Thromboxane A2 Generation Research Articles

Morin hydrate inhibits platelet activation and clot retraction by regulating integrin αIIbβ3, TXA2, and cAMP levels

Morin hydrate is an active constituent of Morus alba L, Prunus dulcis, and Cudrania tricuspidata and has been reported to inhibit platelet activation in vivo and in vitro, but no reports have been issued on its regulation of αIIbβ3, a platelet-specific integrin and thromboxane A2 (TXA2), positive feedback molecule.In this study, we investigated the anti-platelet activity of morin hydrate in collagen- and thrombin-induced human platelets and attempted to identify the mechanism responsible for integrin αIIbβ3 activation and TXA2 generation. Our results demonstrated that morin hydrate (25–100 μM) inhibited collagen- and thrombin-induced platelet aggregation, granule secretion (P-selectin expression, ATP, and serotonin release), calcium mobilization, TXA2 production, integrin αIIbβ3 activation, and clot retraction. Additionally, morin hydrate attenuated the phosphorylations of phospholipase Cγ2 (PLCγ2), cytosolic phospholipase A2 (cPLA2), phosphoinositide 3-kinase (PI3K), Akt, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), and enhanced the phosphorylations of inositol trisphosphate receptor (IP3 receptor) and cyclic adenosine monophosphate (cAMP) generation. However, it had no effect on the coagulation pathway. Taken together, these observations indicate morin hydrate inhibits platelet-mediated thrombosis by down-regulating TXA2 production and integrin αIIbβ3 activation, and by upregulating cAMP generation, and thus, inhibits clot retraction. These results suggest morin hydrate may have therapeutic potential as a treatment for platelet-activation-related diseases.

The protein tyrosine phosphatase PTPN7 is a negative regulator of ERK activation and thromboxane generation in platelets

Protein tyrosine phosphatase nonreceptor type 7 (PTPN7), also called hematopoietic protein tyrosine phosphatase, controls extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase in T lymphocytes. Because ERK1/2 plays an important role in regulating thromboxane A2 (TXA2) generation in platelets, we investigated the function of PTPN7 in these cells. Using immunoblot analysis, we detected PTPN7 in both human and mouse platelets but not in PTPN7-null mice. PTPN7 KO mouse platelets exhibited increased platelet functional responses, including aggregation, dense granule secretion, and TXA2 generation, compared with platelets from WT littermates, upon stimulation with both G protein-coupled receptor (GPCR) and glycoprotein VI (GPVI) agonists. Using the GPCR agonist AYPGKF in the presence of the COX inhibitor indomethacin, we found that PTPN7 KO mouse platelets aggregated and secreted to the same extent as WT platelets, suggesting that elevated TXA2 is responsible for the potentiation of platelet functional responses in PTPN7-KO platelets. Phosphorylation of ERK1/2 was also elevated in PTPN7 KO platelets. Stimulation of platelets with the GPVI agonist collagen-related peptide along with the COX inhibitor indomethacin did not result in phosphorylation of ERK1/2, indicating that GPVI-mediated ERK phosphorylation occurs through TXA2 Although bleeding times did not significantly differ between PTPN7-null and WT mice, time to death was significantly faster in PTPN7-null mice than in WT mice in a pulmonary thromboembolism model. We conclude that PTPN7 regulates platelet functional responses downstream of GPCR agonists, but not GPVI agonists, through inhibition of ERK activation and thromboxane generation.

Aspirin eugenol ester inhibits agonist-induced platelet aggregation in vitro by regulating PI3K/Akt, MAPK and Sirt 1/CD40L pathways

Aspirin eugenol ester (AEE) was a promising drug candidate for treating inflammation, pain and fever and preventing cardiovascular diseases with fewer side effects than its precursors. Previous researches indicated that AEE could markedly inhibit agonist-induced platelet aggregation in vitro and ex vivo, however, the anti-platelet aggregation mechanisms of AEE remain to be defined. Here, AEE in vitro effects on agonist-induced granule-secretion, intercellular Ca2+ mobilization and thromboxane A2 (TXA2) generation were examined. Vasodilator-stimulated phosphoprotein (VASP), mitogen-activated protein kinase (MAPK), Akt, Sirt 1 and CD40L expressions were also studied.In agonist-activated platelets in vitro, AEE markedly attenuated granule secretion markers (P-selectin expression and ATP release), intercellular Ca2+ mobilization and thromboxane B2 (TXB2) formation. AEE also attenuated CD40L activation, suppressed extracellular-signal-regulated protein kinase 2 (ERK2), c-Jun N-terminal kinase 1 (JNK1) and Akt phosphorylation, and recovered Sirt1 expression, but the activation of p38, VASPSer157 and VASPSer239, and the levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were not affected by AEE. Overall, this study demonstrates that AEE inhibits agonist-induced platelet aggregation in vitro by regulating PI3K/Akt, MAPK and Sirt 1/CD40L pathways.

