Washed Platelets Research Articles

Platelet CD40L Expression Response to Mixing of pRBCs and Washed Platelets but no Causality Association between Platelet ROS Generation and CD40L Expression: An In Vitro Study.

Platelets play a role in transfusion reaction via reactive oxygen species (ROS) generation and CD40 ligand (CD40L) expression. In this study, we aimed to test the hypothesis that the mixing of packed red blood cells (pRBCs) and washed platelets has a causal effect on platelet ROS generation and CD40L expression. Thus, a better understanding of this causality relationship may help interrupt the chain of events and avoid an uncontrollable transfusion reaction. We simulated transfusion in vitro by mixing pRBCs and washed platelets. Donor cross-matched stored pRBCs) from our blood bank and recipient whole blood from patients undergoing coronary artery bypass graft surgery prepared into washed platelets were used. Briefly, donor pRBCs were added to washed recipient platelets to form 1%, 5%, or 10% (v/v) mixtures. The mixed blood sample was used to determine platelet ROS generation (dichlorofluorescein fluorescence levels) and CD40L expression. The effect of antioxidants (20 mM glutamine and 20 mM dipeptiven) on ROS generation and CD40L expression was also evaluated. Platelet ROS generation was not significantly associated with the mixing of pRBCs and washed platelets (p = 0.755), glutamine treatment (p = 0.800), or dipeptiven treatment (p = 0.711). The expression of CD40L by platelets increased significantly (p < 0.001), and no significant difference was noted after treatment with glutamine (p = 0.560) or dipeptiven (p = 0.618). We observed that the mixing pRBCs and washed platelets had no effect via ROS, whereas CD40L could directly induce transfusion reactions. Furthermore, platelets did not causally express ROS or CD40L after being mixed with pRBCs. Although antioxidants are more accessible than anti-CD40L antibodies, platelet ROS may not serve as a therapeutic target for antioxidants. Nevertheless, CD40L expression may be a valuable therapeutic target for managing transfusion reactions.

Platelet-Mediated Transfer of Cardioprotection by Remote Ischemic Conditioning and Its Abrogation by Aspirin But Not by Ticagrelor

PurposeThe role of platelets during myocardial ischemia/reperfusion (I/R) is ambivalent. They contribute to injury but also to cardioprotection. Repeated blood flow restriction and reperfusion in a tissue/organ remote from the heart (remote ischemic conditioning, RIC) reduce myocardial I/R injury and attenuate platelet activation. Whether or not platelets mediate RIC’s cardioprotective signal is currently unclear.Methods and ResultsVenous blood from healthy volunteers (without or with pretreatment of 500/1000 mg aspirin or 180 mg ticagrelor orally, 2–3 h before the study, n = 18 each) was collected before and after RIC (3 × 5 min blood pressure cuff inflation at 200 mmHg on the left upper arm/5 min deflation). Washed platelets were isolated. Platelet-poor plasma was used to prepare plasma-dialysates. Platelets (25 × 103/µL) or plasma-dialysates (1:10) prepared before and after RIC from untreated versus aspirin- or ticagrelor-pretreated volunteers, respectively, were infused into isolated buffer-perfused rat hearts. Hearts were subjected to global 30 min/120 min I/R. Infarct size was stained. Infarct size was less with infusion of platelets/plasma-dialysate after RIC (18 ± 7%/23 ± 9% of ventricular mass) than with platelets/plasma-dialysate before RIC (34 ± 7%/33 ± 8%). Aspirin pretreatment abrogated the transfer of RIC’s cardioprotection by platelets (after/before RIC, 34 ± 7%/33 ± 7%) but only attenuated that by plasma-dialysate (after/before RIC, 26 ± 8%/32 ± 5%). Ticagrelor pretreatment induced an in vivo formation of cardioprotective factor(s) per se (platelets/plasma-dialysate before RIC, 26 ± 7%/26 ± 7%) but did not impact on RIC’s cardioprotection by platelets/plasma-dialysate (20 ± 7%/21 ± 5%).ConclusionPlatelets serve as carriers for RIC’s cardioprotective signal through an aspirin-sensitive and thus cyclooxygenase-dependent mechanism. The P2Y12 inhibitor ticagrelor per se induces a humoral cardioprotective signal.Graphical abstract

Human platelet‐mediated transfer of cardioprotection by remote ischemic conditioning is abrogated by aspirin, but not by ticagrelor

