Platelet Arachidonic Acid Research Articles

COVID-19, Blood Lipid Changes, and Thrombosis

Although there is increasing evidence that oxidative stress and inflammation induced by COVID-19 may contribute to increased risk and severity of thromboses, the underlying mechanism(s) remain to be understood. The purpose of this review is to highlight the role of blood lipids in association with thrombosis events observed in COVID-19 patients. Among different types of phospholipases A2 that target cell membrane phospholipids, there is increasing focus on the inflammatory secretory phospholipase A2 IIA (sPLA2-IIA), which is associated with the severity of COVID-19. Analysis indicates increased sPLA2-IIA levels together with eicosanoids in the sera of COVID patients. sPLA2 could metabolise phospholipids in platelets, erythrocytes, and endothelial cells to produce arachidonic acid (ARA) and lysophospholipids. Arachidonic acid in platelets is metabolised to prostaglandin H2 and thromboxane A2, known for their pro-coagulation and vasoconstrictive properties. Lysophospholipids, such as lysophosphatidylcholine, could be metabolised by autotaxin (ATX) and further converted to lysophosphatidic acid (LPA). Increased ATX has been found in the serum of patients with COVID-19, and LPA has recently been found to induce NETosis, a clotting mechanism triggered by the release of extracellular fibres from neutrophils and a key feature of the COVID-19 hypercoagulable state. PLA2 could also catalyse the formation of platelet activating factor (PAF) from membrane ether phospholipids. Many of the above lipid mediators are increased in the blood of patients with COVID-19. Together, findings from analyses of blood lipids in COVID-19 patients suggest an important role for metabolites of sPLA2-IIA in COVID-19-associated coagulopathy (CAC).

Dietary Arachidonate Enhances Tissue Arachidonate Levels and Eicosanoid Production in Syrian Hamsters

Eicosanoids are oxidative derivatives of arachidonic acid. When produced in excess many of them are proinflammatory agents. This study investigates whether dietary arachidonic acid enhances arachidonic acid phospholipid content of various tissues and whether this enrichment increases eicosanoid production. Male Syrian hamsters were divided into four groups and fed diets supplemented with ethyl esters of oleic acid, linoleic acid, arachidonic acid or eicosapentaenoic acid. Differences in the composition of the phospholipid fatty acids were monitored in liver, lung, heart, spleen, kidney, testes, macrophages and platelets. In all tissues analyzed, the phospholipid content of arachidonic acid was significantly higher in the arachidonic dietary group compared with all other dietary groups (average > 50% higher). In contrast, increasing dietary linoleic acid by 50% had little effect on altering tissue arachidonic acid levels. Following in vitro stimulation, macrophages and platelets from animals maintained on arachidonic acid produced, in general, the highest levels of eicosanoids compared with cells from animals fed the other diets. Significant differences were observed in prostaglandin E2 (macrophages) and thromboxane B2 (platelets) formation when compared with the oleic acid and eicosapentaenoic acid dietary groups. The data demonstrate that including low to moderate levels of arachidonic acid in the diet increases macrophage and platelet arachidonic acid levels and may augment eicosanoid production.

Effects of sub-chronic, in vivo administration of sigma-1 receptor ligands on platelet and aortic arachidonate cascade in streptozotocin-induced diabetic rats.

Diabetes mellitus is a chronic metabolic disorder which induces endothelial dysfunction and platelet activation. Eicosanoids produced from arachidonic acid regulate cellular and vascular functions. Sigma-1 receptors (S1R) are expressed in platelets and endothelial cells and S1R expression is protective in diabetes. Our aim was to examine the influence of sub-chronic, in vivo administered S1R ligands PRE-084, (S)-L1 (a new compound) and NE-100 on the ex vivo arachidonic acid metabolism of platelets and aorta in streptozotocin-induced diabetic rats. The serum level of the S1R ligands was detected by LC-MS/MS before the ex vivo analysis. Sigma-1 receptor and cyclooxygenase gene expression in platelets were determined by RT-qPCR. The eicosanoid synthesis was examined with a radiolabelled arachidonic acid substrate and ELISA. One month after the onset of STZ-induced diabetes, in vehicle-treated, diabetic rat platelet TxB2 and aortic 6-k-PGF1α production dropped. Sub-chronic in vivo treatment of STZ-induced diabetes in rats for one week with PRE-084 enhanced vasoconstrictor and platelet aggregator and reduced vasodilator and anti-aggregator cyclooxygenase product formation. (S)-L1 reduced the synthesis of vasodilator and anti-aggregator cyclooxygenase metabolites and promoted the recovery of physiological platelet function in diabetic rats. The S1R antagonist NE-100 produced no significant changes in platelet arachidonic acid metabolism. (S)-L1 decreased the synthesis of vasoconstrictor and platelet aggregator cyclooxygenase metabolites, whereas NE-100 increased the quantity of aortic vasodilator and anti-aggregator cyclooxygenase products and promoted the recovery of diabetic endothelial dysfunction in the aorta. The novel S1R ligand, (S)-L1 had similar effects on eicosanoid synthesis in platelets as the agonist PRE-084 and in aortas as the antagonist NE-100. S1R ligands regulate cellular functions and local blood circulation by influencing arachidonic acid metabolism. In diabetes mellitus, the cell-specific effects of S1R ligands have a compensatory role and aid in restoring physiological balance between the platelet and vessel.

