothermic CPB induces platelet dysfunction and that supplemental fibrinogen can restore in vitro thrombus formation. MethodsBlood from 18 patients, undergoing first-time elective isolated aortic valve surgery was drawn before CPB, 30 min after initiation of CPB, and after CPB and protamine administration, respectively. Platelet aggregation was quantified by optical aggregometry, platelet activation by flow-cytometric detection of platelet surface expression of P-selectin, annexin V, and activated glycoprotein IIb/IIIa, thrombus formation under flow and effect of supplemental fibrinogen (4 mg ml−1) on in vitro thrombogenesis. ResultsPost-CPB adenosine–diphosphate and TRAP-6-induced aggregation decreased by 40% and 10% of pre-CPB levels, respectively (P<0.0001). Although CPB did not change glycoprotein IIb/IIIa receptor expression, it increased the percentage of unstimulated P-selectin (1.2% vs 7%, P<0.01) positive cells and annexin V mean fluorescence intensity (15.5 vs 17.2, P<0.05), but decreased percentage of stimulated P-selectin (52% vs 26%, P<0.01) positive cells and annexin V mean fluorescence intensity (508 vs 325, P<0.05). Thrombus area decreased from 6820 before CPB to 5230 after CPB (P<0.05, arbitrary units [a.u.]). Supplemental fibrinogen increased thrombus formation to 20 324 and 11 367 a.u. before CPB and after CPB, respectively (P<0.001), thereby restoring post-CPB thrombus area to levels comparable with or higher than pre-CPB baseline. ConclusionsSingle valve surgery using moderately hypothermic CPB induces partial platelet dysfunction. Thrombus formation was restored in an experimental study design by ex vivo supplementation of fibrinogen.

Platelet function and soluble P-selectin in patients with primary immune thrombocytopenia

BackgroundThe bleeding phenotype in immune thrombocytopenia (ITP) is heterogeneous, but usually mild and only partly dependent on the severity of thrombocytopenia. Platelet reactivity has previously been suggested to underly the mild phenotype. MethodsPlatelet function was assessed as basal and agonist-induced surface expression of P-selectin and activation of GPIIb/IIIa via flow cytometry, and soluble (s)P-selectin levels were assessed in plasma of 77 patients with primary ITP, 19 hemato-oncologic thrombocytopenic controls (TC) and 20 healthy controls (HC). The association of platelet function with laboratory and clinical parameters such as bleeding manifestations at inclusion and previous thrombosis was analyzed. ResultsITP patients showed tendency towards increased surface P-selectin and elevated levels of activated GPIIb/IIIa. Platelet activation after stimulation with all agonists including TRAP-6, ADP, arachidonic acid and CRP was decreased compared to HC. Compared to TC, only GPIIb/IIIa activation but not surface P-selectin was higher in ITP. Levels of soluble (s)P-selectin were significantly higher in ITP patients compared to TC, but similar to HC. Higher sP-selectin levels were associated with blood group O and current therapy, with highest levels in TPO-RA treated patients. Platelet reactivity was not associated with platelet count or size, platelet antibodies, treatment regime, or blood group. No correlation between platelet activation with the bleeding phenotype or previous thrombotic events could be observed. ConclusionITP patients did not have hyper-reactive platelets compared to HC, but partly higher reactivity compared to TC. Further studies are needed to understand the underlying mechanism behind the bleeding and pro-thrombotic phenotype in ITP.250/250.

NLRP3 inflammasome activation in neutrophils directs early inflammatory response in murine peritonitis.

NLR family pyrin domain containing 3 (NLRP3) inflammasome mediates caspase-1-dependent processing of inflammatory cytokines such as IL-1β, an essential endothelial activator, and contributes to the pathology of inflammatory diseases. To evaluate the role of NLRP3 in neutrophils in endothelial activation, which is still elusive, we used the thioglycollate-induced peritonitis model characterized by an early neutrophil influx, on Nlrp3-/- and Nlrp3+/+ mice. Nlrp3-/- mice recruited fewer neutrophils than Nlrp3+/+ into the peritoneum and showed lower IL-1β in peritoneal lavage fluid. The higher production of IL-1β in Nlrp3+/+ was neutrophil-dependent as neutrophil depletion prevented the IL-1β production. The Nlrp3+/+ neutrophils collected from the peritoneal fluid formed significantly more filaments (specks) than Nlrp3-/- neutrophils of ASC (apoptosis-associated speck-like protein containing a caspase activating and recruitment domain), a readout for inflammasome activation. Intravital microscopy revealed that leukocytes rolled significantly slower in Nlrp3+/+ venules than in Nlrp3-/-. Nlrp3-/- endothelial cells isolated from mesenteric vessels demonstrated a lower percentage of P-selectin-positive cells with lower intensity of surface P-selectin expression than the Nlrp3+/+ endothelial cells evaluated by flow cytometry. We conclude that neutrophils orchestrate acute thioglycollate-induced peritonitis by producing IL-1β in an NLRP3-dependent manner. This increases endothelial P-selectin expression and leukocyte transmigration.