BackgroundRepeated ischemia/reperfusion in a tissue/organ remote from the heart (remote ischemic conditioning, RIC) protects against injury from sustained myocardial ischemia/reperfusion (I/R). Platelets are considered as mediators/targets of RIC´s protection. We have recently reported that infusion of washed platelets, taken from healthy volunteers after RIC, reduced infarct size (IS) in isolated rat hearts (FASEB Journal 2021; 35; S1; DOI: 10.1096/fasebj.2021.35.S1.03444). Anti‐platelet drugs are potential confounders of RIC.AimTo study the impact of anti‐platelet drugs, the cyclooxygenase inhibitor aspirin and the P2Y12 inhibitor ticagrelor, on the platelet‐mediated transfer of cardioprotection.MethodsWe included 18 healthy volunteers. As a contemporary control, volunteers were subjected to RIC through 3 x 5 min blood pressure cuff inflation at 200 mmHg on the left upper arm/ 5 min deflation. After at least 2 weeks, these volunteers were again subjected to RIC 3 hours after pretreatment with oral aspirin (500‐1000 mg) and again at least another 2 weeks later 2 hours after pretreatment with oral ticagrelor (180 mg). Venous blood samples were taken before RIC and 60 min after RIC, respectively. Washed platelets were prepared using apyrase, prostaglandin E1 and citrated Tyrode‐buffer. The platelet count was adjusted to 2.5 x 105platelets/µL. The functionality of washed platelets was tested in response to thrombin (1U/ml), aggregation measured with turbodimetric light transmission aggregometry and expressed as the increase in light transmission in percent of control. Platelet preparations with an aggregation &lt;60% were discarded. Male Lewis rats were sacrificed, their hearts isolated and perfused at constant pressure with buffer. Washed platelets before and after RIC, respectively, were infused for 8 min (2.5 x 104 platelets/µL) into rat hearts before global 30 min/ 120 min I/R. IS was demarcated by triphenyl tetrazolium chloride staining and calculated in percent of ventricular mass.ResultsWe confirm, that infusion of platelets after RIC reduces IS in isolated hearts with global I/R (Figure). This platelet‐mediated transfer of cardioprotection was abrogated by aspirin‐pretreatment of the volunteers (Figure). Platelets before RIC from ticagrelor‐pretreated volunteers reduced IS per se; nevertheless, platelets after RIC further reduced IS (Figure).ConclusionHuman platelets serve as carriers for RIC’s cardioprotective signal through an aspirin‐sensitive and thus cyclooxygenase‐dependent mechanism. The P2Y12 inhibitor ticagrelor per se induces a cardioprotective platelet‐dependent signal, which is further enhanced by RIC. The precise signal transfer from the platelets to the myocardium and the nature of the platelet‐derived factors remain to be further identified.

Prenylcysteine Oxidase 1 (PCYOX1), a New Player in Thrombosis.

Prenylcysteine Oxidase 1 (PCYOX1) is an enzyme involved in the degradation of prenylated proteins. It is expressed in different tissues including vascular and blood cells. We recently showed that the secretome from Pcyox1-silenced cells reduced platelet adhesion both to fibrinogen and endothelial cells, suggesting a potential contribution of PCYOX1 into thrombus formation. Here, we show that in vivo thrombus formation after FeCl3 injury of the carotid artery was delayed in Pcyox1−/− mice, which were also protected from collagen/epinephrine induced thromboembolism. The Pcyox1−/− mice displayed normal blood cells count, vascular procoagulant activity and plasma fibrinogen levels. Deletion of Pcyox1 reduced the platelet/leukocyte aggregates in whole blood, as well as the platelet aggregation, the alpha granules release, and the αIIbβ3 integrin activation in platelet-rich plasma, in response to adenosine diphosphate (ADP) or thrombin receptor agonist peptide (TRAP). Washed platelets from the Pcyox1−/− and WT animals showed similar phosphorylation pathway activation, adhesion ability and aggregation. The presence of Pcyox1−/− plasma impaired agonist-induced WT platelet aggregation. Our findings show that the absence of PCYOX1 results in platelet hypo-reactivity and impaired arterial thrombosis, and indicates that PCYOX1 could be a novel target for antithrombotic drugs.

Antithrombotic Effect of Sorbus commixta in an Arterial Thrombosis Mouse Model

Sorbus commixta Hedlund ( S. commixta) is known to possess cardiovascular protective activity. However, its effect on thrombosis has not been reported, although thrombosis plays a primary role in diverse circulatory diseases. Thus, this study was performed to assess the antithrombotic activity of dried S. commixta fruit extract. Mice were fed with a diet containing 8.3% extract for 4 weeks, which corresponded to the consumption of approximately 1000 mg extract/kg/day. The antithrombotic effect was tested in a FeCl3-induced arterial thrombosis model. Additionally, the effects on platelet aggregation and plasma coagulation were examined in vitro using washed platelets (WP) and isolated blood plasma, respectively. Thrombotic vascular occlusion time was significantly prolonged by the 4-week administration of the extract. Treatment of WP with the extract resulted in a decrease in thrombin-induced aggregation, which was concentration-dependent over 10 to 100 μg/mL and was not attributed to the direct inhibition of thrombin. The extract did not affect blood plasma coagulation, which was assessed by measuring prothrombin time and activated partial thromboplastin time. S. commixta is capable of preventing arterial thrombosis and such an effect may be attributed to the inhibition of platelet aggregation.

Quantification of Cyclic Amp and Cyclic Gmp Levels in Krebs-Henseleit's Solution by Lc-Ms/Ms: Application in Washed Platelet Aggregation Samples

Quantification of Cyclic Amp and Cyclic Gmp Levels in Krebs-Henseleit's Solution by Lc-Ms/Ms: Application in Washed Platelet Aggregation Samples

Practical Considerations of Dissolved Oxygen Levels for Platelet Function under Hypoxia.