Fifty years with aspirin and platelets.

In 2021, we reached the 50th anniversary of the publication of Sir John Vane's seminal paper in Nature New Biology describing the experiments supporting his mechanistic hypothesis that inhibition of prostaglandin synthesis might explain the main pharmacological effects of aspirin and aspirin-like drugs, that is, reduction in pain, fever and inflammation. Bengt Samuelsson's subsequent discoveries elucidating the cyclooxygenase pathway of platelet arachidonic acid metabolism motivated my research interest towards measuring platelet thromboxane A2 biosynthesis as a tool to investigate the clinical pharmacology of cyclooxygenase inhibition by aspirin in health and disease. What followed was a long, winding road of clinical research leading to the characterization of low-dose aspirin as a life-saving antiplatelet drug that still represents the cornerstone of antithrombotic therapy. Having witnessed and participated in these 50 years of aspirin research, I thought of providing a personal testimony of how things developed and eventually led to a remarkable success story of independent research.

Evaluation of inhibitors of the arachidonic acid cascade with intact platelets using an on-line dilution and on-line solid phase extraction HPLC–MS method

An automatic on-line dilution/on-line solid phase extraction (SPE) system has been developed for the detection of metabolites of the arachidonic acid cascade in platelets. The method allows the direct injection of larger quantities of centrifugates from cell suspensions previously treated with an equal volume of an acetonitrile/methanol mixture for protein precipitation. The method was used to study the effect of inhibitors of platelet arachidonic acid cascade enzymes (cytosolic phospholipase A2α, cyclooxygenase-1, thromboxane synthase, 12-lipoxygenase) and related targets (cyclooxygenase-2, microsomal prostaglandin E synthase-1, 5-lipoxygenase) in intact platelets after stimulation with calcium ionophore A23187. In addition to enzyme inhibition, the cell-damaging properties of the test compounds was determined by measuring the release of serotonin from the platelets into the incubation buffer.

ROLE OF ANTIOXIDANT THERAPY IN PREVENTION OF LIPOPEROXIDATION AND PROCOAGULANT ACTIVITY OF PLATELETS IN SARS-COV-2 VIRAL INFECTIONS

Based on a metanalysis of clinical observations in different countries with practical experience in the treatment of pneumonia complicated by the syndrome of multiple organ failure caused by the COVID-19 coronavirus, it is impossible to draw an unambiguous conclusion about the effectiveness of various therapies in patients. viruses modify the apparatus of biosynthesis of cells in the body as a whole. Therefore, finding drugs that have an exceptional effect on viruses without infecting the body is a difficult task. Various antiviral drugs (ribaverin, interferons and glucocorticosteroids, antiparasitic, antimalarials (Chloroquine, Hydroxychloroquine) are effective at certain stages of the infectious process, however, they are toxic and therapy with these drugs has risks of developing severe cytopenia, hepatotoxicity, neuropsychiatric disorders and severe ischemia Interleukin antagonists (Tocilizumab, Anakindra, Levilimab) are effective in severe cases with an increase in IL-6, as they limit the cascade of activation of cells of the immune system, reduce the effect of "cytokine storm" in the formation of acute respiratory distress syndrome and endothelial dysfunction of the pulmonary vessels.
 Discussion. In an experimental study R.G. Alborov, in the framework of the dissertation for the degree of candidate of medical sciences (2001), in which nonlinear white rats were used, it was demonstrated that: activators of lipid peroxidation (LPO), different in nature and biological activity, inhibition of the transformation of arachidonic acid in platelets reduces, along with proaggregant activity, the intensity of LPO processes in them. Based on the experimental data obtained, it can be considered appropriate to study the effect of antioxidants as agents that enhance the effects of antiplatelet inhibitors of the transformation of arachidonate and platelets. It can be expected that antioxidants, by reducing the procoagulant activity of platelets, due to inhibition of LPO, can enhance the effect of antiplatelet agents.
 Findings. 1. The effectiveness of antioxidant therapy for infection with SAPS-COV-2 viruses, including COVID-19, is beyond doubt. It is important that antioxidant therapy was prescribed before the development of lipid peroxidation and "cytokine storm" and an increase in the procoagulant activity of platelets, that is, at the initial stage of infection.
 