Endothelial Cell Activation Enhances Thromboinflammation in Vitt

Aim: Vaccine induced thrombosis thrombocytopenia (VITT) syndrome is a rare complication of ChAdOx1 vaccination for COVID19. VITT shares similarities with heparin induced thrombocytopenia (HIT). In both VITT and HIT, patients develop antibodies against platelet factor 4 (PF4) which promote platelet activation and thrombosis. Involvement of the endothelium in the pathogenic process has been described for HIT. Binding of HIT antibodies to PF4/glycosaminoglycan complexes on the surface of endothelial cells leads to their activation and increased procoagulant activity (Madeeva, J Thromb Haemost, 2016). However, the contribution of the endothelium to VITT has not been described. Here, we evaluate the effect of VITT plasma or serum on endothelial cell activation and thromboinflammation using an endothelialized biochip. Method: Serum and plasma samples were received from patients fulfilling clinical criteria of VITT including venous and/or arterial thrombosis, thrombocytopenia <150 x 10^3/uL, D-dimer >5-fold of upper limit of normal within 4-42 days after 1st dose of ChAdOx1 vaccine (termed "Clinical VITT") and from healthy, age and sex matched, ChAdOx1 vaccinated individuals within the same timeframe from vaccination (termed "Vaccine controls"). PF4 ELISA and VITT functional assays were performed on all samples (Lee, Blood Adv, 2022). Human umbilical vein endothelial cells (HUVECs) were treated with heat inactivated serum or plasma in the presence of peptide GPRP. Polydimethylsiloxane microfluidic straight channels were seeded with HUVECs (Endochip) and treated with VITT or control serum and compared with TNF alpha treatment. The endothelial surface expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), P-selectin, tissue factor (TF) and von Willebrand factor (vWF) were measured by immunostaining in the presence or absence of native platelet factor 4 (PF4). In some experiments HUVECs were treated with ChAdOx1 vaccine or vehicle for 48-120h. Thromboinflammation was measured by fluorescence area of antibody-stained platelets, neutrophils and fibrin on the Endochip after perfusion of recalcified blood from HIT antibody-reactive healthy volunteers (Morel-Kopp, J Thromb Haemost, 2016) at a shear rate of 100s-1. Results: Samples from 10 patients presenting with clinically confirmed VITT were paired with samples from 10 vaccinated individuals. Clinical VITT samples were separated into negative and positive for both PF4 ELISA and VITT functional assay (defined as VITT ELISA positive and VITT ELISA negative, Panel A). Endothelial cell surface expression of P-selectin was increased after 12 h incubation with 25% v/v VITT serum or plasma compared with samples from AZ vaccinated non-VITT individuals (median integrated intensity/cell 8065 vs 3515 a.u, p=0.03). The expression of TF was significantly increased by addition of PF4 to the VITT plasma (median integrated intensity/cell 42038 vs 14881 a.u., p=0.01 by Mann Whitney test). ChAdOx1 treatment of HUVECs at 1:1000 dilution induced the expression of spike protein but did not significantly enhance thromboinflammation compared with media, as assessed by the perfusion of whole blood through the Endochip. Treatment of the Endochip with VITT serum, without the addition of PF4, significantly increased the adhesion of platelets compared with control serum (median 2.2-fold, p=0.002), neutrophils (2.5-fold, p=0.003) and fibrin (5.4-fold, p=0.002 by Wilcoxon paired samples test (Panel B). There was no difference in the thromboinflammatory response between ELISA negative and positive group (Panel B). Conclusion: Endothelial inflammation contributes to the prothrombotic tendency in VITT in an in vitro model of thromboinflammation. VITT serum induces the endothelial expression of P-selectin and TF. Endothelial inflammation may explain thrombotic events in cerebral or splanchnic veins of vaccinated individuals without detectable anti-PF4 antibodies by ELISA or platelet-based VITT functional assays. Endothelialised biochips have diagnostic potential for detecting immune prothrombotic tendencies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cross-Talk between HLA Class I and TLR4 Mediates P-Selectin Surface Expression and Monocyte Capture to Human Endothelial Cells.