Investigating human platelet function in low-oxygen environments is important in multiple settings, including hypobaric hypoxia (e.g., high altitude), sea level hypoxia-related disease, and thrombus stability. These studies often involve drawing blood from which platelets are isolated and analysed at atmospheric conditions or re-exposed to low oxygen levels in hypoxia chambers before testing. However, it remains unknown how the in vitro handling of the samples itself changes their dissolved oxygen concentration, which might affect platelet function and experimental results. Here, we prepared healthy donor platelet-rich plasma and washed platelet (WP) suspensions and exposed them to 2% oxygen. We found that the use of hypoxia pre-equilibrated tubes, higher platelet concentrations (>2 × 108/mL versus 2 × 107/mL), smaller volumes (600 µL versus 3 mL), and presence of plasma reduced the time for samples to reach 2% oxygen. Notably, oxygen levels decreased below 2% in most suspensions, but also in WP maintained at atmospheric 21% oxygen. Additionally, platelet spreading on fibrinogen was decreased when using hypoxic fibrinogen-coated culture plates regardless of the oxygen percentage (2% or 21%) in which platelet incubation took place. Thus, sample handling and experimental conditions should be carefully monitored in platelet-hypoxia studies as they might compromise results interpretation and comparison across studies.

Heme-Induced Platelet Mitochondrial Oxidant Production Regulates Platelet Granule Release

Background: Patients with Sickle Cell Disease (SCD) are at greater risk for thrombosis and the development of chronic vasculopathy, both of which are major contributors to morbidity and mortality in these patients. While thrombosis and vasculopathy are associated with hemolysis in SCD, the molecular mechanisms by which hemolysis propagates these conditions remains unclear. At the cellular level, we and others have shown that hemolytic components, including hemoglobin and its degradation product, free heme, directly activate platelets. Notably, activated platelets are not only central to thrombosis, but are also implicated in vasculopathy through their degranulation, which results in the release of vasoactive molecules. Though heme and hemoglobin-induced platelet activation has been widely studied, the effect of hemolytic products on platelet degranulation and the identity of the resulting platelet secretome remains less clear. We hypothesize that free heme activates a platelet signaling cascade resulting in platelet degranulation and the release of specific “secretome” molecules.Methods: Washed platelets were prepared from whole blood collected from healthy human volunteers in 0.32% sodium citrate (n=6). Platelets were treated with heme (2.5µM) in the presence or absence of MitoTEMPO (10 µM) - a mitochondrial oxidant (mtROS) scavenger, or ARQ092 (10 µM), a small molecule that prevents phosphorylation of the serine/threonine kinase Akt at S473. Platelet mtROS was measured by fluorescence spectroscopy using MitoSOX Red. Thrombospondin-1 (TSP1), CXCL7, Fibroblast Growth Factor (FGF) basic, TGFβ, IL-1β, PDGF-B, angiostatin, kininogen, CD40L and PAI-1 were quantified in heme treated platelet releasates in the presence and absence of MitoTEMPO by dot blot. The enzymatic activity of mitochondrial electron transport complex V was measured spectrophotometrically by kinetic assay.Results:We found that heme stimulated the release of a specific set of molecules from the α-granule of platelets, including TSP1, CXCL7, FGF basic, TGFβ, IL-1β, PDGF-B, angiostatin, and kininogen; but did not stimulate the release of CD40L or PAI-1. Mechanistic studies demonstrate that the release of several of these molecules was dependent on heme-induced activation of platelet Akt which inhibits mitochondrial complex V, resulting in mtROS production. Consistent with this mechanism, the heme-stimulated release of TSP1, CXCL7, FGF basic, IL-1β, PDGF-B, and angiostatin were significantly attenuated by preventing Akt phosphorylation at Ser473 with ARQ092, which also prevented complex V inhibition and mtROS production. Direct scavenging of mtROS with MitoTEMPO also attenuated heme-induced release of these molecules.Conclusion: These data, for the first time, begin to characterize the platelet secretome released in response to free heme. Further, they demonstrate a novel molecular pathway in which extracellular heme induces the activation of platelet Akt to inhibit mitochondrial complex V, ultimately inducing mtROS. Notably, this study suggests that release of a proportion of the heme-induced secretome is regulated by mtROS production and can be suppressed by mtROS scavengers. Ongoing studies are further characterizing the hemolysis-induced platelet secretome, the downstream effects of secretome products on vascular function, and the extent of regulation of the secretome by mtROS. These studies provide a mechanistic link between hemolysis and platelet degranulation, by which the release of mitogens can lead to the pathogenesis of vascular wall dysfunction. These studies also suggest that platelet mtROS may represent a novel therapeutic target to attenuate vascular dysfunction in hemolytic disorders including SCD.Note: The finding discussed in the above abstract are available as preprint in bioRxiv; doi: https://doi.org/10.1101/2021.08.02.454816 DisclosuresNo relevant conflicts of interest to declare.

