Thromboxane A2 Generation Research Articles

Prostacyclin Synthase Deficiency Leads to Exacerbation or Occurrence of Endothelium-Dependent Contraction and Causes Cardiovascular Disorders Mainly via the Non-TxA2 Prostanoids/TP Axis.

Prostaglandin I2 synthesized by endothelial COX (cyclooxygenase) evokes potent vasodilation in some blood vessels but is paradoxically responsible for endothelium-dependent constriction (EDC) in others. Prostaglandin I2 production and EDC may be enhanced in diseases such as hypertension. However, how PGIS (prostaglandin I2 synthase) deficiency affects EDC and how this is implicated in the consequent cardiovascular pathologies remain largely unknown. Experiments were performed with wild-type, Pgis knockout (Pgis-/-) and Pgis/thromboxane-prostanoid receptor gene (Tp) double knockout (Pgis-/-Tp-/-) mice and Pgis-/- mice transplanted with unfractionated wild-type or Cox-1-/- bone marrow cells, as well as human umbilical arteries. COX-derived prostanoids were measured by high-performance liquid chromatography-mass spectrometry. Vasomotor responses of distinct types of arteries were assessed by isometric force measurement. Parameters of hypertension, vascular remodeling, and cardiac hypertrophy in mice at different ages were monitored. PGF2α, PGE2, and a trace amount of PGD2, but not thromboxane A2 (TxA2), were produced in response to acetylcholine in Pgis-/- or PGIS-inhibited arteries. PGIS deficiency resulted in exacerbation or occurrence of EDC ex vivo and in vivo. Endothelium-dependent hyperpolarization was unchanged, but phosphorylation levels of eNOS (endothelial nitric oxide synthase) at Ser1177 and Thr495 were altered and NO production and the NO-dependent relaxation evoked by acetylcholine were remarkably reduced in Pgis-/- aortas. Pgis-/- mice developed high blood pressure and vascular remodeling at 16 to 17 weeks and subsequently cardiac hypertrophy at 24 to 26 weeks. Meanwhile, blood pressure and cardiac parameters remained normal at 8 to 10 weeks. Additional ablation of TP (TxA2 receptor) not only restrained EDC and the downregulation of NO signaling in Pgis-/- mice but also ameliorated the cardiovascular abnormalities. Stimulation of Pgis-/- vessels with acetylcholine in the presence of platelets led to increased TxA2 generation. COX-1 disruption in bone marrow-derived cells failed to affect the development of high blood pressure and vascular remodeling in Pgis-/- mice though it largely suppressed the increase of plasma TxB2 (TxA2 metabolite) level. Our study demonstrates that the non-TxA2 prostanoids/TP axis plays an essential role in mediating the augmentation of EDC and cardiovascular disorders when PGIS is deficient, suggesting TP as a promising therapeutic target in diseases associated with PGIS insufficiency.

Optimizing aspirin dose for colorectal cancer patients through deep phenotyping using novel biomarkers of drug action.

Background: Low-dose aspirin's mechanism of action for preventing colorectal cancer (CRC) is still debated, and the optimal dose remains uncertain. We aimed to optimize the aspirin dose for cancer prevention in CRC patients through deep phenotyping using innovative biomarkers for aspirin's action. Methods: We conducted a Phase II, open-label clinical trial in 34 CRC patients of both sexes randomized to receive enteric-coated aspirin 100mg/d, 100mg/BID, or 300mg/d for 3 ± 1weeks. Biomarkers were evaluated in blood, urine, and colorectal biopsies at baseline and after dosing with aspirin. Novel biomarkers of aspirin action were assessed in platelets and colorectal tissues using liquid chromatography-mass spectrometry to quantify the extent of cyclooxygenase (COX)-1 and COX-2 acetylation at Serine 529 and Serine 516, respectively. Results: All aspirin doses caused comparable % acetylation of platelet COX-1 at Serine 529 associated with similar profound inhibition of platelet-dependent thromboxane (TX)A2 generation ex vivo (serum TXB2) and in vivo (urinary TXM). TXB2 was significantly reduced in CRC tissue by aspirin 300mg/d and 100mg/BID, associated with comparable % acetylation of COX-1. Differently, 100mg/day showed a lower % acetylation of COX-1 in CRC tissue and no significant reduction of TXB2. Prostaglandin (PG)E2 biosynthesis in colorectal tumors and in vivo (urinary PGEM) remained unaffected by any dose of aspirin associated with the variable and low extent of COX-2 acetylation at Serine 516 in tumor tissue. Increased expression of tumor-promoting genes like VIM (vimentin) and TWIST1 (Twist Family BHLH Transcription Factor 1) vs. baseline was detected with 100mg/d of aspirin but not with the other two higher doses. Conclusion: In CRC patients, aspirin 300mg/d or 100mg/BID had comparable antiplatelet effects to aspirin 100mg/d, indicating similar inhibition of the platelet's contribution to cancer. However, aspirin 300mg/d and 100mg/BID can have additional anticancer effects by inhibiting cancerous tissue's TXA2 biosynthesis associated with a restraining impact on tumor-promoting gene expression. EUDRACT number: 2018-002101-65. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT03957902.