Type 2 diabetes mellitus in the Goto-Kakizaki rat impairs microvascular function and contributes to premature skeletal muscle fatigue.

Despite extensive investigation into the impact of metabolic disease on vascular function and, by extension, tissue perfusion and organ function, interpreting results for specific risk factors can be complicated by the additional risks present in most models. To specifically determine the impact of type 2 diabetes without obesity on skeletal muscle microvascular structure/function and on active hyperemia with elevated metabolic demand, we used 17-wk-old Goto-Kakizaki (GK) rats to study microvascular function at multiple levels of resolution. Gracilis muscle arterioles demonstrated blunted dilation to acetylcholine (both ex vivo proximal and in situ distal arterioles) and elevated shear (distal arterioles only). All other alterations to reactivity appeared to reflect compromised endothelial function associated with increased thromboxane (Tx)A2 production and oxidant stress/inflammation rather than alterations to vascular smooth muscle function. Structural changes to the microcirculation of GK rats were confined to reduced microvessel density of ~12%, with no evidence for altered vascular wall mechanics. Active hyperemia with either field stimulation of in situ cremaster muscle or electrical stimulation via the sciatic nerve for in situ gastrocnemius muscle was blunted in GK rats, primarily because of blunted functional dilation of skeletal muscle arterioles. The blunted active hyperemia was associated with impaired oxygen uptake (V̇o2) across the muscle and accelerated muscle fatigue. Acute interventions to reduce oxidant stress (TEMPOL) and TxA2 action (SQ-29548) or production (dazmegrel) improved muscle perfusion, V̇o2, and muscle performance. These results suggest that type 2 diabetes mellitus in GK rats impairs skeletal muscle arteriolar function apparently early in the progression of the disease and potentially via an increased reactive oxygen species/inflammation-induced TxA2 production/action on network function as a major contributing mechanism. NEW & NOTEWORTHY The impact of type 2 diabetes mellitus on vascular structure/function remains an area lacking clarity. Using diabetic Goto-Kakizaki rats before the development of other risk factors, we determined alterations to vascular structure/function and skeletal muscle active hyperemia. Type 2 diabetes mellitus reduced arteriolar endothelium-dependent dilation associated with increased thromboxane A2 generation. Although modest microvascular rarefaction was evident, there were no other alterations to vascular structure/function. Skeletal muscle active hyperemia was blunted, although it improved after antioxidant or anti-thromboxane A2 treatment.

Functional redundancy between RAP1 isoforms in murine platelet production and function

AbstractRAP GTPases, important regulators of cellular adhesion, are abundant signaling molecules in the platelet/megakaryocytic lineage. However, mice lacking the predominant isoform, RAP1B, display a partial platelet integrin activation defect and have a normal platelet count, suggesting the existence of a RAP1-independent pathway to integrin activation in platelets and a negligible role for RAP GTPases in megakaryocyte biology. To determine the importance of individual RAP isoforms on platelet production and on platelet activation at sites of mechanical injury or vascular leakage, we generated mice with megakaryocyte-specific deletion (mKO) of Rap1a and/or Rap1b. Interestingly, Rap1a/b-mKO mice displayed a marked macrothrombocytopenia due to impaired proplatelet formation by megakaryocytes. In platelets, RAP isoforms had redundant and isoform-specific functions. Deletion of RAP1B, but not RAP1A, significantly reduced α-granule secretion and activation of the cytoskeleton regulator RAC1. Both isoforms significantly contributed to thromboxane A2 generation and the inside-out activation of platelet integrins. Combined deficiency of RAP1A and RAP1B markedly impaired platelet aggregation, spreading, and clot retraction. Consistently, thrombus formation in physiological flow conditions was abolished in Rap1a/b-mKO, but not Rap1a-mKO or Rap1b-mKO, platelets. Rap1a/b-mKO mice were strongly protected from experimental thrombosis and exhibited a severe defect in hemostasis after mechanical injury. Surprisingly, Rap1a/b-mKO platelets were indistinguishable from controls in their ability to prevent blood–lymphatic mixing during development and hemorrhage at sites of inflammation. In summary, our studies demonstrate an essential role for RAP1 signaling in platelet integrin activation and a critical role in platelet production. Although important for hemostatic/thrombotic plug formation, platelet RAP1 signaling is dispensable for vascular integrity during development and inflammation.