Abstract P119: The 15-lipoxygenase-derived Oxylipins 15-HETrE And 15-HETE Inhibit Platelet Activation In Part Through Activation Of PPARs

Cardiovascular disease is the leading cause of mortality in the US annually. The underlying cause of mortality in cardiovascular disease is the formation of platelet-rich clots that occlude blood vessels due to aberrant platelet activation. While antiplatelet therapeutic intervention has significantly reduced the risk of an occlusive thrombotic event, many patients remain at risk for a myocardial infarction or stroke. The antiplatelet and antithrombotic effects of omega-6 polyunsaturated fatty acids are primarily attributed to its metabolism to bioactive metabolites by oxygenases such as lipoxygenases (LOX). LOXs are a group of lipid-peroxidizing enzymes named according to the specific position where they add an oxygen to arachidonic acid: 5-LOX, 12-LOX, and 15-LOX. Previous studies from our group have demonstrated that dihomo-γ-linolenic acid and arachidonic acid regulate platelet function through their respective 12-LOX-derived oxylipins, 12(S)-hydroxyeicosatrienoic (12-HETrE) and 12(S)-hydroxyeicosatetraenoic acid (12-HETE). While the expression of 15-LOX in platelets is controversial, platelets have demonstrated the ability to generate the 15-LOX-derived bioactive metabolites. In this study we sought to elucidate the mechanistic effects of 15(S)-hydroxyeicosatrienoic acid (15-HETrE) and 15(S)-hydroxyeicosatetraenoic (15-HETE) on platelet reactivity. Washed human platelets were treated with 15-HETrE or 15-HETE and platelet aggregation, integrin αIIbβ3 activation, calcium mobilization, and granule secretion were quantified. Both 15-HETrE and 15-HETE were shown to inhibit platelet aggregation mediated by collagen. In comparison to vehicle-treated platelets, treatment with 15-HETrE or 15-HETE inhibited agonist-induced intracellular signalling events, including PKC activation, calcium mobilization, and granule secretion. Surprisingly, while 15-HETrE was shown to inhibit platelets through a signalling cascade involving the activation of PPARβ, 15-HETE’s inhibitory effect was shown to involve the activation of PPARα and the inhibition of 12-HETE production. A better understanding of the effects of 15-LOX oxylipins in platelets could lead to the identification of novel antiplatelet therapies.

Fluorometric Quantification of Human Platelet Polyphosphate Using 4',6-Diamidine-2-phenylindole Dihydrochloride: Applications in the Japanese Population.

Polyphosphate (polyP), a biopolymer of inorganic phosphate, is widely distributed in living organisms. In platelets, polyP is released upon activation and plays important roles in coagulation and tissue regeneration. However, the lack of a specific quantification method has delayed the in-depth study of polyP. The fluorescent dye 4′,6-diamidine-2-phenylindole dihydrochloride (DAPI) has recently received attention as a promising probe for the visualization and quantification of cellular polyP levels. In this study, we further optimized quantification conditions and applied this protocol in quantification of platelet polyP levels in a Japanese population. Blood samples were collected from non-smoking, healthy Japanese subjects (23 males, 23 females). Washed platelets were fixed and probed with DAPI for fluorometric determination. PolyP levels per platelet count were significantly higher in women than that in men. A moderate negative correlation between age and polyP levels was found in women. Responsiveness to CaCl2 stimulation was also significantly higher in women than that in men. Overall, our optimized protocol requires neither purification nor degradation steps, reducing both the time and bias for reproducible quantification. Thus, we suggest that despite its low specificity, this DAPI-based protocol would be useful in routine laboratory testing to quantify platelet polyP levels efficiently and economically.

Phosphorylation of Y311 on Pkcδ Regulates Protease Activated Receptor 4-Mediated Signaling in Platelets

Background: Protein kinase C (PKC) is known to regulate various aspects of platelet functions. In platelets, PKCδ is known to both positively and negatively regulate dense granule secretion in response to PAR and GPVI agonists, respectively. Hence, we speculate that the differential regulation of PKCδ in platelets is due to differences in tyrosine phosphorylation of PKCδ.Results: We investigated several phosphorylation sites on PKCδ and found that Y311 is phosphorylated downstream of PAR agonists to a greater extent than GPVI agonists in human and murine platelets when feedback is blocked. Hence, the focus of this study is to characterize the function of PKCδ tyrosine Y311 in platelets. Therefore, we generated PKCδY311F knock-in mice to study the contribution of Y311 on PKCδ to PAR-4-mediated platelet activation. PKCδY311F mice are viable and have no gross abnormalities. Washed platelets from PKCδY311F mice have reduced aggregation and dense granule secretion after stimulation with AYPGKF, a PAR-4 agonist (Fig. 1) indicating its importance in PAR-4 mediated signaling. In an in vivo tail-bleeding assay, PKCδY311 mice have significantly enhanced tail-bleeding times compared to wild-type (WT) littermate controls (Fig. 2). Finally, PKCδY311F mice exhibit longer time to occlusion compared to WT control using a ferric chloride in vivo thrombosis model. This indicates that the phosphorylation of PKCδ Y311 is pro-thrombotic. At the molecular level, PKCδY311 was phosphorylated in Fyn-/- null platelets but not in Lyn-/- platelets by AYPGKF, indicating that Lyn phosphorylates this tyrosine residue.Conclusions: Phosphorylation of PKCδ on Y311 is important for regulation of platelet functional responses initiated by thrombin. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pharmacologic targeting of Cdc42 GTPase by a small molecule Cdc42 activity-specific inhibitor prevents platelet activation and thrombosis