Adjustment for Renal Function Improves the Prognostic Performance of Urinary Thromboxane Metabolites.

Systemic thromboxane A2 generation, assessed by quantifying the concentration of stable thromboxane B2 metabolites (TXB2-M) in the urine adjusted for urinary creatinine, is strongly associated with mortality risk. We sought to define optimal TXB2-M cutpoints for aspirin users and nonusers and determine if adjusting TXB2-M for estimated glomerular filtration rate (eGFR) in addition to urinary creatinine improved mortality risk assessment. Urinary TXB2-M were measured by competitive ELISA in 1363 aspirin users and 1681 nonusers participating in the Framingham Heart Study. Cutpoints were determined for TXB2-M and TXB2-M/eGFR using log-rank statistics and used to assess mortality risk by Cox proportional hazard modeling and restricted mean survival time. Multivariable models were compared using the Akaike Information Criterion (AIC). A cohort of 105 aspirin users with heart failure was used for external validation. Optimized cutpoints of TXB2-M were 1291 and 5609 pg/mg creatinine and of TXB2-M/eGFR were 16.6 and 62.1 filtered prostanoid units (defined as pg·min/creatinine·mL·1.73 m2), for aspirin users and nonusers, respectively. TXB2-M/eGFR cutpoints provided more robust all-cause mortality risk discrimination than TXB2-M cutpoints, with a larger unadjusted hazard ratio (2.88 vs 2.16, AIC P < 0.0001) and greater differences in restricted mean survival time between exposure groups (1.46 vs 1.10 years), findings that were confirmed in the external validation cohort of aspirin users. TXB2-M/eGFR cutpoints also provided better cardiovascular/stroke mortality risk discrimination than TXB2-M cutpoints (unadjusted hazard ratio 3.31 vs 2.13, AIC P < 0.0001). Adjustment for eGFR strengthens the association of urinary TXB2-M with long-term mortality risk irrespective of aspirin use.

Chymase-independent vascular Ang-(1–12)/Ang II pathway and TXA2 generation are involved in endothelial dysfunction in the murine model of heart failure

The angiotensin (Ang)-(1–12)/Ang II pathway contributes to cardiac pathology. However, its involvement in the development of peripheral endothelial dysfunction associated with heart failure (HF) remains unknown. Therefore, this study aimed to characterise the effect of exogenous Ang-(1–12) and its conversion to Ang II on endothelial function using the murine model of HF (Tgαq*44 mice), focusing on the role of chymase and vascular-derived thromboxane A2 (TXA2).Ex vivo myographic assessments of isolated aorta showed impaired endothelium-dependent vasodilation in late-stage HF in 12-month-old Tgαq*44 mice. However, endothelium-dependent vasodilation was fully preserved in the early stage of HF in 4-month-old Tgαq*44 mice and 4- and 12-month-old FVB control mice. Ang-(1–12) impaired endothelium-dependent vasodilation in 4- and 12-month-old Tgαq*44 mice, that was associated with increased Ang II production. The chymase inhibitor chymostatin did not inhibit this response. Interestingly, TXA2 production reflected by TXB2 measurement was upregulated in response to Ang-(1–12) and Ang II in aortic rings isolated from 12-month-old Tgαq*44 mice but not from 4-month-old Tgαq*44 mice or age-matched FVB mice. Furthermore, in vivo magnetic resonance imaging showed that Ang-(1–12) impaired endothelium-dependent vasodilation in the aorta of Tgαq*44 mice and FVB mice. However, this response was inhibited by angiotensin I converting enzyme (ACE) inhibitor; perindopril, angiotensin II receptor type 1 (AT1) antagonist; losartan and TXA2 receptor (TP) antagonist-picotamide in 12-month-old-Tgαq*44 mice only.In conclusion, the chymase-independent vascular Ang-(1–12)/Ang II pathway and subsequent TXA2 overactivity contribute to systemic endothelial dysfunction in the late stage of HF in Tgαq*44 mice. Therefore, the vascular TXA2 receptor represents a pharmacotherapeutic target to improve peripheral endothelial dysfunction in chronic HF.

The Inhibitory Effects of Protaetia brevitarsis seulensis Larvae Extract on Human Platelet Aggregation and Glycoprotein IIb/IIIa Expression.