Dichloroacetate, an inhibitor of pyruvate dehydrogenase kinases, inhibits platelet aggregation and arterial thrombosis

AbstractResting platelets rely on oxidative phosphorylation (OXPHOS) and aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen) to generate adenosine triphosphate, whereas activated platelets exhibit a high level of aerobic glycolysis, suggesting the existence of metabolic flexibility in platelets. Mitochondrial pyruvate dehydrogenase kinases (PDK 1-4) play a pivotal role in metabolic flexibility by inhibiting pyruvate dehydrogenase complex. We determined whether metabolic reprogramming, diverting metabolism from aerobic glycolysis back to OXPHOS, would inhibit platelet function. PDKs activity in human and mouse platelets was inhibited with dichloroacetic acid (DCA), a potent inhibitor of all 4 forms of PDK. Human and mouse platelets pretreated with DCA exhibited decreased platelet aggregation to suboptimal doses of collagen, convulxin, thrombin, and adenosine diphosphate concomitant with decreased glucose uptake. Bioenergetics profile revealed that platelets pretreated with DCA exhibited decreased aerobic glycolysis in response to convulxin only. Furthermore, DCA inhibited ATP secretion, thromboxane A2 generation, and tyrosine phosphorylation of Syk and PLCγ2 in response to collagen or convulxin in human and mouse platelets (P < .05 vs vehicle treated). In the flow chamber assay, human and mouse blood pretreated with DCA formed smaller thrombi when perfused over collagen for 10 minutes at an arterial shear rate of 1500 s−1 (P < .05 vs control). Wild-type mice pretreated with DCA were less susceptible to thrombosis in the FeCl3-induced carotid and laser injury–induced mesenteric artery thrombosis models (P < .05 vs vehicle control), without altering hemostasis. Targeting metabolic plasticity with DCA may be explored as a novel strategy to inhibit platelet function.

L‐arginine Supplementation and Thromboxane Synthase Inhibition Reverse Cerebral Ischemia in Murine Cerebral Malaria by Plasmodium berghei ANKA

Cerebral malaria (CM) is one of the most severe and lethal complications of Plasmodium falciparum infection. The mainstay treatment for CM is intravenous artesunate, nevertheless 15–20% of the patients receiving this drug still die. Vascular dysfunction, with vasoconstriction, leads to decreased cerebral blood flow, ischemia, tissue hypoxia and death in CM. Nitric oxide (NO) and arachidonic acid metabolites are important regulators of physiological cerebral blood flow through their vasodilator or vasoconstrictive properties. The objective of this study was to determine the effects of L‐arginine supplementation and pharmacological inhibition of thromboxane A2 (TXA2) synthesis on the reversal of ischemia in experimental cerebral malaria (ECM). C57BL/6 mice infected with Plasmodium berghei ANKA (PbA) and showing clinical signs of ECM were treated subcutaneously with the NO precursor L‐arginine (50mg/kg) and/or the thromboxane synthase inhibitor ozagrel (100mg/kg). Cerebral blood flow was measured by Laser Speckle Contrast Imaging (LSCI). The adjuvant effect of L‐arginine in combination with artesunate on survival of mice with late‐stage ECM was evaluated. Animals that developed ECM showed a significant decrease (25%) in cerebral blood flow compared to non‐infected control animals. L‐arginine induced a marked increase (22.5%) in cerebral blood flow in mice with ECM one hour after administration. The administration of ozagrel, which prevents the generation of the vasoconstrictor metabolite TXA2, also led to increased (16.5%) cerebral blood flow in animals with ECM, and this was consistent with decreased levels of brain TXA2 in ozagrel‐treated compared to saline‐treated animals. The combination of ozagrel and L‐arginine showed no synergistic effect on cerebral blood flow. Treatment of animals with late‐stage ECM with artesunate plus L‐arginine increased survival compared to artesunate plus saline. Conclusion: L‐arginine and ozagrel increase cerebral blood flow, and L‐arginine increase survival in mice with cerebral malaria and show potential as adjuvant therapy for this malaria complication.Support or Funding InformationCAPES, FAPERJ, IOC‐FiocruzThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibitory effects of thromboxane A2 generation by ginsenoside Ro due to attenuation of cytosolic phospholipase A2 phosphorylation and arachidonic acid release

BackgroundThromboxane A2 (TXA2) induces platelet aggregation and promotes thrombus formation. Although ginsenoside Ro (G-Ro) from Panax ginseng is known to exhibit a Ca2+-antagonistic antiplatelet effect, whether it inhibits Ca2+-dependent cytosolic phospholipase A2 (cPLA2α) activity to prevent the release of arachidonic acid (AA), a TXA2 precursor, is unknown. In this study, we attempted to identify the mechanism underlying G-Ro-mediated TXA2 inhibition. MethodsWe investigated whether G-Ro attenuates TXA2 production and its associated molecules, such as cyclooxygenase-1 (COX-1), TXA2 synthase (TXAS), cPLA2α, mitogen-activated protein kinases, and AA. To assay COX-1 and TXAS, we used microsomal fraction of platelets. ResultsG-Ro reduced TXA2 production by inhibiting AA release. It acted by decreasing the phosphorylation of cPLA2α, p38-mitogen-activated protein kinase, and c-Jun N-terminal kinase1, rather than by inhibiting COX-1 and TXAS in thrombin-activated human platelets. ConclusionG-Ro inhibits AA release to attenuate TXA2 production, which may counteract TXA2-associated thrombosis.