Gene targeting of Cdc42 GTPase has been shown to inhibit platelet activation. In this study, we investigated a hypothesis that inhibition of Cdc42 activity by CASIN, a small molecule Cdc42 Activity-Specific INhibitor, may down regulate platelet activation and thrombus formation. We investigated the effects of CASIN on platelet activation in vitro and thrombosis in vivo. In human platelets, CASIN, but not its inactive analog Pirl7, blocked collagen induced activation of Cdc42 and inhibited phosphorylation of its downstream effector, PAK1/2. Moreover, addition of CASIN to washed human platelets inhibited platelet spreading on immobilized fibrinogen. Treatment of human platelets with CASIN inhibited collagen or thrombin induced: (a) ATP secretion and platelet aggregation; and (b) phosphorylation of Akt, ERK and p38-MAPK. Pre-incubation of platelets with Pirl7, an inactive analog of CASIN, failed to inhibit collagen induced aggregation. Washing of human platelets after incubation with CASIN eliminated its inhibitory effect on collagen induced aggregation. Intraperitoneal administration of CASIN to wild type mice inhibited ex vivo aggregation induced by collagen but did not affect the murine tail bleeding times. CASIN administration, prior to laser-induced injury in murine cremaster muscle arterioles, resulted in formation of smaller and unstable thrombi compared to control mice without CASIN treatment. These data suggest that pharmacologic targeting of Cdc42 by specific and reversible inhibitors may lead to the discovery of novel antithrombotic agents.

Phosphorylation of protein kinase Cδ Tyr311 positively regulates thromboxane generation in platelets

Platelets are key mediators of physiological hemostasis and pathological thrombosis, whose function must be carefully balanced by signaling downstream of receptors such as protease-activated receptor (PAR)4. Protein kinase C (PKC) is known to regulate various aspects of platelet function. For instance, PKCδ is known to regulate dense granule secretion, which is important for platelet activation. However, the mechanism by which PKCδ regulates this process as well as other facets of platelet activity is unknown. We speculated that the way PKCδ regulates platelet function may be because of the phosphorylation of tyrosine residues on PKCδ. We investigated phosphorylation of PKCδ following glycoprotein VI-mediated and PAR4-mediated platelet activation and found that Y311 is selectively phosphorylated when PAR4 is activated in human platelets. Therefore, we generated PKCδ Y311F knock-in mice, which are viable and have no gross abnormalities. However, PKCδY311F mice have significantly enhanced tail-bleeding times compared with WT littermate controls, which means hemostasis is interrupted. Furthermore, PKCδY311F mice exhibit longer time to carotid artery occlusion compared with WT control using a ferric chloride in vivo thrombosis model, indicating that the phosphorylation of PKCδ Y311 is prothrombotic. Washed platelets from PKCδY311F mice have reduced reactivity after stimulation with a PAR-4 agonist indicating its importance in platelet signaling. The phenotype observed in Y311F mouse platelets is because of reduced thromboxane generation, as an inhibitor of thromboxane generation equalizes the PKCδY311F platelet response to that of WT. Therefore, phosphorylation of PKCδ on Y311 is important for regulation of platelet function and specifically thromboxane generation, which reinforces platelet activation.

Heme Induced Platelet Mitochondrial Oxidant Production Regulates Thrombospondin-1 Release from Platelets