The white-spotted flower chafer, Protaetia brevitarsis seulensis, is used as a traditional remedy against liver cirrhosis, hepatitis, and hepatic cancer. In this study, we investigated if P. brevitarsis extract (PBE) inhibited platelet aggregation via integrin αIIb/β3 regulation. We observed that PBE inhibited αIIb/β3 activation by regulating the cyclic nucleotides, cyclic adenosine monophosphate and cyclic guanosine monophosphate. Additionally, PBE affected phosphatidylinositol-3 kinase, Akt, SYK, glycogen synthase kinase-3α/β, cytosolic phospholipase A2, and p38 expression, which are signal transduction molecules expressed by platelets, and consequently suppressed αIIbβ3 activity and thromboxane A2 generation. Taken together, PBE showed strong antiplatelet effects and may be used to block thrombosis- and platelet-mediated cardiovascular diseases.

Acetyl-CoA carboxylase 1 deletion reduces platelet phospholipid and alters thrombus formation

Acetyl-CoA carboxylase (ACC) is the key enzyme regulating de novo lipogenesis (DNL). In recent years, ACC inhibitors has been of great interest to handle a wide range of diseases, especially cancers and cardiovascular diseases. However, the impact of its inhibition on hemostasis and thrombosis remains under-investigated. We previously demonstrated that ACC1 activation promotes thrombus formation by increasing platelet phospholipid content and thromboxane A2 generation. Here, we sought to evaluate whether ACC1 platelet-specific deletion may affect platelet functions and by decreasing phospholipid content. To test this hypothesis we generated a new Cre transgenic mouse strain in which ACC1 is specifically deleted in megakaryocyte and platelet (GpIbCre±x ACC1 flx/flx mouse). Lipidomics analysis was carried out on the commercial Lipidyzer platform. Thromboxane A2 secretion was evaluated by ELISA. Platelet functions were assessed in vitro by aggregometry and ex vivo by flow chamber assay. Thrombus growth was analyzed in vivo with intravital microscopy. As expected, ACC1 deletion was restricted to megakaryocytic lineage. Hematological parameters show a platelet count decrease (30%) and a platelet volume increase (31%) in ACC1 knockout mice (ACC1 pKO) compared to the control mice. In agreement with our hypothesis, lipidomics analyses highlight a significant reduction in arachidonic acid containing phosphotidylethanolamine plasmalogen in ACC1 KO platelets. Logically, thromboxane A2 generation (upon thrombin or CRP stimulation) is also altered in ACC1 pKO mice in comparison to controls. This decrease in phospholipid content is associated with a default in thrombin and CRP-induced platelet aggregation and with impaired dense granules secretion. In line with these results, platelet-specific ACC1 deletion reduces thrombus growth and fibrinogen receptor activation. Platelet-specific ACC1 deletion leads to a decrease in phospholipid content which, in turn, decreases platelet thromboxane A2 generation, dense granules secretion and platelet aggregation. Thrombus formation is also reduced in platelet-specific ACC1 knockout mice, highlighting the importance of de novo lipogenesis and ACC-produced lipids in the regulation of platelet functions.

Antiplatelet Effect of Daphnetin Is Regulated by cPLA2-Mediated Thromboxane A2 Generation in Mice.

Coumarin derivatives have been recognized for their antithrombotic, anti-inflammatory, and antioxidant properties, and daphnetin is one of the natural coumarin derivatives isolated from Daphne Koreana Nakai. Although the pharmacological value of daphnetin is well documented in diverse biological activities, its antithrombotic effect has not been studied to date. Here, we characterized the role and underlying mechanism of daphnetin in the regulation of platelet activation using murine platelets. In order to check the effect of daphnetin on platelet function, we first measured the effect of daphnetin on platelet aggregation and secretion. Collagen-induced platelet aggregation and dense granule secretion were partially inhibited by daphnetin. Interestingly, 2-MeSADP-induced secondary waves of aggregation and secretion were completely inhibited by daphnetin. It is known that 2-MeSADP-induced secretion and the resultant secondary wave of aggregation are mediated by the positive feedback effect of thromboxane A2 (TxA2) generation, suggesting the important role of daphnetin on TxA2 generation in platelets. Consistently, daphnetin did not affect the 2-MeSADP-induced platelet aggregation in aspirinated platelets where the contribution of TxA2 generation was blocked. Additionally, platelet aggregation and secretion induced by a low concentration of thrombin, which is affected by the positive feedback effect of TxA2 generation, were partially inhibited in the presence of daphnetin. Importantly, 2-MeSADP- and thrombin-induced TxA2 generation was significantly inhibited in the presence of daphnetin, confirming the role of daphnetin on TxA2 generation. Finally, daphnetin significantly inhibited 2-MeSADP-induced cytosolic phospholipase A2 (cPLA2) and ERK phosphorylation in non-aspirinated platelets. Only cPLA2 phosphorylation, not ERK phosphorylation, was significantly inhibited by daphnetin in aspirinated platelets. In conclusion, daphnetin plays a critical role in platelet function by inhibiting TxA2 generation through the regulation of cPLA2 phosphorylation.