 Antioxidant therapy presupposes adequate dosages according to the type of "saturating phase of treatment" with subsequent transfer to maintenance doses.
 
 Currently, no drugs have been found that have an exceptional effect on viruses without affecting the body. Antioxidants are protective at the cellular level. In addition, they practically do not cause side effects. It is advisable to prescribe antioxidant drugs in large doses in the complex therapy of viral infections.

Does the Degree of Platelet Adenosine Diphosphate and Arachidonic Acid Receptor Inhibition Correlate With the Severity of Injury in Non-Brain-Injured Trauma Patients?

Direct correlations between platelet adenosine diphosphate (ADP) and arachidonic acid (AA) receptor inhibition have been described in the traumatic brain injury (TBI) population. Our goal was to evaluate the percent inhibition of ADP receptor inhibition (ADPri) and AA receptor inhibition (AAri) receptors in non-TBI patients and correlate injury severity and outcomes. We performed a retrospective review of non-TBI patients admitted to our trauma center, who received thromboelastography with platelet mapping prior to blood transfusion. Exclusion criteria included patients younger than 18years, current antiplatelet therapy, or history of renal failure. Univariate descriptive statistics and bivariate comparisons were performed on patient demographic and outcomes. Multivariable linear regression models were constructed to quantify any association between ADPri and AAri with injury outcomes. High ADP inhibition was defined >20% and high AA inhibition >7%. 117 patients met inclusion criteria. Mean age was 53years with 61% male. Mean ADPri was 64% and AAri 42%. On bivariate analysis, no statistically significant differences with respect to injury severity measures or outcomes were identified. On multivariable linear regression, AAri was associated with longer hospital length of stay. There was a high degree of platelet dysfunction in this cohort of severely injured patients without TBI. Despite this, the only correlation identified between injury severity and outcomes was AAri correlating with hospital length of stay. Irrespective of injury severity or outcomes, these patients' results were far from reported "normal" values. Further, research is needed to determine the significance and clinical implications of thromboelastography with platelet mapping use in trauma care.

Large triglyceride-rich lipoproteins from fasting patients with type 2 diabetes activate platelets

AimsType 2 diabetes (T2D) patients present with risk factors for atherothrombosis such as fasting hypertriglyceridaemia and platelet hyperactivity. Our study objective was to determine the effect of large triglyceride-rich lipoproteins (TGRL) from fasting T2D patients on platelet aggregation and, if any, to identify the signaling pathway involved. MethodsLarge TGRL were isolated from the plasma of 25 T2D patients by ultracentrifugation (density < 1.000 g/mL). Platelets were isolated from healthy blood donors (HBD) and suspended in buffer, then preincubated in the presence or absence of TGRL and stimulated with either collagen or thrombin. Platelet aggregation and the arachidonic acid (AA) signaling pathway were studied. ResultsFasting T2D large TGRL were mostly of hepatic origin (apoB100/apoB48 ratio: 42 ± 7) and rich in triglycerides (TG/total apoB ratio: 4.2 ± 0.5), and able to potentiate agonist-stimulated platelet aggregation (collagen: +68%, P < 0.05; thrombin: +771%, P < 0.05). It should also be mentioned that TGRL from the plasma of HBD (n = 7) had no effect on platelet aggregation. In addition, T2D large TGRL increased thromboxane B2 (TxB2) concentration in platelets stimulated with either collagen (+34%, P < 0.05) or thrombin (+37%, P < 0.05) compared with platelets stimulated with either of these agonists without TGRL. Phosphorylation of p38 MAPK and cytosolic phospholipase A2 (cPLA2) was enhanced after incubation of platelets with T2D TGRL and thrombin (+87% and +32%, respectively, P < 0.05) compared with platelets incubated with thrombin only. ConclusionLarge TGRL from fasting T2D patients may play a role in the development of atherothrombosis by increasing platelet aggregation and activating the platelet AA signaling pathway.