Background: Sickle Cell Disease (SCD) patients develop chronic vasculopathy leading to conditions such as pulmonary hypertension (PH), a major cause of morbidity and mortality. Vasculopathy is tightly associated with hemolysis in SCD. We and others have shown that in SCD, hemolysis activates platelets, and activated platelets are implicated in vasculopathy through their release of vasoactive molecules, such as thrombosponding-1 (TSP1), from storage granules. In SCD, free heme is accumulated in the circulation as a consequence of oxidative degradation of hemoglobin secondary to intravascular hemolysis. Under homeostatic conditions, albumin, haptoglobin, hemopexin, and high- and low-density lipoproteins clear plasma heme from the circulation. However excessive hemolysis can overwhelm these clearance mechanisms, resulting in elevated levels of free heme, which can lead to platelet activation, aggregation and thrombus formation. The role of activated platelets in these thrombotic events is well studied, but the mechanisms by which heme induces platelet activation and platelet-dependent signaling to the vessel wall remain unclear. We previously showed that hemolysis induces platelet mitochondrial reactive oxygen species (mtROS) production, but the mechanism by which heme induces mtROS production and downstream consequences are less clear. We hypothesize that heme induces platelet mtROS through Toll-like Receptor 4 (TLR4) signaling, which regulates platelet activation and granule secretion. Methods: Washed platelets were prepared from whole blood collected from healthy human volunteers in 0.32% sodium citrate anticoagulant (n=6). Platelets were treated with heme (2.5µM) in presence or absence of TLR4 neutralizing antibody, IRAK1/4 inhibitor, TBK1/IKKε inhibitor, MitoTEMPO or DS16570511 (Mitochondrial calcium uniporter inhibitor). Platelet activation was measured using flow cytometry by staining for CD62P (P-selectin) on the platelet surface and the active confirmation of GPIIb/IIIa. Platelet mtROS was measured by fluorescence spectroscopy using MitoSOX Red. The granule secretion function of platelets was measured by assessing TSP-1 and Platelet Factor 4 (PF4) levels in platelet releasates by ELISA. Results: We found that heme activates platelets and induces mtROS production, which can be mitigated by TLR4 neutralizing antibody, a molecule that makes platelet surface TLR4 unavailable to heme. In heme-treated platelets, pharmacological inhibition of the interleukin-1 receptor kinase-4 (IRAK4), a key kinase recruited by the TLR4 adaptor protein MyD88, had no significant effect on platelet activation. In contrast, inhibition of tank binding kinase-1 (TBK1), a TLR4 effector molecule independent of MyD88 signaling, significantly attenuated heme-mediated platelet activation and mtROS production. Scavenging mtROS with MitoTEMPO did not alter platelet activation, instead it decreased the TSP1 release from platelets, indicating heme induced platelet mtROS tightly regulates the granule secretion function of platelets. In addition, blocking mitochondrial calcium uptake by Mitochondrial calcium uniporter (MCU) inhibitor significantly decreased mtROS production and TSP-1 release from heme treated platelets. Conclusion: Our data demonstrate that heme activates human platelets and induces mtROS production via a TLR4 -MyD88-independent signaling pathway. In addition, heme also induces mitochondrial calcium uptake that enhances mtROS production in platelets. Heme induced platelet mtROS tightly regulates platelet granule secretion function. Ongoing studies are further elucidating this pathway and determining the role of mtROS in heme-dependent platelet function. These studies will provide a mechanistic link between hemolysis and thrombosis, as well as platelet-vascular wall signaling. These studies suggest that platelet mtROS may potentially be a novel therapeutic target to prevent thrombotic events and vascular dysfunction in hemolytic disorders including SCD. Disclosures No relevant conflicts of interest to declare.

Heparin Induced Thrombocytopenia in Patients with COVID-19

BackgroundThe Coronavirus disease-2019 (COVID-19) is a global pandemic caused by novel coronavirus SARS-CoV-2. Acute respiratory and renal failure as well as systemic coagulopathy are critical aspects of the morbidity and mortality in patients with COVID-19. Heparin Induced Thrombocytopenia (HIT) occurs when IgG antibodies form against platelet factor 4-Heparin complex, resulting in platelet activation and removal, leading to a prothrombotic state. HIT is suspected when there is a platelet count decrease of more than 50% after exposure to heparin, along with hypercoagulability. Clinical Scoring systems like 4T Score (Thrombocytopenia, Timing, Thrombosis, no other cause of Thrombocytopenia) have been developed to assess the pretest probability of HIT. The use of unfractionated heparin, post-orthopedic and post-cardiac surgery state, female gender, and old age are recognized as risk factors for HIT. There is a nine-fold increased risk of developing HIT in patients requiring hemodialysis. ICU patients and patients with VTE without thrombocytopenia are considered to have low pre-test probability for HIT. Studies have shown that only 6% who are investigated serologically for HIT actually have the diagnosis. We conducted this study to assess the incidence and risk factors for HIT in COVID-19 positive patients and its impact on mortality.MethodsA retrospective analysis was performed on all patients who were COVID-19 positive and hospitalized between March 1, 2020 and June 26, 2020 at our institution. Patients with intermediate or high suspicion for HIT, based on 4T score of 4 or higher, underwent IgG-specific platelet factor 4(PF4)-dependent enzyme immune assay (EIA). Washed platelet assays such as serotonin release assay (SRA) and heparin-induced platelet aggregation (HIPA) were used as confirmatory tests in cases with intermediate or low optical density (OD) with EIA. The incidence of HIT, its impact on mortality, and positivity of IgG-specific PF4-EIA in COVID-19 patients were studied, and statistical analysis was done with X2 testing. Subgroup analysis was performed based on demographic factors and risk factors for HIT, including exposure to heparin or low molecular weight heparin (LMWH), history of or cancer, recent orthopedic or cardiac surgery, exposure to renal replacement therapy (RRT), and severity of disease (D-dimer >6 ULN, Acute Kidney Injury, ICU admission, and mechanical ventilation requirement). These factors were analyzed by Fisher’s exact test to determine their impact on mortality. The hospital course for HIT antibody-positive patients was further analyzed to study the impact of COVID-19 related therapy, such as Remdesivir, Tocilizumab, Hydroxychloroquine, Steroids, and anticoagulation after diagnosis of HIT.ResultsWEight out of 1265 hospitalized COVID-19 positive patients tested positive for IgG-specific platelet factor 4(PF4)-dependent enzyme immune assay (EIA+). Incidence of EIA+ in COVID-19 patients was 0.6%, which is significantly higher than in the general population 0.2% (p<.0001, 95% CI 0.25-1.20%). The incidence of thromboembolic events in EIA+ patients was 87.5%, significantly higher than the rate of 10.90% in all COVID-19 patients (p<.0001, CI 41.96- 86.98%). The mortality rate in EIA+ patients was 50%, significantly greater than the mortality rate of 12% in all hospitalized COVID-19 patients (p=.0011, CI 9.46-66.53). Serological confirmation of HIT diagnosis was 37.5% (2 had OD>1, 1 was SRA positive) which is significantly higher than confirmation of HIT in nonCOVID-19 patients 6% (p<.0001, 95% CI 29.57-85.32%). All 3 confirmed HIT patients (100%) had severe disease (3/3 required ICU admission, 2/3 required mechanical ventilation, the one not requiring mechanical ventilation required RRT).ConclusionOur study indicates incidence of HIT is higher in the COVID-19 population. The incidence of positive EIA for patients with intermediate to high 4T scores is also higher in COVID-19 positive patients. This can be attributed to the cytokine storm and severe sepsis seen in critically ill COVID-19 patients. Our study also suggests that development of HIT can contribute to increased risk for thromboembolic events as well as mortality of COVID-19 patients, however, our study is limited due to small sample size. Therefore, prospective studies are needed to analyze the impact of HIT on morbidity, mortality and long-term outcomes in COVID-19 patients.TableDisclosuresNo relevant conflicts of interest to declare.