Sulforaphane attenuates platelet granule secretion through down-regulating glycoprotein VI-mediated p38 MAPK/cPLA2 signaling pathway

ABSTRACT Glycoprotein VI (GPVI) is the major collagen receptor on the platelet surface and is important in mediating granule secretion and atherothrombosis. Sulforaphane (SFN), a dietary isothiocyanate enriched in cruciferous vegetables, exerts multiple biological activities. This study aimed to investigate the efficacy of SFN on GPVI-mediated signaling and platelet granule secretion in vitro. We herein demonstrated that SFN greatly attenuated granule secretion in human gel-filtered platelets in response to collagen, including inhibiting CD62P and CD63 expression and PF4, CCL5, and ATP release. Mechanistically, these inhibitory effects of SFN were mainly mediated by its attenuation of cytosolic phospholipase A2 (cPLA2) phosphorylation and thromboxane A2 (TxA2) generation. Moreover, SFN did not further decrease the inhibitory effects of p38 MAPK inhibitor SB203580 on cPLA2 phosphorylation, TxA2 generation and granule secretion. Thus, SFN attenuates platelet granule secretion via down-regulating GPVI-mediated p38 MAPK/cPLA2/TxA2 signaling, which may play important preventive roles in cardiovascular diseases.

Pharmacological Actions of 5-Hydroxyindolin-2 on Modulation of Platelet Functions and Thrombus Formation via Thromboxane A2 Inhibition and cAMP Production

Platelets play a very significant role in hemostasis while simultaneously posing a risk for the development of various cardiovascular diseases. Platelet-mediated issues can occur in blood vessels and trigger various medical problems. Therefore, controlling platelet function is important in the prevention of thrombosis. In this regard, we need to find compounds that provide potent antiplatelet activity with minimum side effects. Therefore, we examined the effect of 5-hydroxyindolin-2-one isolated from Protaetia brevitarsis larvae having antiplatelet properties and investigated different pathways that mediate the antiplatelet activity. We examined the effect of 5-hydroxyindolin-2-one (5-HI) on the regulation of phosphoproteins, thromboxane A2 generation, and integrin αIIbβ3 action. Our data showed that human platelet aggregation was inhibited by 5-HI (75, 100, 150, 200 μM) without cytotoxicity, and it suppressed intracellular Ca2+ concentration through the regulation of inositol 1, 4, 5-triphosphate receptor I (Ser1756) and extracellular signal-regulated kinase (ERK). Moreover, collagen-elevated thromboxane A2 production and αIIbβ3 action were inhibited by 5-HI through the regulation of cytosolic phospholipase A2 (cPLA2), mitogen-activated protein kinase p38 (p38MAPK), vasodilator-stimulated phosphoprotein (VASP), phosphoinositide 3-kinase (PI3K), and Akt (protein kinase B). Therefore, we suggested that 5-HI could be a potential substance for the prevention of thrombosis-mediated thrombosis.

Characterization of molecular basis of anti‐platelet effect of daphnetin

Coumarin derivates have been known for their antithrombotic, anti‐inflammatory, and antioxidant activities and daphnetin is one of the natural coumarin derivatives isolated from Daphne Korean Nakai. Although the pharmacological value of daphnetin is well documented in diverse biological activities, its antithrombotic effect is not studied till date.We investigated the functional role of daphnetin and its molecular basis on the regulation of platelet activation using murine platelets.In order to check the effect of daphnetin on GPVI‐mediated platelet response, we first measured the effect of daphnetin on collagen‐induced platelet aggregation and secretion. Collagen‐induced platelet aggregation and dense granule secretion were partially inhibited in the presence of daphnetin. Interestingly, 2‐MeSADP‐induced secondary wave of aggregation and secretion were completely inhibited in the presence of daphnetin. It has been known that 2‐MeSADP‐induced secretion and the resultant secondary wave of aggregation are mediated by positive‐feedback effect of thromboxane A2 (TxA2) generation, suggesting the important role of daphnetin on TxA2 generation in platelets. Consistently, daphnetin did not affect the 2‐MeSADP‐induced platelet aggregation in aspirin‐treated platelets where the contribution of TxA2 generation is blocked. Additionally, AYPGKF‐induced platelet aggregation and secretion were inhibited in the presence of daphnetin by blocking the positive‐feedback effect of TxA2 generation. Moreover, 2‐MeSADP‐induced TxA2 generation was completely inhibited in the presence of daphnetin confirming the role of daphnetin on TxA2 generation. Finally, 2‐MeSADP‐induced ERK phosphorylation was inhibited in the presence of daphnetin.We conclude that daphnetin plays a role in platelet function by inhibiting the TxA2 generation through the regulation of ERK phosphorylation.