Iodine contrast agents do not influence Platelet-Rich Plasma function at an early time point in vitro

BackgroundIodine contrast agents (ICAs) are routinely used by radiologists to help guide intra-articular infiltrations. The aim of this study was to assess the in vitro effects of ICA on platelet function of human autologous Platelet-Rich Plasma (PRP).MethodsOne hundred thirty-seven consecutive patients with symptomatic femoral-patellar osteoarthritis were included. All were addressed to our institution for a fluoroscopy-guided intra-articular PRP infiltration of the pathological femoral-patellar joint. For each patient, 500 μl of PRP were sampled before intra-articular injection. First, PRP samples were mixed with 50 μl of 2 widely used ICA: Visipaque270® (Iodixanol, n = 58) and Iopamiron200® (Iopamidol, n = 69). PRP concentration ([PRP]) was measured at different delays of incubation (t = 0, 5, 10, 15, 20 and 30 min) enabling to calculate PRP ratio (defined as [PRP](t)/[PRP](0mn)) at each delay, for each mixture, in order to quantitatively assess the influence of ICA on PRP ratio. Second, the PRP samples of 10 additional patients were mixed with Visipaque270®, Visipaque270®, Iopamiron200® and phosphate buffer saline (PBS: control solution) in order to qualitatively assess the influence of ICA on platelet aggregation, using ADP, Collagen, Arachidonic acid and TRAP tests. The surface expression of human P-selectin, a marker of α-granule release, in the PRP + Visipaque270® and PRP + Iopamiron200® mixtures was finally compared. Repeated-measures ANOVA, classical 2-way ANOVA and Wilcoxon matched-pairs test were used to study the influence of ICA on PRP quality.ResultsThere was no significant change in PRP ratio during the first 30mn of incubation (p = 0.991) whatever the ICA (p = 0.926). Whatever the aggregation test, there was no significant difference in the percentage of platelet aggregation between PRP + PBS, PRP + Visipaque270® and PRP + Iopamiron200® (p = 0.998), nor between PRP + PBS and PRP + Visipaque320® (p = 0.470). Finally, there was no significant difference in P-selectin expression between the PRP + Visipaque270® and PRP + Iopamiron200® mixtures (p = 0.500).ConclusionAt early delays of incubation, Visipaque® and Iopamiron®, which are two widely used ICA for intra-articular infiltrations, did not influence the in vitro platelet function nor the quality of PRP.

Effects of platelet dysfunction and platelet transfusion on outcomes in traumatic brain injury patients

ABSTRACTObjective: Platelet inhibition in traumatic brain injury (TBI) may be due to injury or antiplatelet medication use pre-injury. This study aims to identify factors associated with increased platelet arachidonic acid (AA) and adenosine diphosphate (ADP) inhibition and determine if platelet transfusion reduces platelet dysfunction and affects outcome.Methods: Prospective thromboelastography (TEG) assays were collected on adult patients with TBI with intracranial injuries detected by computed tomography (CT). Outcomes included in-hospital mortality, and CT lesion expansion.Results: Of 153 patients, ADP inhibition was increased in moderate and severe TBI compared to mild TBI (p = 0.0011). P2Y12 inhibiting medications had increased ADP inhibition (p = 0.0077). Admission ADP inhibition was not associated with in-hospital mortality (p = 0.24) or CT lesion expansion (p = 0.94). Mean reduction of ADP inhibition from platelet transfusion (−15.1%) relative to no transfusion (+ 11.7%) was not statistically different (p = 0.0472).Conclusions: Mild TBI results in less ADP inhibition compared to moderate and severe TBI, suggesting a dose response relationship between TBI severity and degree of platelet dysfunction. Further, study is warranted to determine efficacy and parameters for platelet transfusion in patients with TBI.

Effects of storage conditions on two platelet agonists for whole blood impedance platelet aggregometry in dogs.