The sodium-glucose cotransporter-2 (SGLT2) inhibitors synergize with nitric oxide and prostacyclin to reduce human platelet activation

Gliflozins (canagliflozin, dapagliflozin and empagliflozin) are the newest anti-hyperglycemic class and have offered cardiovascular and renal benefits. Because platelets are involved in the atherothrombosis process, this study is aimed to evaluate the direct effect of gliflozins on platelet reactivity. Platelet-rich plasma (PRP) or washed platelets (WP) were obtained from healthy volunteers. Aggregation, flow cytometry for glycoprotein IIb/IIIa, cyclic nucleotides and intracellular calcium levels, Western blot, thromboxane B2 (TXB2) measurement and COX-1 activity were performed in the presence of gliflozins (1–30 μM) alone or in combination with sodium nitroprusside (SNP, 10 or 100 nM) + iloprost (ILO, 0.1 or 1 nM). SGLT2 protein is not expressed on human platelets. Gliflozins produced little inhibitory effect in agonists-induced aggregation and this effect was greatly potentiated by ~10-fold in the presence of SNP + ILO, accompanied by lower levels of TXB2 (58.1 ± 5.1%, 47.1 ± 7.2% and 43.4 ± 9.2% inhibition for canagliflozin, dapagliflozin and empagliflozin, respectively). The activity of COX-1 was not affected by gliflozins. Collagen increased Ca2+ levels and α(IIb)β(3) activation, both of which were significantly reduced by gliflozins + SNP + ILO. The intracellular levels of cAMP and cGMP and the protein expression of p-VASPSer157 and p-VASPSer239 were not increased by gliflozins while the expression of the serine-threonine kinase, AktSer473 was markedly reduced. Our results showed that the antiplatelet activity of gliflozins were greatly enhanced by nitric oxide and prostacyclin, thus suggesting that the cardiovascular protection seen by this class of drugs could be in part due to platelet inhibition.

Washed Equine Platelet Extract as an Anti-Inflammatory Biologic Pharmaceutical

Mammalian platelets participate in the immediate tissue injury response by initiating coagulation and further promoting tissue injury mitigation and repair. The latter properties are deployed following platelet release of presynthetized morphogens, cytokines, and growth and chemotactic factors, which launch a tissue regenerative, angiogenic, and anti-inflammatory program. Several blood-derived biologic products, like platelet-rich plasma (PRP) and platelet lysate (PL), are currently on the market to allow proper healing and tissue regeneration. However, not all growth factors are released from the platelets and the final products contain plasma proteins such as albumin, fibrinogen, complement, and immunoglobulins, increasing the risks of serum sickness or allergic reaction. To address this problem, we developed a new platelet extract where equine blood platelets are concentrated, washed, and thereafter lysed by detergent Triton X-114. Distinct from PRP, this extract is devoid of albumin, fibrinogen, and immunoglobulins and is 266-fold enriched in platelet-derived growth factor content relative to PRP. Washed equine platelet extract (WEPLEX) is amenable to lyophilization without loss of biological activity. In vitro, WEPLEX significantly inhibits human and equine T cell proliferative response to phytohemagglutinin and also polarizes murine CD45+/CD11b+ peritoneal macrophages to an IL-10+ M2-like phenotype. In vivo, WEPLEX substantially improves clinical outcome of murine experimental dextran sulfate sodium colitis. We propose that equine-sourced, zoonosis-free WEPLEX may serve as an anti-inflammatory biological therapy across mammalian species.

