Platelet Thromboxane Generation Research Articles

MAPK-interacting kinase 1 regulates platelet production, activation, and thrombosis

AbstractThe MAPK-interacting kinase (Mnk) family includes Mnk1 and Mnk2, which are phosphorylated and activated in response to extracellular stimuli. Mnk1 contributes to cellular responses by regulating messenger RNA (mRNA) translation, and mRNA translation influences platelet production and function. However, the role of Mnk1 in megakaryocytes and platelets has not previously been studied. The present study investigated Mnk1 in megakaryocytes and platelets using both pharmacological and genetic approaches. We demonstrate that Mnk1, but not Mnk2, is expressed and active in human and murine megakaryocytes and platelets. Stimulating human and murine megakaryocytes and platelets induced Mnk1 activation and phosphorylation of eIF4E, a downstream target of activated Mnk1 that triggers mRNA translation. Mnk1 inhibition or deletion significantly diminished protein synthesis in megakaryocytes as measured by polysome profiling and [35S]-methionine incorporation assays. Depletion of Mnk1 also reduced megakaryocyte ploidy and proplatelet forming megakaryocytes in vitro and resulted in thrombocytopenia. However, Mnk1 deletion did not affect the half-life of circulating platelets. Platelets from Mnk1 knockout mice exhibited reduced platelet aggregation, α granule secretion, and integrin αIIbβ3 activation. Ribosomal footprint sequencing indicated that Mnk1 regulates the translation of Pla2g4a mRNA (which encodes cPLA2) in megakaryocytes. Consistent with this, Mnk1 ablation reduced cPLA2 activity and thromboxane generation in platelets and megakaryocytes. In vivo, Mnk1 ablation protected against platelet-dependent thromboembolism. These results provide previously unrecognized evidence that Mnk1 regulates mRNA translation and cellular activation in platelets and megakaryocytes, endomitosis and thrombopoiesis, and thrombosis.

PTPN7 Tyrosine Phosphatase Negatively Regulates Thromboxane Generation and ERK Phosphorylation in Platelets

Background: Thromboxane is a short-lived lipid mediator which recruits more platelets to site of vascular injury. Dysregulation of thromboxane generation can lead to several thrombotic complications such as stroke and myocardial infarction. In platelets, thromboxane generation is regulated by extracellular signal-regulated protein kinase 1/2 (ERK1/2) and therefore it is important to understand how ERK is regulated in platelets. In lymphocytes, ERK phosphorylation is regulated by a non-transmembrane protein tyrosine phosphatase PTPN7. In this study we investigated the presence of PTPN7 in platelets and effect of PTPN7 deficiency on platelet activation.Results: Western blot analysis revealed that PTPN7 is present in both human and murine platelets (Fig. 1). PTPN7 KO murine platelets show increased platelet functional responses such as aggregation, dense granule secretion and thromboxane generation upon stimulation with both GPCR and GPVI agonists (Fig. 2). In the presence of COX inhibitor indomethacin, the PTPN7 KO murine platelets aggregate and secrete to the same extent as the WT. This suggests that elevated thromboxane A2 levels are responsible for potentiation of platelet functional responses. ERK phosphorylation is also potentiated in PTPN7 KO (Fig.3) and the activity of signaling molecules upstream of ERK such as MEK is unaffected. This shows that specificity of the PTPN7 towards ERK in platelets. Lastly, in pulmonary embolism model PTPN7 KO mice have shorter time to death due to pulmonary thrombosis.Conclusions: PTPN7 is a negative regulator ERK phosphorylation and thromboxane generation in platelets. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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