Anti‐platelet effect of prednisolone is regulated by ERK‐mediated thromboxane generation

Endogenous hypercortisolism is associated with increased risk of blood‐related diseases such as high blood pressure and hyperglycemia that result in fatal cardiovascular disorders. Platelet plays a major role in hemostasis and thrombosis. Although it is well known that glucocorticoids exert a significant effect on hemostasis, the effect of endogenous cortisol on platelet function is much less clear.We aimed to characterize the in vitro effect of prednisolone and its molecular basis on regulation of platelet function using murine platelets.We first evaluated the concentration‐dependent effect of prednisolone on 2‐MeSADP‐induced platelet function and found that 2‐MeSADP‐induced secondary wave of aggregation and dense granule secretion were completely inhibited from 500 nM prednisolone. Since 2‐MeSADP‐induced secretion and the resultant secondary wave of aggregation is mediated by thromboxane A2 (TxA2) generation, this result suggested a role of prednisolone on TxA2generation in platelets. Consistently, prednisolone did not affect the 2‐MeSADP‐induced aggregation in aspirin‐treated platelets, where the secondary wave of aggregation and secretion were blocked by eliminating the contribution of TxA2 generation by aspirin. In addition, AYPGKF‐ and thrombin‐induced platelet aggregation and secretion were inhibited in the presence of prednisolone by inhibiting the positive‐feedback effect of TxA2 generation on platelet function. Furthermore, prednisolone completely inhibited 2‐MeSADP‐induced TxA2 generation confirming the role of prednisolone on TxA2 generation. Finally, Western blot analysis revealed that prednisolone inhibited the 2‐MeSADP‐induced ERK phosphorylation.In conclusion, prednisolone affects platelet function by the inhibition of TxA2 generation through the regulation of ERK phosphorylation, thereby shedding light on the clinical characterization and its treatment efficacy in patients with cardiovascular disorders in the future.

Protein disulfide isomerase‐A1 regulates intraplatelet reactive oxygen species–thromboxane A2‐dependent pathway in human platelets

BackgroundPlatelet‐derived protein disulfide isomerase 1 (PDIA1) regulates thrombus formation, but its role in the regulation of platelet function is not fully understood. AimsThe aim of this study was to characterize the role of PDIA1 in human platelets. MethodsProteomic analysis of PDI isoforms in platelets was performed using liquid chromatography tandem mass spectometry, and the expression of PDIs on platelets in response to collagen, TRAP‐14, or ADP was measured with flow cytometry. The effects of bepristat, a selective PDIA1 inhibitor, on platelet aggregation, expression of platelet surface activation markers, thromboxane A2 (TxA2), and reactive oxygen species (ROS) generation were evaluated by optical aggregometry, flow cytometry, ELISA, and dihydrodichlorofluorescein diacetate‐based fluorescent assay, respectively. ResultsPDIA1 was less abundant compared with PDIA3 in resting platelets and platelets stimulated with TRAP‐14, collagen, or ADP. Collagen, but not ADP, induced a significant increase in PDIA1 expression. Bepristat potently inhibited the aggregation of washed platelets induced by collagen or convulxin, but only weakly inhibited platelet aggregation induced by TRAP‐14 or thrombin, and had the negligible effect on platelet aggregation induced by arachidonic acid. Inhibition of PDIA1 by bepristat resulted in the reduction of TxA2 and ROS production in collagen‐ or thrombin‐stimulated platelets. Furthermore, bepristat reduced the activation of αIIbβ3 integrin and expression of P‐selectin. ConclusionsPDIA1 acts as an intraplatelet regulator of the ROS‐TxA2 pathway in collagen‐GP VI receptor‐mediated platelet activation that is a mechanistically distinct pathway from extracellular regulation of αIIbβ3 integrin by PDIA3.