Anti-thrombotic efficacy of S007-867: Pre-clinical evaluation in experimental models of thrombosis in vivo and in vitro

Pharmacological inhibition of platelet collagen interaction is a promising therapeutic strategy to treat intra-vascular thrombosis. S007-867 is a novel synthetic inhibitor of collagen-induced platelet aggregation. It has shown better antithrombotic protection than aspirin and clopidogrel with minimal bleeding tendency in mice. The present study is aimed to systematically investigate the antithrombotic efficacy of S007-867 in comparison to aspirin and clopidogrel in vivo and to delineate its mechanism of action in vitro. Aspirin, clopidogrel, and S007-867 significantly reduced thrombus weight in arterio-venous (AV) shunt model in rats. In mice, following ferric chloride induced thrombosis in either carotid or mesenteric artery; S007-867 significantly prolonged the vessel occlusion time (1.2-fold) and maintained a sustained blood flow velocity for >30 min. Comparatively, clopidogrel showed significant prolongation in TTO (1.3-fold) while aspirin remained ineffective. Both S007-867 and aspirin did not alter bleeding time in either kidney or spleen injury models, and thus maintained hemostasis, while clopidogrel showed significant increase in spleen bleeding time (1.7-fold). The coagulation parameters namely thrombin time, prothrombin time or activated partial thromboplastin time remained unaffected even at high concentration of S007-867 (300 µM), thus implying its antithrombotic effect to be primarily platelet mediated. S007-867 significantly inhibited collagen-mediated platelet adhesion and aggregation in mice ex-vivo. Moreover, when blood was perfused over a highly thrombogenic combination of collagen mimicking peptides like CRP-GFOGER-VWF-III, S007-867 significantly reduced total thrombus volume or ZV50 (53.4 ± 5.7%). Mechanistically, S007-867 (10–300 μM) inhibited collagen-induced ATP release, thromboxane A2 (TxA2) generation, intra-platelet [Ca+2] flux and global tyrosine phosphorylation including PLCγ2. Collectively the present study highlights that S007-867 is a novel synthetic inhibitor of collagen induced platelet activation, that effectively maintains blood flow velocity and delays vascular occlusion. It inhibits thrombogenesis without compromising hemostasis. Therefore, S007-867 may be further developed for the treatment of thrombotic disorders in clinical settings.

Ask1 regulates murine platelet granule secretion, thromboxane A2 generation, and thrombus formation

AbstractMitogen-activated protein kinases (MAPKs) are expressed in platelets and are activated downstream of physiological agonists. Pharmacological and genetic evidence indicate that MAPKs play a significant role in hemostasis and thrombosis, but it is not well understood how MAPKs are activated upon platelet stimulation. Here, we show that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAP3K family, is expressed in both human and murine platelets. ASK1 is rapidly and robustly activated upon platelet stimulation by physiological agonists. Disruption of Ask1 (Ask1−/−) resulted in a marked functional defect in platelets. Ask1−/− platelets showed an impaired agonist-induced integrin αIIbβ3 activation and platelet aggregation. Although there was no difference in Ca2+ rise, platelet granule secretion and thromboxane A2 (TxA2) generation were significantly attenuated in Ask1−/− platelets. The defective granule secretion observed in Ask1−/− platelets was a consequence of impaired TxA2 generation. Biochemical studies showed that platelet agonists failed to activate p38 MAPK in Ask1−/− platelets. On the contrary, activation of c-Jun N-terminal kinases and extracellular signal-regulated kinase 1/2 MAPKs was augmented in Ask1−/− platelets. The defect in p38 MAPK results in failed phosphorylation of cPLA2 in Ask1−/− platelets and impaired platelet aggregate formation under flow. The absence of Ask1 renders mice defective in hemostasis as assessed by prolonged tail-bleeding times. Deletion of Ask1 also reduces thrombosis as assessed by delayed vessel occlusion of carotid artery after FeCl3-induced injury and protects against collagen/epinephrine-induced pulmonary thromboembolism. These results suggest that the platelet Ask1 plays an important role in regulation of hemostasis and thrombosis.