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

Functional properties of platelets in piglets when changing methods of nutrition

A functionally significant element in ensuring homeostasis of the internal environment of an animal organism is platelets. The state of their activity greatly influences the rheology of blood in small vessels and, thus, the metabolism in tissues. It becomes clear that the process of growth and development of piglets at any age substantially depends on the level of their activity. In this regard, the assessment of agerelated changes in platelet activity in piglets during their early ontogenesis is of great importance. In the work performed, it was found that in piglets during the dairy and vegetable nutrition phase there is an increase in the adhesive, aggregation and secretory properties of platelets. The leading cause of these changes can be the enhancement of receptor processes and activation of the work of post-receptor mechanisms of information transfer in platelets. This is noted in piglets during the milk-plant nutrition phase simultaneously for both strong and weak aggregation inducers. The growth of platelet activity in piglets found during the observation period apparently was due to the intensification of thromboxane generation in platelets as a result of the increased activity of their cyclooxygenase and thromboxane synthetase, as well as an increase in the secretion of ADP molecules from them. The increase in the severity of the hemostatic properties of platelets in piglets during the milk-plant nutrition phase seems to be a serious regulator of their microcirculation and metabolism processes in any environmental conditions.

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.

Differential Impact of Serial Measurement of Nonplatelet Thromboxane Generation on Long-Term Outcome After Cardiac Surgery.

BackgroundSystemic thromboxane generation, not suppressible by standard aspirin therapy and likely arising from nonplatelet sources, increases the risk of atherothrombosis and death in patients with cardiovascular disease. In the RIGOR (Reduction in Graft Occlusion Rates) study, greater nonplatelet thromboxane generation occurred early compared with late after coronary artery bypass graft surgery, although only the latter correlated with graft failure. We hypothesize that a similar differential association exists between nonplatelet thromboxane generation and long‐term clinical outcome.Methods and ResultsFive‐year outcome data were analyzed for 290 RIGOR subjects taking aspirin with suppressed platelet thromboxane generation. Multivariable modeling was performed to define the relative predictive value of the urine thromboxane metabolite, 11‐dehydrothromboxane B2 (11‐dhTXB2), measured 3 days versus 6 months after surgery on the composite end point of death, myocardial infarction, revascularization or stroke, and death alone. 11‐dhTXB2 measured 3 days after surgery did not independently predict outcome, whereas 11‐dhTXB2 >450 pg/mg creatinine measured 6 months after surgery predicted the composite end point (adjusted hazard ratio, 1.79; P=0.02) and death (adjusted hazard ratio, 2.90; P=0.01) at 5 years compared with lower values. Additional modeling revealed 11‐dhTXB2 measured early after surgery associated with several markers of inflammation, in contrast to 11‐dhTXB2 measured 6 months later, which highly associated with oxidative stress.ConclusionsLong‐term nonplatelet thromboxane generation after coronary artery bypass graft surgery is a novel risk factor for 5‐year adverse outcome, including death. In contrast, nonplatelet thromboxane generation in the early postoperative period appears to be driven predominantly by inflammation and did not independently predict long‐term clinical outcome.

Cross talk between serine/threonine and tyrosine kinases regulates ADP-induced thromboxane generation in platelets

ADP-induced thromboxane generation depends on Src family kinases (SFKs) and is enhanced with pan-protein kinase C (PKC) inhibitors, but it is not clear how these two events are linked. The aim of the current study is to investigate the role of Y311 phosphorylated PKCδ in regulating ADP-induced platelet activation. In the current study, we employed various inhibitors and murine platelets from mice deficient in specific molecules to evaluate the role of PKCδ in ADP-induced platelet responses. We show that, upon stimulation of platelets with 2MeSADP, Y311 on PKCδ is phosphorylated in a P2Y1/Gq and Lyn-dependent manner. By using PKCδ and Lyn knockout murine platelets, we also show that tyrosine phosphorylated PKCδ plays a functional role in mediating 2MeSADP-induced thromboxane generation. 2MeSADP-induced PKCδ Y311 phosphorylation and thromboxane generation were potentiated in human platelets pre-treated with either a pan-PKC inhibitor, GF109203X or a PKC α/β inhibitor and in PKC α or β knockout murine platelets compared to controls. Furthermore, we show that PKC α/β inhibition potentiates the activity of SFK, which further hyper-phosphorylates PKCδ and potentiates thromboxane generation. These results show for the first time that tyrosine phosphorylated PKCδ regulates ADP-induced thromboxane generation independent of its catalytic activity and that classical PKC isoforms α/β regulate the tyrosine phosphorylation on PKCδ and subsequent thromboxane generation through tyrosine kinase, Lyn, in platelets.

Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Calcium Mobilization and Thromboxane Generation in Platelets

Members of the protein kinase C (PKC) family are shown to positively and negatively regulate platelet activation. Although positive regulatory roles are extensively studied, negative regulatory roles of PKCs are poorly understood. We investigated the mechanism and specific isoforms involved in PKC-mediated negative regulation of ADP-induced functional responses. A pan-PKC inhibitor, GF109203X, potentiated ADP-induced cPLA(2) phosphorylation and thromboxane generation as well as ERK activation and intracellular calcium (Ca(2+)(i)) mobilization, 2 signaling molecules, upstream of cPLA(2) activation. Thus, PKCs inhibit cPLA(2) activation by inhibiting ERK and Ca(2+)(i) mobilization. Because the inhibitor of classic PKC isoforms, GO-6976, did not affect ADP-mediated thromboxane generation, we investigated the role of novel class of PKC isoforms. ADP-induced thromboxane generation, calcium mobilization, and ERK phosphorylation were potentiated in PKCε null murine platelets compared with platelets from wild-type littermates. Interestingly, when thromboxane release is blocked, ADP-induced aggregation in PKCε knockout and wild-type was similar, suggesting that PKCε does not affect ADP-induced aggregation directly. PKCε knockout mice exhibited shorter times to occlusion in an FeCl(3)-induced arterial injury model and shorter bleeding times in tail-bleeding experiments. We conclude that PKCε negatively regulates ADP-induced thromboxane generation in platelets and offers protection against thrombosis.

Effects of Aspirin Responsiveness and Platelet Reactivity on Early Vein Graft Thrombosis After Coronary Artery Bypass Graft Surgery

The purpose of this study was to determine if an incomplete response to or inadequate antiplatelet effect of aspirin, or both, contribute to saphenous vein graft (SVG) occlusion after coronary artery bypass graft (CABG) surgery. Thrombosis is the predominant cause of early SVG occlusion. Aspirin, which inhibits cyclooxygenase-1 activity and thromboxane generation in platelets, reduces early SVG occlusion by one-half. Aspirin responsiveness and platelet reactivity were characterized 3 days and 6 months after coronary artery bypass graft surgery in 229 subjects receiving aspirin monotherapy by platelet aggregation to arachidonic acid, adenosine diphosphate, collagen and epinephrine, Platelet Function Analyzer-100 (Siemens Healthcare Diagnostics, Newark, Delaware) closure time (CT) using collagen/epinephrine agonist cartridge and collagen/adenosine diphosphate (CADP) agonist cartridge, VerifyNow Aspirin assay (Accumetrics, Inc., San Diego, California), and urine levels of 11-dehydro-thromboxane B(2) (UTXB(2)). SVG patency was determined 6 months after surgery by computed tomography coronary angiography. Inhibited arachidonic acid-induced platelet aggregation, indicative of aspirin-mediated cyclooxygenase-1 suppression, occurred in 95% and >99% of subjects 3 days and 6 months after surgery, respectively. Despite this, 73% and 31% of subjects at these times had elevated UTXB(2). Among tested parameters, only UTXB(2) and CADP CT measured 6 months after surgery correlated with outcome. By multivariate analysis, CADP CT of ≤88 s (odds ratio: 2.85, p = 0.006), target vessel diameter of ≤1.5 mm (odds ratio: 2.38, p = 0.01), and UTXB(2) of ≥450 pg/mg creatinine (odds ratio: 2.59, p = 0.015) correlated with SVG occlusion. CADP CT and UTXB(2) in combination further identified subjects at particularly high and low risk for SVG occlusion. Aspirin-insensitive thromboxane generation measured by UTXB(2) and shear-dependent platelet hyper-reactivity measured by Platelet Function Analyzer-100 CADP CT are novel independent risk factors for early SVG thrombosis after coronary artery bypass graft surgery.

Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Thromboxane Generation by Regulating cPLA2 Activation and Calcium Mobilization In Platelets

AbstractAbstract 2020Positive regulatory role of Protein Kinase C (PKC) isoforms in platelets have been extensively studied. However, negative regulatory roles of PKCs in platelets are poorly understood. In this study we investigated the mechanism by which PKCs negatively regulate ADP-induced thromboxane generation and identified PKC isoforms involved in this process. Pan PKC inhibition with GF109203X potentiated ADP-induced cPLA2 phosphorylation suggesting that PKCs negatively regulate thromboxane generation by regulating cPLA2 activation. Inhibition of PKCs potentiated ADP-induced ERK activation and intracellular calcium mobilization, two upstream signaling molecules of cPLA2.These data suggest that PKCs negatively regulate thromboxane by regulating ERK activation and calcium mobilization, which inturn regulate cPLA2 activation. Pan-PKC inhibition potentiated ADP-induced, P2Y1 receptor-mediated calcium mobilization in platelets independent of P2Y12-receptor. Pretreatment of platelets with GF109203X, a Pan PKC inhibitor, but not Go-6976, a classical PKC isoform inhibitor, potentiated ADP-induced thromboxane generation. Thus, we investigated the role of various novel class of PKC isoforms (nPKCs) in platelets. We have previously demonstrated that nPKC η, θ, δ positively regulates agonist-induced thromboxane generation in platelets. Thus, we investigated if the role of nPKC ε in ADP-induced thromboxane generation using PKC ε knockout mice (PKCε KO). ADP-induced thromboxane generation in PKC ε KO murine platelets was ten-fold higher than that of wild type platelets. Furthermore, PKC ε KO mice exhibited shorter times to occlusion in FeCl3-induced arterial injury model and shorter bleeding times in Tail bleeding experiments. We conclude that PKCε negatively regulates ADP-induced thromboxane generation in platelets and thereby offers protection against thrombosis. Disclosures:No relevant conflicts of interest to declare.

Mechanism of Activation and Functional Role of Protein Kinase Cη in Human Platelets

The novel class of protein kinase C (nPKC) isoform eta is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCeta using pharmacological and gene knock-out approaches. nPKCeta was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1 receptor antagonist, or YM-254890, a G(q) blocker, abolished 2MeSADP-induced phosphorylation of nPKCeta. Similarly, ADP failed to activate nPKCeta in platelets isolated from P2Y1 and G(q) knock-out mice. However, pretreatment of platelets with P2Y12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCeta phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCeta was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin alpha(IIb)beta3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a alpha(IIb)beta3 receptor antagonist, nPKCeta dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cgamma, a catalytic subunit of serine/threonine phosphatase, alpha(IIb)beta3 failed to dephosphorylate nPKCeta. Thus, we conclude that ADP activates nPKCeta via P2Y1 receptor and is subsequently dephosphorylated by PP1gamma phosphatase activated by alpha(IIb)beta3 integrin. In addition, pretreatment of platelets with eta-RACK antagonistic peptides, a specific inhibitor of nPKCeta, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCeta positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.