Whole blood impedance platelet aggregometry (Multiplate-) can be performed with different agonists to evaluate platelet function. Although the manufacturer recommends disposal of stored reagents after 1month at -20°C or 24hours at 4°C, reagent integrity after reconstitution under different storage conditions is unknown. If reagent integrity is stable for longer periods, assay costs could decrease dramatically. This study aimed to determine the stability of reconstituted arachidonic acid (AA) and adenosine diphosphate (ADP) platelet agonists stored at -20°C and -80°C for up to 6months. Aliquots of reconstituted AA and ADP were stored at -20°C and -80°C each month for a total of 6months. Six healthy staff-owned dogs were enrolled in the study. A physical examination, CBC, diagnostic panel, urinalysis, and baseline platelet aggregometry assessment was performed on all of the dogs. Platelet aggregometry was performed using fresh and stored aliquots of AA and ADP reagents on the same day. The area under the curve (AUC) was recorded from each platelet aggregometry analysis. Repeated measures (RM) analysis (one-way ANOVA) was performed and subsequent time points (1, 2, 3, 4, 5, and 6months) were compared with fresh AUC results. All dogs were clinically healthy, and all diagnostic tests were normal. There were no differences in AUC obtained from fresh samples at any time point or either temperature for AA or ADP. Whole blood impedance platelet aggregometry reagents, AA and ADP, were stable for up to 6months when stored at -20°C or -80°C, obviating the need to discard viable reagents, and decreasing assay costs.

Antiplatelet and antithrombotic effects of cordycepin-enriched WIB-801CE from Cordyceps militaris ex vivo, in vivo, and in vitro.

BackgroundA species of the fungal genus Cordyceps has been used as a complementary and alternative medicine of traditional Chinese medicine, and its major component cordycepin and cordycepin-enriched WIB-801CE are known to have antiplatelet effects in vitro. However, it is unknown whether they have also endogenous antiplatelet and antithrombotic effects. In this study, to resolve these doubts, we prepared cordycepin-enriched WIB-801CE, an ethanol extract from Cordyceps militaris-hypha, then evaluated its ex vivo, in vivo, and in vitro antiplatelet and antithrombotic effects.MethodsEx vivo effects of WIB-801CE on collagen- and ADP-induced platelet aggregation, serotonin release, thromboxane A2 (TXA2) production and its associated activities of enzymes [cyclooxygenase-1 (COX-1), TXA2 synthase (TXAS)], arachidonic acid (AA) release and its associated phosphorylation of phospholipase Cβ3, phospholipase Cγ2 or cytosolic phospholipase A2, mitogen-activated protein kinase (MAPK) [p38 MAPK, extracellular signal-regulated kinase (ERK)], and blood coagulation time in rats were investigated. In vivo effects of WIB-801CE on collagen plus epinephrine-induced acute pulmonary thromboembolism, and tail bleeding time in mice were also inquired. In vitro effects of WIB-801CE on cytotoxicity, and fibrin clot retraction in human platelets, and nitric oxide (NO) production in RAW264.7 cells or free radical scavenging activity were studied.ResultsCordycepin-enriched WIB-801CE inhibited ex vivo platelet aggregation, TXA2 production, AA release, TXAS activity, serotonin release, and p38 MAPK and ERK2 phosphorylation in collagen- and ADP-activated rat platelets without affecting blood coagulation. Furthermore, WIB-801CE manifested in vivo inhibitory effect on collagen plus epinephrine-induced pulmonary thromboembolism mice model. WIB-801CE inhibited in vitro NO production and fibrin clot retraction, but elevated free radical scavenging activity without affecting cytotoxicity against human platelets.ConclusionWIB-801CE inhibited collagen- and ADP-induced platelet activation and its associated thrombus formation ex vivo and in vivo. These were resulted from down-regulation of TXA2 production and its related AA release and TXAS activity, and p38 MAPK and ERK2 activation. These results suggest that WIB-801CE has therapeutic potential to treat platelet activation-mediated thrombotic diseases in vivo.

Involvement of Nitric Oxide on Calcium Mobilization and Arachidonic Acid Pathway Activation during Platelet Aggregation with different aggregating agonists

Platelet aggregation by different aggregating agonists is essential in the normal blood coagulation process, the excess of which caused acute coronary syndrome (ACS). In all cases, the activation of arachidonic acid by cycloxygenase was needed for the synthesis of thromboxane A2 (TXA2) but the mechanism of arachidonic acid release in platelets remains obscure. Studies were conducted to determine the role of nitric oxide (NO), if any, on the release of arachidonic acid in platelets. The cytosolic Ca2+ was visualized and quantitated by fluorescent spectroscopy by using QUIN-2. NO was measured by methemoglobin method. Arachidonic acid was determined by HPLC. TXA2 was measured as ThromboxaneB2 (TXB2) by ELISA. Treatment of platelets in platelet-rich plasma (PRP) with different aggregating agents resulted in the inhibition of nitric oxide synthase (NOS) which inhibited the production of NO synthesis and increased TXA2 synthesis. Furthermore, the treatment of washed PRP with different platelet aggregating agents resulted in the increase of [Ca2+] in nM ranges. In contrast, the pre-treatment of washed PRP with aspirin increased platelet NO level and inhibited the Ca2+ mobilization and TXA2 synthesis. These results indicated that the aggregation of platelets by different aggregating agonists was caused by the cytosolic Ca2+ mobilization due to the inhibition of NOS.