Donor-specific HLA Abs contribute to Ab-mediated rejection (AMR) by binding to HLA molecules on endothelial cells (ECs) and triggering intracellular signaling, leading to EC activation and leukocyte recruitment. The molecular mechanisms involving donor-specific HLA Ab-mediated EC activation and leukocyte recruitment remain incompletely understood. In this study, we determined whether TLRs act as coreceptors for HLA class I (HLA I) in ECs. We found that human aortic ECs express TLR3, TLR4, TLR6, and TLR10, but only TLR4 was detected on the EC surface. Consequently, we performed coimmunoprecipitation experiments to examine complex formation between HLA I and TLR4. Stimulation of human ECs with HLA Ab increased the amount of complex formation between HLA I and TLR4. Reciprocal coimmunoprecipitation with a TLR4 Ab confirmed that the crosslinking of HLA I increased complex formation between TLR4 and HLA I. Knockdown of TLR4 or MyD88 with small interfering RNAs inhibited HLA I Ab-stimulated P-selectin expression, von Willebrand factor release, and monocyte recruitment on ECs. Our results show that TLR4 is a novel coreceptor for HLA I to stimulate monocyte recruitment on activated ECs. Taken together with our previous published results, we propose that HLA I molecules form two separate signaling complexes at the EC surface, that is, with TLR4 to upregulate P-selectin surface expression and capture of monocytes to human ECs and integrin β4 to induce mTOR-dependent firm monocyte adhesion via ICAM-1 clustering on ECs, two processes implicated in Ab-mediated rejection.

Riociguat inhibits ultra-large VWF string formation on pulmonary artery endothelial cells from chronic thromboembolic pulmonary hypertension patients.

Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by elevated pulmonary arterial pressure and organized thrombi within pulmonary arteries. Riociguat is a soluble guanylate cyclase stimulator and is approved for patients with inoperable CTEPH or residual pulmonary hypertension after pulmonary endarterectomy (PEA). Previous work suggested that riociguat treatment is associated with an increased risk of bleeding, although the mechanism is unclear. The aim of this study is to assess how riociguat affects primary hemostasis by studying its effect on the interaction between platelets and endothelial cells derived from CTEPH patients. Pulmonary artery endothelial cells (PAECs) were isolated from thrombus-free regions of PEA material. Purified PAECs were cultured in flow chambers and were stimulated with 0.1 and 1 µM riociguat for 24 h before flow experiments. After stimulation with histamine, PAECs were exposed to platelets under shear stress. Platelet adhesion and expression of von Willebrand Factor (VWF) were evaluated to assess the role of riociguat in hemostasis. Under dynamic conditions, 0.1 and 1.0 µM of riociguat suppressed platelet adhesion on the surface of PAECs. Although riociguat did not affect intracellular expression and secretion of VWF, PAECs stimulated with riociguat produced fewer VWF strings than unstimulated PAECs. Flow cytometry suggested that decreased VWF string formation upon riociguat treatment may be associated with suppressed cell surface expression of P-selectin, a protein that stabilizes VWF anchoring on the endothelial surface. In conclusion, Riociguat inhibits VWF string elongation and platelet adhesion on the surface of CTEPH-PAECs, possibly by reduced P-selectin cell surface expression.

Decreased Platelet Specific Receptor Expression of P-Selectin and GPIIb/IIIa Predict Future Non-Surgical Bleeding in Patients after Left Ventricular Assist Device Implantation.

Non-surgical bleeding (NSB) is one of the major clinical complications in patients under continuous-flow left ventricular assist device (LVAD) support. The increased shear stress leads to an altered platelet receptor composition. Whether these changes increase the risk for NSB is unclear. Thus, we compared the platelet receptor composition of patients with (bleeder group, n = 18) and without NSB (non-bleeder group, n = 18) prior to LVAD implantation. Blood samples were obtained prior to LVAD implantation and after bleeding complications in the post-implant period. Platelet receptor expression of GPIbα, GPIIb/IIIa, P-selectin and CD63 as well as intra-platelet oxidative stress levels were quantified by flow cytometry. Bleeders and non-bleeders were comparable regarding clinical characteristics, von Willebrand factor diagnostics and the aggregation capacity before and after LVAD implantation (p > 0.05). LVAD patients in the bleeder group suffered from gastrointestinal bleeding (33%; n = 6), epistaxis (22%; n = 4), hematuria or hematoma (17%; n = 3, respectively) and cerebral bleeding (11%; n = 2). Prior to LVAD implantation, a restricted surface expression of the platelet receptors P-selectin and GPIIb/IIIa was observed in the bleeder group (P-selectin: 7.2 ± 2.6%; GPIIb/IIIa: 26,900 ± 13,608 U) compared to non-bleeders (P-selectin: 12.4 ± 8.1%, p = 0.02; GPIIb/IIIa: 36,259 ± 9914 U; p = 0.02). We hypothesized that the reduced platelet receptor expression of P-selectin and GPIIb/IIIa prior to LVAD implantation may be linked to LVAD-related NSB.

Platelet Priming and Activation in Naturally Occurring Thermal Burn Injuries and Wildfire Smoke Exposure Is Associated With Intracardiac Thrombosis and Spontaneous Echocardiographic Contrast in Feline Survivors.