NPKC Epsilon Negatively Regulates Platelet Functional Responses

Protein Kinase C (PKC) are family of serine threonine kinases, known to regulate various platelet functional responses. Among them novel class of PKC isoforms (nPKC) including delta(δ), theta(θ), eta(η), and epsilon(ε) are expressed in platelets. Although, the role of nPKC ε and η in platelets is fairly understood, not much is known about nPKC ε and η in platelets. In this study, we investigated the role of nPKC ε in platelet functional responses using ADP-induced signaling as our stereotype. ADP causes platelet activation via Gq-coupled P2Y1 receptor and Gi-coupled P2Y12 receptor. Thus, we primarily studied the role of P2Y1 receptor in nPKC ε activation. ADP activated nPKC ε in time- and concentration- dependent manner. In the presence of P2Y1 receptor antagonist MRS-2179 and in P2Y1 knockout (KO) murine platelets ADP failed to activate nPKC ε, suggesting that ADP activates nPKC ε via P2Y1 receptor. We further investigated the functional role of nPKC ε using specific nPKC ε inhibitory RACK peptide (ε V1-2). ε V1-2 is a peptide designed to compete with native nPKC ε to bind ε-Receptors for activated C Kinase (ε-RACK) and thereby inhibits nPKC ε catalytic activity due to decreased substrate accessibility. ADP-induced thromboxane generation in human platelets pretreated with ε V1-2 peptide was more compared to the platelets pretreated with control peptide. Similarly, ADP-induced thromboxane generation in platelets derived from nPKC ε KO mouse was more compared to the wild type (WT) littermates. However, ADP- induced alpha granule secretion and aggregation in aspirin treated platelets derived from PKC ε KO mice was not significantly different from platelets derived from wild type littermates. These data suggest that nPKC e regulates an unknown pathway, which primarily regulates thromboxane generation with minimal effects on aggregation and alpha granule secretion. Furthermore, we also investigated the role of nPKC ε in PAR- and GPVI- mediated platelet aggregation and dense granule secretion. Interestingly, in both aspirin-treated and non-aspirin-treated platelets PAR- and GPVI- mediated platelet aggregation and dense granule secretion were potentiated. Consistent with ex vivo studies, FeCl3-induced arterial thrombosis was enhanced in nPKC ε KO mice compared to WT littermates.

Incomplete Inhibition of Thromboxane Biosynthesis by Acetylsalicylic Acid

Incomplete inhibition of platelet thromboxane generation, as measured by elevated urinary 11-dehydro thromboxane B(2) concentrations, has been associated with an increased risk of cardiovascular events. We aimed to determine the external validity of this association in aspirin-treated patients enrolled in the Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management and Avoidance (CHARISMA) trial and to determine whether there are any modifiable factors or interventions that lower urinary 11-dehydro thromboxane B(2) concentrations that could thereby reduce cardiovascular risk. Urinary 11-dehydro thromboxane B(2) concentrations were measured in 3261 aspirin-treated patients at least 1 month after they had been randomly assigned to placebo or clopidogrel. Baseline urinary 11-dehydro thromboxane B(2) concentrations in the highest quartile were associated with an increased risk of stroke, myocardial infarction, or cardiovascular death compared with the lowest quartile (adjusted hazard ratio 1.66, 95% CI 1.06 to 2.61, P=0.03). Increasing age, female sex, history of peripheral artery disease, current smoking, and oral hypoglycemic or angiotensin-converting enzyme inhibitor therapy were independently associated with higher urinary concentrations of 11-dehydro thromboxane B(2), whereas aspirin dose > or =150 mg/d, history of treatment with nonsteroidal antiinflammatory drugs, history of hypercholesterolemia, and statin treatment were associated with lower concentrations. Randomization to clopidogrel (versus placebo) did not reduce the hazard of cardiovascular events in patients in the highest quartile of urinary 11-dehydro thromboxane B(2) levels. In aspirin-treated patients, urinary concentrations of 11-dehydro thromboxane B(2) are an externally valid and potentially modifiable determinant of stroke, myocardial infarction, or cardiovascular death in patients at risk for atherothrombotic events.