Platelet-specific deletion of acetyl-CoA carboxylase 1 decreases phospholipid content and impairs platelet functions

Abstract Background Acetyl-CoA carboxylase (ACC), the first enzyme regulating lipid synthesis, promotes thrombus formation by increasing platelet phospholipid content and thromboxane A2 generation. Purpose Our study sought to evaluate whether ACC1 platelet-specific deletion may affect platelet functions by decreasing phospholipid content. Methods We generated a new Cre transgenic mouse strain that allows megakaryocyte/platelet specific ACC1 deletion (GpIbCre+/− x ACC1 flx/flx mouse). In vitro, platelet functions were assessed by aggregometry and flow cytometry. In vivo, hemostasis was assessed via the measurement of bleeding time. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Thromboxane A2 secretion was evaluated by ELISA. Results As expected, ACC1 deletion was restricted to the megakaryocytic lineage. Hematological parameters in platelet-specific ACC1 knockout mice showed a decrease in platelet count by 30% and an increase in platelet volume by 31%, compared to ACC1 flx/flx platelets. In vitro, platelets from platelet-specific ACC1 knockout mice displayed a decrease in thrombin and CRP-induced platelet aggregation, associated with impaired dense granules secretion. In contrast, ADP-induced platelet aggregation was higher in the absence of ACC1. In vivo, platelet-specific ACC1 knockout mice showed a normal bleeding time. In agreement with our hypothesis, lipidomics analyses showed that ACC1 deletion in platelets was associated with a significant decrease in arachidonic acid-contaning phosphatidylethanolamine plasmalogen, and subsequently with a reduced production of thromboxane A2 upon thrombin or CRP stimulation. Conclusion Platelet-specific ACC1 deletion led to a decrease in phospholipid content which, in turn, decreased platelet thromboxane A2 generation, dense granules secretion and aggregation upon thrombin and CRP, but not ADP stimulation. Further studies are needed to elucidate the impact of ADP on platelet functions Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Fonds pour la formation à la Recherche dans l'Industrie et l'Agriculture (FRIA)

Tetrahydrocurcumin Downregulates MAPKs/cPLA2 Signaling and Attenuates Platelet Thromboxane A2 Generation, Granule Secretion, and Thrombus Growth.

Platelet granule secretion plays a key role in atherothrombosis. Curcumin, a natural polyphenol compound derived from turmeric, exerts multiple biological activities. The current study sought to investigate the efficacy of tetrahydrocurcumin (THC, the major active metabolite of curcumin) on platelet granule secretion in vitro and thrombus formation in vivo. We found that THC significantly attenuated agonist-induced granule secretion in human gel-filtered platelets in vitro, including CD62P and CD63 expression and platelet factor 4, CCL5, and adenosine triphosphate release. These inhibitory effects of THC were partially mediated by the attenuation of cytosolic phospholipase A2 (cPLA2) phosphorylation, leading to a decrease in thromboxane A2 (TxA2) generation. Moreover, the MAPK (Erk1/2, JNK1/2, and p38 MAPK) signaling pathways were downregulated by THC treatment, resulting in reduced cPLA2 activation, TxA2 generation, and granule secretion. Additionally, THC and curcumin attenuated murine thrombus growth in a FeCl3-induced mesenteric arteriole thrombosis model in C57BL/6J mice without prolonging the tail bleeding time. THC exerted more potent inhibitory effects on thrombosis formation than curcumin. Through blocking cyclooxygenase-1 activity and thus inhibiting platelet TxA2 synthesis and granule secretion with aspirin, we found that THC did not further decrease the inhibitory effects of aspirin on thrombosis formation. Thus, through inhibiting MAPKs/cPLA2 signaling, and attenuating platelet TxA2 generation, granule secretion, and thrombus formation, THC may be a potent cardioprotective agent.

Cytoplasmic Phospholipase A2 Is Essential in GPVI Signaling Initiated Procoagulant Platelet Formation