Anti-platelet eff ects of nimesulide in isoproterenol-induced myocardial ischaemia and infarction in rabbits

Background The present study was designed to determine the eff ects of cyclooxygenase-2 (COX-2) inhibitor-nimesulide on platelet activating factor (PAF) and arachidonic acid (AA)-induced platelet aggregation during myocardial infarction (MI) and ischaemia in rabbits.Methods Rabbits were divided into three groups; group I served as control and contains normal rabbits that received saline, group II rabbits received saline before induction of MI, and group III rabbits received nimesulide (25 mg/kg) before MI induction. Myocardial infarction was induced by isoproterenol (ISP) (65 mg/kg) and confi rmed by biochemical, electrophysiological and histopathological methods. Platelet aggregation was induced with arachidonic acid (AA) and platelet activating factor (PAF), and monitored by using light transmission aggregometery.Results When platelets were challenged with two diff erent aggregating agents, nimesulide-treated infarcted rabbits showed resistance to platelet aggregation while non-treated infarcted rabbits were found prone to aggregation with both aggregating agents (P >0.05).Conclusion These results suggest platelet inhibitory eff ects of nimesulide during experimental ischaemia and infarction in rabbits.

Platelet Arachidonic Acid Deficiency May Contribute to Abnormal Platelet Function During Parenteral Fish Oil Monotherapy in a Piglet Model.

Fish oil monotherapy has been an advance for treating intestinal failure-associated liver disease (IFALD). However, such patients are at risk of bleeding complications from liver disease and because fish oil can inhibit thrombosis. We have previously reported abnormal platelet function in neonatal piglets given fish oil monotherapy during parenteral nutrition (PN). The purpose of this study was to determine if abnormal fatty acid composition of the platelets could explain the prior observed antiplatelet effect. Neonatal piglets were assigned to 2 treatments: PN with fish oil monotherapy (FO; n = 4) or PN with soy oil (SO; n = 5). On day 14, plasma was collected and platelets isolated by centrifuging. The fatty acid content in plasma and platelet plug were measured using gas liquid chromatography and compared with controls (CON; n = 5). The arachidonic acid (AA) content in the FO group was on average half that of the SO group, in both the platelets (FO, 3.5% vs SO, 7.6%; P = .021; CON, 4.5%-11%) and the plasma (FO, 3.8% vs SO, 9.2%; P = .002; CON, 6.1%-9.5%). No bleeding complications were observed for any piglets during PN treatment. Using platelet mapping, we have previously shown that neonatal piglets given fish oil monotherapy have abnormal platelet function in the AA pathway. This report demonstrates that such an abnormality can be explained by platelet AA deficiency. Platelet mapping and platelet fatty acid analysis should be undertaken in human infants treated with fish oil monotherapy during PN.

Liquid chromatography–tandem mass spectrometry for simultaneous measurement of thromboxane B2 and 12(S)-hydroxyeicosatetraenoic acid in serum

Arachidonic acid (AA) is metabolized in human platelets by two main pathways: via cyclooxygenase (COX-1) to prostaglandins and thromboxane (TX)A2 and via 12-lipoxygenase (12-LOX) to 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). While COX products are known to regulate platelet reactivity, the role of 12-LOX metabolites is still controversial. To better understand the platelet enzymatic pathways, we developed a simple and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for the simultaneous measurement of both platelet metabolites in human serum. After the addition of deuterated d4-TXB2 and d8-12(S)-HETE as internal standards and the solid-phase extraction of serum samples, analytes were resolved using reversed-phase C18 column and quantified using negative ion electrospray ionization-tandem mass spectrometry. Intra and interassay imprecisions were less than 10% for both analytes. The lower limits of quantification were 0.244ng/ml and 0.976ng/ml for TXB2 and 12(S)-HETE, respectively. This method was applied to measure platelet metabolites in healthy subjects (n=35). LC–MS/MS allows rapid, simultaneous, sensitive and accurate quantification of both platelet AA products in human serum with a small sample volume required and a minimal sample preparation.