The MAP3K Apoptosis Signal-Regulating Kinase 1 (ASK1) Functions As a Calcium-Dependent Regulator of Platelet Activation Downstream of the G(α)q-PLC Signaling Axis

Upon vascular injury, platelets rapidly adhere to sub-endothelial matrix proteins and activate to form a hemostatic platelet plug. Dysregulation of the molecular mechanisms dictating platelet plug formation is responsible for numerous thrombotic disorders. While it is known that MAPK signaling proteins such as p38, JNK, and ERK play a central role in platelet activation, little is known about the upstream MAP3K proteins regulating their activation following receptor stimulation. Our laboratory has identified the presence and activation of Apoptosis Signal-regulating Kinase 1 (ASK1), a member of the MAP3K family in human and mouse platelets. Ablation of Ask1 from mice results in a strong anti-thrombotic phenotype, and Ask1-/- platelets display significant functional defects such as integrin αIIbβ3 activation, granule secretion, and thromboxane A2 (TxA2) generation. To elucidate the signaling pathway leading to ASK1 activation, washed human platelets (4 x 108 platelets/mL) were evaluated for ASK1 phosphorylation at residue T845 (activated ASK1) by western blot analysis. Blocking TxA2 generation with aspirin (1 mM) severely diminished ASK1 activation induced by PAR1 peptide SFLLRN (100 μM), convulxin (10 ng/mL), and ADP (20 μM). Interestingly, PAR4 peptide AYPGKF (100 μM)-induced ASK1 activation was unaffected by aspirin treatment, suggesting AYPGKF-induced ASK1 activation to occur independently of TxA2 generation. The TxA2 mimetic, U46619, also induced strong T845 ASK1 phosphorylation. This data suggests ASK1 activation in human platelets to occur downstream of the TxA2 receptor TP-α and thrombin receptor PAR4. In nucleated cells, ASK1 is primarily activated by reactive oxygen species (ROS) following oxidative stress. The mechanism of ASK1 activation in platelets has never been studied. Treatment of washed human platelets (4 x 108 platelets/mL) with the ROS donor hydrogen peroxide (1 μM) failed to induce ASK1 activation. Additionally, scavenging of intracellular ROS by the ROS-scavenging compound MnTMPyP (200 μM) had no effect on agonist-induced ASK1 activation suggesting that ASK1 is activated by a ROS-independent mechanism. Interestingly, inducing intracellular calcium rise with the SERCA inhibitor, thapsigargin (1 µM), resulted in strong activation of ASK1 in human platelets. Blocking calcium rise using the calcium chelator BAPTA (50 μM) blocked both thapsigargin and agonist-induced ASK1 activation. This apparent dependency on calcium for ASK1 activation was also observed in the megakaryoblastic cell line MEG-01. Inhibition of IP3-mediated calcium release using the PLC inhibitor U73122 (10 μM) was sufficient to block ASK1 activation induced by U46619 (5 μM) and AYPGKF (100 μM), providing further evidence of a calcium-dependent mechanism for ASK1 activation in human platelets. The identification of ASK1 in platelets and the observed defects in Ask1-/- murine platelets as well as the impaired thrombosis highlights the central role of ASK1 in mediating platelet activation which, until this point, has largely been overlooked. In addition, the observation that ASK1 activation in platelets is exclusively calcium-dependent makes platelets to be a unique model for studying calcium-dependent ASK1 signaling that is independent of ROS. Collectively, we strongly believe that ASK1 is a novel target for anti-thrombotic drug therapy. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Two Structurally Distinct Novel Inhibitors of Apoptosis Signal-Regulating Kinase 1 (MAP3K5), As Potent Anti-Platelet Agents

Platelets play a critical role in both hemostasis and thrombosis. Anti-platelet drugs currently available apart from aspirin are directed against platelet agonist receptors or fibrinogen receptor integrin aIIbb3. These antagonists, although having potent anti-thrombotic activities, cause severe bleeding due to their effect on hemostasis. It is therefore of utmost important to develop new drugs that will protect from thrombosis with minimal effect on hemostasis. Apoptosis signal-regulating kinase (ASK1) is a redox sensitive serine/threonine kinase, belonging to the MAP kinase-kinase-kinase family, which is activated in response to stress. However, its role in platelets is not known. We found that ASK1 is rapidly activated downstream of all platelet agonists. Ablation of Ask1 gene results in impaired platelet functions such as granule secretion, thromboxane A2 generation, as well as fibrinogen receptor activation, which translates into attenuated platelet aggregation compared to WT littermates. We also found that thrombin failed to activate p38 in Ask1 null platelets, showing that Ask1 is indispensable for p38 activation by thrombin. FeCl3-induced carotid artery injury model of thrombosis showed a significantly increased (P=0.0003) time of occlusion and unstable thrombus formation in Ask1 null mice. These results indicated that ASK1 plays a central role in regulating platelet function, making it a potential target for combating thrombosis. We therefore synthesized two novel and highly specific ASK1 inhibitors based on the published reports, N-(6-(1H-imidazol-1-yl)imidazo[1,2-a]pyridin-2-yl)-4-(tert-butyl)benzamide (IPTB) and GS-4997. We found that GS-4997 (500nM) and IPTB (5mM) inhibit agonist-induced ASK1 activation in human platelets. They do not affect activities of related protein kinases such as ASK2, MEKK1, TAK1, and ERK1/2. We also found that IPTB and GS-4997 dose-dependently inhibited activation of p38, a downstream effector kinase, induced by a variety of platelet agonists. Furthermore, these compounds dose-dependently inhibited ADP, collagen, convulxin and PAR4 activating peptide AYPGKF-induced platelet aggregation as well as platelet spreading on immobilized fibrinogen. In-vivo carotid artery thrombosis assay revealed that WT mice injected intraperitoneally with either IPTB (100mg/kg) of GS-4997 (100mg/kg), showed a significantly increased time of occlusion (P=0.028 and P=0.005 respectively) and thrombus formed were unstable as compared to control WT mice treated with saline alone. Furthermore, injection of either IPTB or GS-4997 protected mice against collagen/epinephrine-induced pulmonary thromboembolism. Out of 14 saline-treated mice only two survived whereas, 10 out of 11 mice treated with GS-4997 (100mg/kg) survived (P=0.0002). In case of IPTB 1 out of 12 control mice survived as compared to 9 out of 12 treated mice (P=0.0028). Interestingly, tail-bleeding studies revealed that WT mice treated with either IPTB (1mg/kg) of GS-4997 (1mg/kg), did not affect the average bleeding time (100s) seen in the WT mice treated with saline alone, suggesting that both inhibitors had no effect on in-vivo hemostasis. Moreover, pretreatment of the whole blood with these inhibitors significantly reduced thrombus formation under arterial flow (800s-1) without affecting platelet adhesion to collagen as assessed using a microfluidic device. Our results strongly suggest that both IPTB and GS-4997 protect the mice from thrombosis without affecting hemostasis. Further development of these inhibitors as a potential therapeutic agent to combat thrombotic disorders is highly warranted. DisclosuresNo relevant conflicts of interest to declare.