Evaluation of functional assays for the diagnosis of heparin induced thrombocytopenia using 5B9, a monoclonal IgG that mimics human antibodies

BackgroundSerotonin release assay (SRA) is considered as the “gold standard” for detecting pathogenic heparin‐induced thrombocytopenia (HIT) antibodies. However, this method is time consuming, expensive, and uses radioelements. Heparin‐induced multiple electrode aggregometry (HIMEA), light transmission aggregometry (LTA) with platelet rich plasma (PRP) or washed platelets (WP), adenosine triphosphate (ATP) release, and flow cytometry (FC) are available alternatives. ObjectivesTo evaluate the performance of these assays, comparatively with SRA, for detecting HIT antibodies, using 5B9, a monoclonal IgG fully mimicking human HIT antibodies. Patients/MethodsHeparin‐dependent platelet activation induced by 5B9 (50/20/10 µg/mL) was evaluated by all assays performed on the same day using platelets from 20 healthy donors. The three methods exhibiting the highest sensitivity to 5B9 were then assessed by testing samples from patients with either likely (n = 10), or indeterminate/unlikely HIT (n = 10). ResultsAll methods exhibited good sensitivity for detecting 5B9 50 µg/mL, but only SRA and HIMEA were positive with 100% of donors using 5B9 20 µg/mL, followed by FC (83%). SRA detected 5B9 10 μg/mL with 90% of donors, while HIMEA and FC were positive in 45% and 44% of cases, respectively. Whereas SRA was positive with 9/10 samples from likely HIT, HIMEA and FC were positive with 6 and 7 of them, respectively. Neither SRA nor HIMEA was positive with indeterminate/unlikely HIT samples, while FC was positive or doubtful in three cases. ConclusionsSerotonin release assay likely remains the most sensitive and specific assay for detecting platelet activating HIT antibodies, but HIMEA or FC are potential alternatives, despite being less performant.

Diallyl Trisulfide Inhibits Platelet Aggregation through the Modification of Sulfhydryl Groups.

Diallyl trisulfide (DATS) is a secondary metabolite of allicin, a volatile organosulfur flavoring compound generated by the crushing of garlic. These compounds have various medicinal effects such as antiplatelet activity. In this study, we demonstrated for the first time the cellular mechanism involved in the inhibition of platelet aggregation by DATS and dipropyl trisulfide (DPTS), which is a saturated analogue of DATS. Washed murine platelets were incubated with these sulfides, and platelet aggregation was evaluated by light transmission aggregometry. The amount of reaction products produced by DATS, DPTS, and glutathione (GSH) was measured using liquid chromatography-mass spectrometry. Compared with DPTS, DATS potently inhibited platelet aggregation induced by thrombin, U46619, and collagen. N-Ethylmaleimide (NEM), which is commonly used to modify sulfhydryl groups, also suppressed platelet aggregation. The reactivity of DATS with GSH was higher than that of DPTS. These data suggested that DATS inhibited platelet aggregation through the reaction of sulfhydryl groups.

Epinephrine restores platelet functions inhibited by ticagrelor: A mechanistic approach

Ticagrelor, an antagonist of the platelet adenosine diphosphate (ADP)-P2Y12 receptor is recommended for patients with acute coronary syndromes. However, ticagrelor exposes to a risk of bleeding, the management of which is challenging because platelet transfusion is ineffective, and no antidote is yet available. We hypothesized that the vasopressor drug epinephrine could counter the antiplatelet effects of ticagrelor and restore platelet functions. We assessed in vitro the efficiency of epinephrine in restoring platelet aggregation inhibited by ticagrelor and investigated the underlying mechanisms. Washed platelet aggregation and secretion were measured upon stimulation by epinephrine alone or in combination with ADP, in the presence or absence of ticagrelor. Mechanistic investigations used P2Y1 and phosphoinositide 3-kinase (PI3K) inhibitors and included vasodilator-stimulated phosphoprotein (VASP) and Akt phosphorylation assays as well as measurement of Ca2+ mobilisation. We found that epinephrine restored ADP-induced platelet aggregation, but not dense granule release. Epinephrine alone failed to induce aggregation whereas it fully induced VASP dephosphorylation and Akt phosphorylation regardless of the presence of ticagrelor. In the presence of ticagrelor, blockage of the P2Y1 receptor prevented restoration of platelet aggregation by the combination of epinephrine and ADP, as well as intracellular Ca2+ mobilisation. In combination with ADP, epinephrine induced platelet aggregation of ticagrelor-treated platelets through inhibition of the cAMP pathway and activation of the PI3K pathway, thus enabling the P2Y1 receptor signalling and subsequent Ca2+ mobilisation. This proof-of-concept study needs to be challenged in vivo for the management of bleeding in ticagrelor-treated patients.