Familial Essential Thrombocythemia Among Qatari Tribes

Essential thrombocythaemia (ET) is a chronic myeloproliferative syndrome due to sustained proliferation of megakaryocytes,which results in elevated numbers of circulating platelets, thrombotic or haemorrhagic episodes and occasional leukaemic transformation.Familial essential thrombocythemia,also called hereditary or inherited thrombocythemia or thrombocytosis, is a rare disorder transmitted in an autosomal dominant with clinical presentation and complications resembling sporadic essential thrombocythaemia (ET).Mutations in the thrombopoietin (TPO) gene causing overproduction of TPO and elevated TPO serum levels familial essential thrombocythemia is a heterogeneous disorder as families have been described with an activating mutation in the gene for the TPO receptor (c-MPL), the thrombopoietin gene, a germline mutation in JAK2 V617I or without a mutation in any of these genesOccurrence of Essential thrombocythemia among arab population never been reported before. In this study, essential thrombocythemia is documented in 25 families from 5 major tribes in Qatar Familial cases were defined when two or more individuals within the same pedigree were affected with MPNs. Clinical records of affected relatives were reviewed systematically to confirm MPNs diagnosis. Patients without family history of were defined as having sporadic. MPNs Diagnosis of MPNs was made in accordance with the WHO criteria 2008Ninety eight members of both sexes from 18 to 62 years of age in three successive generations. The propositus had a persistent elevation of the platelet count, splenomegaly, a normal hemoglobin level, a normal white blood cell count, and abnormal platelet aggregation. Platelet arachidonic acid metabolites assayed by high-performance liquid chromatography and serum thrombopoietin levels were normal. Megakaryocytes were increased in number and size. Both mature and early immature megakaryocytes, but no atypical megakaryocytes, were identified by surface immunofluorescence. Bone marrow cultures showed normal myeloid and erythroid colony formation, and chromosome studies revealed a normal The analysis of mutations of JAK2 and MPL may improve our ability to identify these conditions. In a consecutive series of patients observed in our Institution from January 2011 to June 2013 total No of 300 MPNs patients were diagnosed, out of 300 patient, 119 had PV, 160 had ET, 15 had PMF and six patient had unclassified atypical MPN case. Out of 119 PV pts, 116 (97.4%) were positive for the JAK2 V617F mutation and 3 cases met the WHO criteria for PV but were negative for JAK2 V617F and JAK2 exon 12 mutations. Out of 160 ET pts, 80 (50%) were positive for JAK2 V617F, one had MPL S505N mutation and 79 cases met the WHO criteria for ET but were negative for both JAK2 617F and MPL mutations. Out of 15 PMF pts 5 (33.3%) were positive for JAK2 V617F and the unclassified case which was characterized by Deep Vein Thrombosis had JAK2 exon 13 mutation (R564L).25 Arab families were described as having familial ET (5 major tribes were screened )20 were having JAK2V617F positive in three consequetive generations ,4 families were having Familial ET in three generations but both Jak2v617F and MPL were negativeMPL S505N mutation was found in one family( Father and daughter).there was no difference in clinical presentation and response to therapy in sporadic versus familial cases. ConclusionIn our series of familial cases clinical presentation, therapeutic approach type and severity of complications were comparable to that of sporadic cases. Furthermore, the present study indicates the relevant possibility of familial MPNs, thus suggesting the opportunity of a detailed family history as part of the initial work-up of patients with MPNs; in addition it also suggests the usefulness of an accurate biological studies Disclosures:Yassin:qatar national research fund: Patents & Royalties, Research Funding. Al-Dewik:qatar national research fund: Patents & Royalties, Research Funding. Cassinat:qatar national research fund: Patents & Royalties, Research Funding.