Procoagulant platelets (PP), a subpopulation of highly activated platelets, are essential in hemostasis by supporting active tenase and prothrombinase complexes on their phosphatidylserine (PSer) externalized surface. When platelets are stimulated with a GPVI agonist, such as convulxin (CVX) or collagen related peptide (CRP), both extracellular calcium entry and mitochondrial permeability transition pore (mPTP) formation are required to mediate the transition to the procoagulant phenotype. Activation of phospholipase A2 (PLA2) is an essential prerequisite for GPVI stimulated thromboxane A2 (TXA2) generation that induces paracrine platelet activation and aggregation. Active PLA2 cleaves the arachidonic acid from the second carbon group of glycerol within the phospholipid membrane. The resultant arachidonic acid, upon downstream enzymatic modification, is modified into TXA2, leukotrienes, and other metabolites. Inhibitors of cyclooxygenases (i.e. aspirin), which prevent arachidonic acid conversion to TXA2 are widely used clinically as antiplatelet agents. Here we investigate the role of PLA2 and its metabolites in inducing GPVI-induced PP formation.Human platelets (106/mL) were activated in the presence of three distinct PLA2 inhibitors: non-specific (PLA2; oleyloxyethyl phosphorylcholine, 1 μM), and specific to inducible (iPLA2; bromoenol lactone, 1 μM) and Ca2+-dependent cytosolic PLA2 (cPLA2; PACOCF3, 1 μM). Examination of platelets in these dilute conditions facilitates both the assessment of autocrine effects of PLA2 and its metabolites and flow cytometry analysis. Platelets were stimulated with different concentrations of GPVI agonists (CRP and CVX), stained (Annexin V for PSer, PAC1 for integrin activation and P-Selectin to assess granule release) and fixed. Additionally pharmacologic inhibitors of the TXA2 receptor (SQ29548, 0.5 μM) and lipoxygenases were utilized.As expected, PP formation in this setting was GPVI signaling and mPTP mediated, as dasatinib (SRC kinase inhibitor, 1 μM) and cyclosporin A (cyclophilin D inhibitor, 5 μM) completely inhibited PP formation. Analysis of platelet activation by flow cytometry demonstrated no effect of PLA2 inhibition on either platelet aggregatory ability or granule release. In contrast, procoagulant platelet formation was significantly decreased in the presence of the non-specific PLA2 inhibitor oleyloxyethyl phosphorylcholine (~59% decrease) and the specific cPLA2 inhibitor PACOCF3 (~51% decrease). Specific iPLA2 inhibition had no effect on any aspect of platelet activation. These results indicate that cPLA2-mediated events are required for autocrine induction of procoagulant platelet formation by GPVI. To begin to interrogate the downstream effectors mediating cPLA2's effects PP formation was examined in the presence of inhibitors of TXA2-mediated platelet activation (SQ29548) and lipoxygenase inhibitors, specifically zileuton, caffeic acid and 8,11,14-eicosatriynoate (all at 1 μM). Inhibition of either of these metabolite-mediated pathways had no effect on GPVI-induced PP formation. In contrast to TXA2's lack of effect on autocrine PP formation, platelet accretion on collagen, presumably mediated by paracrine stimulation of the TXA2 receptor, was decreased by 70% in the presence of SQ29548.These studies identify a novel pathway, mediated through activation of cPLA2, that is essential for GPVI induced PP formation. This cPLA2-mediated pathway is not mediated through the TXA2 receptor, and is distinct from that required for TXA2-mediated paracrine activation of platelet aggregation. Consistent with the findings presented here, previous studies have demonstrated minimal effect of cyclooxygenase inhibition on procoagulant platelet formation. Thus, our studies identify a novel aspirin-insensitive pathway mediating PP formation, and suggest a novel pharmacologic target that could be used for the treatment of procoagulant platelet related pathologic thromboses without affecting proaggregatory phenotype. DisclosuresNo relevant conflicts of interest to declare.

SARS-CoV-2 vaccination induced cerebral venous sinus thrombosis: Do megakaryocytes, platelets and lipid mediators make up the orchestra?

The COVID-19 vaccines comprised of adenoviral vectors encoding the Spike (S) glycoprotein of SARS-CoV-2 are highly effective but associated with rare thrombotic complications. The adenovirus vector infects epithelial cells expressing the coxsackievirus and adenovirus receptor (CAR). The S glycoprotein expressed locally stimulates neutralizing antibody and cellular immune responses. These vaccines have been associated with thromboembolic events including cerebral venous sinus thrombosis (CVST). S glycoprotein stimulates the expression of cyclooxygenase-2 (COX-2) and leads to massive generation of thromboxane A2 in COVID-19. Megakaryocytes express CAR and we postulate that S glycoprotein stimulated generation of thromboxane A2 leads to megakaryocyte activation, biogenesis of activated platelets and thereby increased thrombogenicity. Cerebral vein sinuses express podoplanin, a natural ligand for CLEC2 receptors on platelets. Platelets traversing through the cerebral vein sinuses could be further activated by thromboxane A2-dependent podoplanin-CLEC2 signaling, leading to CVST. A prothrombotic hormonal milieu, and increased generation of thromboxane A2 and platelet activation in healthy females compared to males is consistent with increased risk for CVST observed in women. We propose that antiplatelet agents targeting thromboxane A2 receptor signaling such as low-dose aspirin merit consideration for chemoprophylaxis when administering the adenovirus based COVID-19 vaccines to young adults at risk of thrombosis provided there are no contraindications.