A randomised controlled trial comparing a dietary antiplatelet, the water-soluble tomato extract Fruitflow, with 75\u2009mg aspirin in healthy subjects

Background/Objectives:Increasing numbers of food ingredients are gaining acknowledgement, via regulated health claims, of benefits to human health. One such is a water-soluble tomato extract, Fruitflow (FF), a dietary antiplatelet. We examined relative platelet responses to FF and to 75 mg aspirin (ASA) in healthy subjects.Subjects/Methods:A total of 47 healthy subjects completed a double-blinded randomised controlled trial following a crossover design. Acute and 7-day treatments with 75 mg ASA were compared with control with and without concomitant FF, over a 5-h timecourse. Platelet aggregation response agonist, platelet thromboxane A2 release, plasma clotting times and time to form a primary haemostatic clot (PFA-100 closure time, TTC) were measured.Results:Administration of all treatments lowered platelet function and thromboxane A2 generation, and extended the TTC, relative to baseline (P<0.001) and to control (P<0.001). Plasma clotting times were not affected. A single 75 mg dose of ASA showed approximately equal efficacy to a dose of FF, whereas daily 75 mg ASA was approximately three times as effective after 7 days (P=0.002). Platelet responses were heterogenous with distinct weak and strong responder groups. Weak ASA responders retained a functional platelet response to collagen agonist and were responsive to FF. Concomitant FF and ASA did not lead to significant additive effects.Conclusions:The suppression of platelet function observed after consuming FF is approximately one-third that of daily 75 mg ASA. The reversible action of FF renders it less likely to overextend the time to form a primary haemostatic clot than ASA, an important safety consideration for primary prevention.

Simultaneous quantification of PGI2 and TXA2 metabolites in plasma and urine in NO-deficient mice by a novel UHPLC/MS/MS method

The balance between vascular prostacyclin (PGI2) generated mainly via cyclooxygenase-2 (COX-2) and its physiological antagonist platelet-derived thromboxane A2 (TXA2) formed by cyclooxygenase-1 (COX-1) determines cardiovascular homeostasis. In the present work, a novel bioanalytical method for simultaneous quantification of stable plasma and urinary metabolites of PGI2 (6-keto-PGF1α, 2,3-dinor-6-keto-PGF1α) and TXA2 (TXB2, 2,3-dinor-TXB2) using ultra high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) was developed. The method was validated using artificial plasma and urine and linearity range, intra- and inter-day precision and accuracy, recovery of analytes, relative and absolute matrix effect and stability of analytes were determined. The use of artificial biofluids improved the method sensitivity as it eliminated the contribution of endogenous metabolites present in mice plasma and urine to validation procedure. The newly developed and validated method allowed to quantify 6-keto-PGF1α and TXB2 in mice plasma as well as 2,3-dinor-6-keto-PGF1α and 2,3-dinor-TXB2 in urine samples with high sensitivity and accuracy. The calibration range was established from 0.1 to 100ng/mL for all analytes using artificial biofluids and the recoveries were greater than 89.9%. All validated parameters met the criteria of acceptance specified in FDA and EMA guidance. This method was successfully employed for profiling of the changes in PGI2 and TXA2 generation in NO-deficient mice. This work demonstrated that NO-deficiency induced by L-NAME, evidenced by a fall in nitrite in plasma and urine, was associated with platelet activation, robust increase in TXB2 and mild increase in 6-keto-PGF1α concentration in plasma. Changes in 2,3-dinor-6-keto-PGF1α and 2,3-dinor-TXB2 concentration in urine were less evident suggesting that the measurements in plasma better reflect modest changes in PGI2/TXA2 homeostasis than measurements in urine.