207 PLATELET COX-1 SUPPORTS THE PRODUCTION OF BOTH PROSTANOIDS AND HETES

<h3>Backround</h3> Prostaglandins (PGs) and hydroxyeicosatetraeinoic acids (HETEs) are synthesized from arachidonic acid (AA), which is released from the membrane phospholipids of cells through the activity of cytosolic phospholipase A_2α (cPLA_2α). It is known that in platelets AA is metabolised through the enzymes cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) leading to the production of a range of eicosanoids. Here we have determined the enzyme sources of the major prostanoid and HETE products released by platelets following activation by selective platelet agonists. To better model platelet activation we used either platelet rich plasma or whole blood within a platelet aggregometer, warmed and stirred at 1200rpm, and assessed whether HETEs originate from platelets or other blood cells. Furthermore, to investigate how the production of these eicosanoids is affected by anti-platelet therapy we examined the effects of the cyclooxygenase inhibitor, aspirin (100 µM), with or without the addition of the P2Y₁₂ receptor blocker, prasugrel (3 µM). <h3>Methods</h3> Blood from healthy volunteers was collected into hirudin and incubated (30 min, 37°C) either intact or following preparation into platelet rich plasma (PRP) in the presence of collagen (30 µg/ml), TRAP-6 (30 µM) or vehicle, in a platelet aggregometer with stirring (1200rpm). Plasma was then separated from the samples and the levels of prostanoids and HETEs were determined by LC/MS/MS analysis. In some experiments, blood and PRP were pre-treated with aspirin (100 µM), prasugrel (3 µM) or aspirin+prasugrel. <h3>Results</h3> LC/MS/MS analysis of collagen- and TRAP-stimulated PRP and blood showed large increases in the levels of thromboxane (TX) A₂, prostaglandin (PG) E₂, and PGD₂, as well as 8-, 11-, 15-and 12-HETE compared to unstimulated samples. Interestingly, as well as inhibiting the production of TXA₂, PGE₂ and PGD₂, aspirin also strongly inhibited the productions of 11-HETE and 15-HETE by both PRP and blood. The production of 12-HETE was reduced by aspirin and prasugrel used in combination. <h3>Conclusions</h3> LC/MS/MS analysis demonstrated that platelets are the main source of both prostanoids (TXA₂, PGE₂ and PGD₂) and HETEs (8-, 11-, 15- and 12) in blood exposed to platelet activators. Interestingly, in addition to abolishing the production of prostanoids, aspirin also blocked the production of 11-HETE and 15-HETE in both PRP and blood, indicating these HETEs are also formed through the activity of platelet COX-1. Aspirin in combination with prasugrel reduced, but did not abolish, the production of 12-HETE, consistent with this drug inhibiting the processes of platelet activation but not directly inhibiting platelet 12-LOX. It is possible these data may well help provide explanations for the beneficial effects of anti-platelet doses of aspirin in a range of cardiovascular diseases and cancers.

Activity of platelet hemostasis in newborn calves

Healthy newborn Simmental and Black Pied calves with a normal number of platelets in bloodstream don’t have reliable dynamics of the aggregation function of blood platelets with adenosine diphosphate, collagen, thrombin, ristocetin, epinephrine, as well as combinations of ADP + epinephrine, ADP + collagen, and collagen + epinephrine inducers. The level of discocytes in blood on days 1–2 was 78.5, not reliably changing until the end of the neonatal stage. In this case, the number of disco-echinocytes, spherocytes, spheroechinocytes, and bipolar forms of platelets in the bloodstream remained stably low. Maintenance during colostrum feeding of a low intensity of metabolism of endogenous arachidonic acid in platelets and of a low content of ATP, ADP, actin, and myosin in them can be considered important mechanisms providing stability of platelet aggregation activity in calves in the neonatal stage.

LDL from obese patients with the metabolic syndrome show increased lipid peroxidation and activate platelets

This study assessed oxidative stress in LDL from obese patients with the metabolic syndrome and compared it with that in LDL from type 2 diabetic patients or control volunteers. It also determined the effect on platelets of LDL from the three groups. The profiles of lipids, fatty acids and fatty acid oxidation products were determined in LDL isolated from plasma of patients with the metabolic syndrome, patients with type 2 diabetes and volunteers (n = 10 per group). The effects of LDL from the participant groups on the platelet arachidonic acid signalling cascade and aggregation were investigated. Compared with LDL from control volunteers, LDL from obese metabolic syndrome and type 2 diabetic patients had lower cholesteryl ester, higher triacylglycerol and lower ethanolamine plasmalogen levels. Proportions of linoleic acid were decreased in phosphatidylcholine and cholesteryl esters in LDL from both patient groups. Among the markers of lipid peroxidation, oxidation products of linoleic acid (hydroxy-octadecadienoic acids) and malondialdehyde were increased by 59% and twofold, respectively in LDL from metabolic syndrome and type 2 diabetic patients. LDL from metabolic syndrome and type 2 diabetic patients were equally potent in activating the platelet arachidonic acid signalling cascade through increased phosphorylation of p38 mitogen-activated protein kinase and cytosolic phospholipase A(2), and through increased thromboxane B(2) formation. LDL from patients with the metabolic syndrome and type 2 diabetes potentiated platelet aggregation by threefold and 3.5-fold respectively, whereas control LDL had no activating effects on platelets. The metabolic syndrome in obese patients, without or with diabetes, is associated with increased oxidative stress in LDL, which triggers platelet activation.