The antiplatelet agent revacept prevents the increase of systemic thromboxane A2 biosynthesis and neointima hyperplasia

Neointima hyperplasia is a crucial component of restenosis after coronary angioplasty. We have hypothesized that enhanced generation of platelet-derived thromboxane (TX)A2 in response to vascular damage plays a critical role in neointimal hyperplasia and that antiplatelet agents may mitigate it. In cocultures of human platelets and coronary artery smooth muscle cells (CASMC), we found that platelets induced morphologic changes and enhanced the migration of CASMC. The exposure of platelets to Aspirin [an inhibitor of cyclooxygenase (COX)-1] reduced the generation of TXA2 and prevented the morphological and functional changes induced by platelets in CASMC. Platelet-derived TXA2 induced COX-2 and enhanced prostaglandin (PG)E2 biosynthesis in CASMC, a known mechanism promoting neointimal hyperplasia. COX-2 induction was prevented by different antiplatelet agents, i.e., Aspirin, the TP antagonist SQ29,548, or Revacept (a dimeric soluble GPVI-Fc fusion protein). The administration of the novel antiplatelet agent Revacept to C57BL/6 mice, beginning three days before femoral artery denudation, and continuing up to seven days after injury, prevented the increase of the systemic biosynthesis di TXA2 and reduced femoral artery intima-to-media area and the levels of markers of cell proliferation and macrophage infiltration. Revacept might serve as a therapeutic agent for percutaneous coronary angioplasty and stent implantation.

Arctigenin attenuates platelet activation and clot retraction by regulation of thromboxane A2 synthesis and cAMP pathway

Pathophysiological reaction of platelets in the blood vessel is an indispensable part of thrombosis and cardiovascular disease, which is the most common cause of death in the world. In this study, we performed in vitro assays to evaluate antiplatelet activity of arctigenin in human platelets and attempted to identify the mechanism responsible for thromboxane A2 (TXA2) generation, integrin αIIbβ3 activation and cAMP pathway. Arctigenin exhibited obvious inhibitory effects on collagen-, thrombin-, and ADP-induced human platelet aggregation, granule secretion, TXA2 generation, integrin αIIbβ3 activation, and clot retraction. Additionally, we found that arctigenin attenuated PI3K/Akt/mTOR/GSK-3β and MAPK signaling pathways, and increased cAMP level. Accordingly, the findings support that arctigenin attenuates thrombotic events through the inhibition of platelet activation and platelet plug formation. Therefore, we suggest that arctigenin may have therapeutic potential as an antiplatelet and antithrombotic agent.

Synthesis, Stability, and Biological Studies of Fluorinated Analogues of Thromboxane A2.

Platelet activation results in the generation of thromboxane A2 (TxA2), which promotes thrombus formation by further amplifying platelet function, as well as causing vasoconstriction. Due to its role in thrombus formation and cardiovascular disease, its production is the target of antiplatelet drugs such as aspirin. However, the study of TxA2-stimulated cellular function has been limited by its instability (t1/2 = 32 s, pH = 7.4). Although more stable analogues such as U46619 and difluorinated 10,10-F2-TxA2 have been prepared, we targeted a closer mimic to TxA2 itself, monofluorinated 10-F-TxA2, since the number of fluorine atoms can affect function. Key steps in the synthesis of F-TxA2 included α-fluorination of a lactone bearing a β-alkoxy group, and a novel synthesis of the strained acetal. F-TxA2 was found to be 105 more stable than TxA2, and surprisingly was only slightly less stable than F2-TxA2. Preliminary biological studies showed that F-TxA2 has similar potency as TxA2 toward inducing platelet aggregation but was superior to F2-TxA2 in activating integrin αIIbβ3.

Catalytic dysregulation of SHP2 leading to Noonan syndromes affects platelet signaling and functions

AbstractSrc homology 2 domain–containing phosphatase 2 (SHP2), encoded by the PTPN11 gene, is a ubiquitous protein tyrosine phosphatase that is a critical regulator of signal transduction. Germ line mutations in the PTPN11 gene responsible for catalytic gain or loss of function of SHP2 cause 2 disorders with multiple organ defects: Noonan syndrome (NS) and NS with multiple lentigines (NSML), respectively. Bleeding anomalies have been frequently reported in NS, but causes remain unclear. This study investigates platelet activation in patients with NS and NSML and in 2 mouse models carrying PTPN11 mutations responsible for these 2 syndromes. Platelets from NS mice and patients displayed a significant reduction in aggregation induced by low concentrations of GPVI and CLEC-2 agonists and a decrease in thrombus growth on a collagen surface under arterial shear stress. This was associated with deficiencies in GPVI and αIIbβ3 integrin signaling, platelet secretion, and thromboxane A2 generation. Similarly, arterial thrombus formation was significantly reduced in response to a local carotid injury in NS mice, associated with a significant increase in tail bleeding time. In contrast, NSML mouse platelets exhibited increased platelet activation after GPVI and CLEC-2 stimulation and enhanced platelet thrombotic phenotype on collagen matrix under shear stress. Blood samples from NSML patients also showed a shear stress–dependent elevation of platelet responses on collagen matrix. This study brings new insights into the understanding of SHP2 function in platelets, points to new thrombopathies linked to platelet signaling defects, and provides important information for the medical care of patients with NS in situations involving risk of bleeding.