Abstract 49: Interaction of the protein kinase C-related kinases with the TPα and TPβ isoforms of the T prostanoid receptor: role in thromboxane-induced neoplastic responses

Abstract Compelling epidemiological evidence concurs that long-term use of aspirin lowers the risk of many prevalent cancers. While such studies do not specify which cyclooxygenase-1/2 metabolite(s) are lowered by aspirin accounting for its prophylactic benefits, many reports show that some of its anti-cancer effects are due to its ability to inhibit thromboxane (TX)A2 generation, a substance more typically known for its role in hemostasis/thrombosis. In humans, TXA2 signals through the TPα and TPβ isoforms of the T prostanoid receptor (TP). There is increasing evidence supporting roles for the TXA2 axis in several neoplasms including prostate cancer, potentially due to the ability of TPα/TPβ to regulate key RhoA- and ERK-mediated signalling cascades that contribute to tumor growth &amp; metastasis. Mechanistically, we recently identified protein kinase C-related kinase (PRK) 1 as a direct interacting partner of TPα/TPβ, a functional interaction essential for TXA2-induced prostate cancer (PCa) cell migration. Moreover, agonist-activation of TPα/TPβ induced phosphorylation of histone H3 at Thr11 (H3Thr11), a PRK1-catalysed marker of androgen-induced chromatin remodelling and transcriptional activation. This was the first demonstration that the TXA2/TP axis can lead to epigenetic changes previously only associated with androgens. As PRK1/PKN1 is a member of a wider subfamily that also includes PRK2/PKNγ and PRK3/PKNβ which, like PRK1, are widely implicated in cancers, we sought to investigate if PRK2 and PRK3 may interact with TPα/TPβ. Both PRK2 and PRK3 were found to interact with TPα and TPβ, interactions dynamically regulated following TP-agonist activation. Activation of the TPs also led to phosphorylation and catalytic activation of all three kinases. Furthermore, PRK1 and PRK2, but not PRK3, interacted with TPα and TPβ in PC-3 cells, while targeted disruption of PRK1 and PRK2, but not PRK3, impaired TP-mediated H3Thr11 phosphorylation, cell proliferation and migration of PC-3 cells. Considering the critical roles of TPα/TPβ and of the PRKs in cancer progression, the discovery of these distinct, yet regulated, interactions is functionally significant, not least in prostate cancer but also in other pathophysiologies where aberrant PRK1-3 &amp;/or TPα/TPβ expression and function are implicated. Funded by the Irish Cancer Society, Health Research Board Ireland and the Movember Foundation (# PCI12KIN, MRCG/2012/3). Citation Format: Eamon P. Mulvaney, Aine G. O'Sullivan, B Therese Kinsella. Interaction of the protein kinase C-related kinases with the TPα and TPβ isoforms of the T prostanoid receptor: role in thromboxane-induced neoplastic responses. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 49. doi:10.1158/1538-7445.AM2015-49

Beneficial effect of persimmon leaves and bioactive compounds on thrombosis

The effects of ethanol extracts of persimmon leaves (EPL) and the major flavonoids catechin, epicatechin, and epicatechin gallate on blood coagulation and platelet aggregation were evaluated. Anticoagulant activities were examined by monitoring the activated partial thromboplastin time (aPTT) and the prothrombin time (PT). EPL significantly prolonged PT and aPTT. The aPTT was significantly increased in the presence of catechin, epicatechin, and epicatechin gallate, compared with controls. In order to determine the antiplatelet activity, arachidonic acid (AA), collagen, and adenosine diphosphate (ADP)-induced platelet aggregation were examined. PL and the major flavonoids significantly reduced AA-induced platelet aggregation, although they did not significantly affect ADP and collagen-induced platelet aggregation, compared with controls. EPL and the major flavonoids significantly inhibited serotonin, thromboxane A2, and soluble P-selectin generation, compared with controls. PL has potential for prevention and improvement of thrombosis by inhibiting blood coagulation and platelet activation.