Wildfires pose a major health risk for humans, wildlife, and domestic animals. We previously discovered pathophysiologic parallels between domestic cats with naturally occurring smoke inhalation and thermal burn injuries and human beings with similar injuries; these were characterized by transient myocardial thickening, cardiac troponin I elevation and formation of intracardiac thrombosis. While the underlying mechanisms remain unclear, results from murine models suggest that platelet priming and activation may contribute to a global hypercoagulable state and thrombosis. Herein, we evaluated and compared the degree of platelet activation, platelet response to physiologic agonists and levels of platelet-derived microvesicles (PDMV) in 29 cats with naturally occurring wildfire thermal injuries (WF), 21 clinically healthy cats with subclinical hypertrophic cardiomyopathy (HCM) and 11 healthy cats without HCM (CC). We also quantified and compared circulating PDMVs in WF cats to CC cats. In addition, we examined the association between thrombotic events, severity of burn injuries, myocardial changes, and the degree of platelet activation in cats exposed to wildfires. Flow cytometric detection of platelet surface P-selectin expression showed that WF cats had increased platelet response to adenosine diphosphate (ADP) and thrombin compared to the two control groups indicating the presence of primed platelets in circulation. In addition, cats in the WF group had increased circulating levels of PDMV, characterized by increased phosphatidylserine on the external leaflet. Cats in the WF group with documented intracardiac thrombosis had elevated platelet activation and platelet priming in the presence of ADP. While high dose arachidonic acid (AA) mostly resulted in platelet inhibition, persistent response to AA was noted among cats in the WF group with intracardiac thrombosis. Univariate and multiple logistic regression analyses demonstrated that increased platelet response to AA was independently associated with thrombotic events. This is the first study reporting the significant association between platelet priming and intracardiac thrombosis in domestic cats with naturally occurring wildfire-related injuries and smoke inhalation. Further studies are required to delineate additional mechanisms between inflammation and thrombosis, especially regarding platelet primers and the cyclooxygenase pathway.One Sentence SummaryPlatelet activation and shedding of platelet-derived microvesicles due to platelet priming is present following naturally occurring wildfire smoke exposure and thermal burn injuries in a population of domestic cats.

Chronic Immune Platelet Activation Is Followed by Platelet Refractoriness and Impaired Contractility.

Autoimmune diseases, including systemic lupus erythematosus (SLE), have a high risk of thrombotic and hemorrhagic complications associated with altered platelet functionality. We studied platelets from the blood of SLE patients and their reactivity. The surface expression of phosphatidylserine, P-selectin, and active integrin αIIbβ3 were measured using flow cytometry before and after platelet stimulation. Soluble P-selectin was measured in plasma. The kinetics of platelet-driven clot contraction was studied, as well as scanning and transmission electron microscopy of unstimulated platelets. Elevated levels of membrane-associated phosphatidylserine and platelet-attached and soluble P-selectin correlated directly with the titers of IgG, anti-dsDNA-antibodies, and circulating immune complexes. Morphologically, platelets in SLE lost their resting discoid shape, formed membrane protrusions and aggregates, and had a rough plasma membrane. The signs of platelet activation were associated paradoxically with reduced reactivity to a physiological stimulus and impaired contractility that revealed platelet exhaustion and refractoriness. Platelet activation has multiple pro-coagulant effects, and the inability to fully contract (retract) blood clots can be either a hemorrhagic or pro-thrombotic mechanism related to altered clot permeability, sensitivity of clots to fibrinolysis, obstructiveness, and embologenicity. Therefore, chronic immune platelet activation followed by secondary platelet dysfunction comprise an understudied pathogenic mechanism that supports hemostatic disorders in autoimmune diseases, such as SLE.

Tirofiban potentiates agonist-induced platelet activation and degranulation, despite effectively inhibiting aggregation

We aimed to investigate the effects of integrin αIIbβ3 inhibitor tirofiban on hallmarks of platelet activation, degranulation, and aggregation during its use to analyze activated but non-complexed platelets via flow cytometry. To do so, we used washed platelets from healthy human donors. We combined aggregometry, an assay of platelet functionality, with flow cytometry and ELISA to detect and correlate, respectively, platelet aggregation, activation, and granule release. While tirofiban effectively inhibited agonist-induced platelet aggregation (thrombin receptor-activating peptide 6 (TRAP), convulxin (CVX), U46619 and IV.3), the surface expression of P-selectin and CD63 and granule release of RANTES were significantly increased, indicating that tirofiban enhances degranulation, uncoupled from aggregation. The results show that tirofiban alters the activation phenotype of platelets, something that should be considered when using tirofiban to enable flow cytometric analysis of activated but unaggregated platelet suspensions.