Impaired Platelet Responses to Thromboxane a2 and Thrombin in Mice Lacking Receptor-Interacting Protein Kinase 3

Objective: Receptor-interacting protein 3 (RIP3) is a member of RIP family with a Ser/Thr protein kinase domain in its amino-terminus which is essential for kinase activity and autophosphorylation. The roles of RIP3 in embryonic development and different disease pathologies, such as inflammation and infections, have been reported in recent years. However, the role of RIP3 in thrombosis and hemostasis remains unknown.Methods: Hematologic analysis was performed and tail bleeding time was monitored. Mouse platelets were isolated from anti-coagulated whole blood. Platelet aggregation and secretion were recorded at real time. Platelet P-selectin exposure and specific fibrinogen binding were detected by flow cytometry. TXA2 generation was measured with enzyme immunoassay (EIA) kit. Protein phosphorylations were detected by western blotting.Result: RIP3-/- mice had tail-bleeding times that were significantly prolonged compared with their wild type littermates. In an in vivo model of mesenteric arteriole thrombosis, mice lacking RIP3 exhibited delayed thrombus formation, fewer accumulated platelets, smaller thrombi, and prolonged occlusion times. RIP3 was expressed in both human and mouse platelets. Deletion of RIP3 in mouse platelets caused a marked defect in aggregation and attenuated dense granule secretion in response to low doses of thrombin or a thromboxane A2 (TXA2) analogue, U46619. The defect in ADP secretion appears responsible for the impaired platelet aggregation, because addition of exogenous ADP rescued the reduced platelet aggregation. Although TXA2 generation and α-granule secretion were not impaired, integrin αIIbβ3 activation was attenuated in RIP3-/- platelets. Moreover, phosphorylation of Akt induced by U46619 or thrombin was markedly reduced in the absence of RIP3. Activation of Akt signaling restored the impaired aggregation of RIP3-/- platelets. ERK and p38 phosphorylation elicited by either U46619 or thrombin was attenuated in RIP3-/- platelets. In contrast, U46619- and thrombin-induced activation of PTEN, PDK1, or Src was not impaired in RIP3-/- platelets.Conclusion: Our data demonstrate a novel role for RIP3 in amplifying U46619- and thrombin-induced platelet activation by mediating Akt-dependent ADP secretion, and in supporting hemostasis and thrombus formation in vivo. RIP3 may represent a novel target to modulate PARs and TP signaling and a potential new target for antithrombotic strategy. DisclosuresNo relevant conflicts of interest to declare.

12-hydroxyeicosatetraenoic acid is associated with variability in aspirin-induced platelet inhibition.

BackgroundAspirin is one of the most widely used non-steroidal anti-inflammatory drugs (NSAIDs). It is also a commonly used anti-platelet drug, which inhibits the formation of the platelet activator, thromboxane A2 (TxA2) via inhibition of cyclooxygenase-1 (COX-1). However, the presence of a patient subset that fails to respond to aspirin despite reduced TxA2 concentrations suggests that the effect of aspirin might be more complex than exclusive COX-1 inhibition.MethodsIn this study we evaluated the impact of in vivo oral administration of a standard anti-platelet dose (75 mg) of aspirin in healthy volunteers on the acute impact of in vitro collagen-mediated platelet aggregation and generation of platelet-derived TxA2 and the 12-lipoxygenase (LOX) metabolite 12-hydroxyeicosatetraenoic acid (12-HETE). The eicosanoids were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS).ResultsLow-dose aspirin administration not only inhibited TxA2 generation but also decreased the production of 12-HETE. Furthermore, a significant correlation was observed between the levels of 12-HETE and collagen-induced platelet aggregation. Pre-treatment of platelets with the 12-LOX inhibitor, baicalein, prior to activation attenuated platelet aggregation.ConclusionsThese findings support a role for 12-HETE as a pro-aggregatory eicosanoid in platelet function and suggest a role for 12-HETE in variable sensitivity to aspirin. The study also highlights a potentially important mechanism by which aspirin impacts upon eicosanoid generation.

Antiplatelet activity of β-blockers: new light on existing data.

Antiplatelet activity of β-blockers: new light on existing data.

Dioxin-induced thrombocyte aggregation in zebrafish.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a canonical member of a group of dioxins which are byproducts of industrial combustion and are dangerous environmental pollutants. TCDD has been shown to cause several abnormalities in humans and wildlife, and recently, some dioxins have been found to activate platelets. However, TCDD-mediated platelet activation pathways are elusive and virtually nothing is known about TCDD activation of fish thrombocytes. To investigate TCDD effect on thrombocyte function, we tested zebrafish blood in presence of TCDD using a thrombocyte functional assay. We found that TCDD activated thrombocytes. Further experiments showed that thrombocytes of fish treated with TCDD formed both aggregates and filopodia. To investigate the mechanism of TCDD-mediated activation of thrombocytes we used inhibitors for Gq, cyclooxygenase-1, aryl hydrocarbon receptor (AHR), c-src, Akt, and ERK1/2. We found that TCDD induces AHR which activates c-src and signals the activation of Akt and ERK1/2 which are ultimately involved in generation of thromboxane A2. Furthermore, we found that ADP potentiates TCDD action, which led to the discovery that ADP itself activates AHR in the absence of TCDD. Taken together, these results resolved the pathway of TCDD activation of thrombocytes and led to the finding that ADP is an activator of AHR.