Hematopoeitic lineage cell-specific protein-1 (HS1) regulates PAR-mediated ERK activation and thromboxane generation in platelets

Thrombin-induced platelet activation leads to tyrosine phosphorylation of hematopoietic lineage cell-specific protein-1 (HS1), a 75 kDa adapter protein expressed exclusively in cells of hematopoietic lineage. We have shown HS1 to be a functionally important signaling molecule downstream of PAR-4 and GPVI collagen receptor. We have thus begun to elucidate PAR signaling pathway of HS1 phosphorylation, and its functional implications. PAR-1 and PAR-4 activating peptides (SFLLRN and AYPGKF, respectively) induced HS1 phosphorylation in a Gq-dependent manner as shown by incubation with the Gq inhibitor, YM254890. Consistently, HS1 phosphorylation was abolished in platelets from Gq deficient mice upon AYPGKF stimulation. Treatment with ADP receptor antagonists did not affect HS1 phosphorylation. Pretreatment of platelets with Src kinase inhibitors abolished HS1 phosphorylation. Further Syk activation, as measured by tyrosine phosphorylation of Syk (residues 525/526), in response to PAR activation was abolished in the presence of Src inhibitors. HS1 null mice show inhibition of PAR-mediated thromboxane A2 generation compared to wild type littermates. Phosphorylation of Erk, a key signaling molecule in thromboxane generation, was also diminished in HS1 null mice platelets. Based on these findings, we conclude that tyrosine phosphorylation of HS1 occurs downstream of both PAR-1 and PAR-4. HS1 phosphorylation is a Gq mediated response regulated by Src kinases. Thus, HS1 may mediate PAR-induced thromboxane generation through regulation of Erk phosphorylation.

Evaluation of [3-(1-methyl-1H-indol-3-yl-methylene)-2-oxo-2, 3-dihydro-1H-indole-5-sulfonamide] (OXSI-2), as a Syk-selective inhibitor in platelets

In the present study, we characterized OXSI-2 [3-(1-Methyl-1H-indol-3-yl-methylene)-2-oxo-2, 3-dihydro-1H-indole-5-sulfonamide], a putative inhibitor of Syk, and determined its specificity and selectivity in platelets. We found that OXSI-2 completely abolished convulxin-induced platelet functional responses. In order to determine whether OXSI-2 inhibited Src family kinase-mediated platelet responses, we evaluated its effect on Src family kinase (SFK)-mediated signaling events in platelets, viz. Lyn-mediated phosphorylation of Y352 on Syk, LAT-Y191 phosphorylation by Syk, and protease-activated receptor (PAR)-mediated phosphorylation of ERK. In the present work, we report that convulxin mediated Syk tyrosine 352 phosphorylation is not inhibited by OXSI-2, whereas piceatannol and PP2 abolished it. Syk-mediated Y191 LAT phosphorylation is abolished by all the three inhibitors. AYPGKF-induced phosphorylation of ERK was marginally inhibited by OXSI-2, whereas treatment with PP2 and piceatannol completely abolished it. However, PAR-mediated thromboxane generation (an event mediated by ERK) was potentiated by OXSI-2 whereas PP2 and piceatannol brought thromboxane to basal levels. Protein kinase C (PKC) inhibitors are known to potentiate PAR-mediated thromboxane generation in platelets. In contrast, OXSI-2, unlike PKC inhibitors, did not inhibit secretion. Therefore, we conclude that OXSI-2 is not a Syk-selective inhibitor in platelets because of its unexplained non-specific effects.

Lyn and Fyn Kinases Positively Regulate Thromboxane Generation in Platelets.

Src family Kinases (SFKs) are important tyrosine kinases in platelets. The family consists of 9 members (viz., Blk, Fgr, Fyn, Hck, Lck, Lyn, Src, Yes and Yrk) and many of the isoforms are expressed in platelets. SFKs are key kinases in glycoprotein (GPVI) mediated platelet activation and we have shown that these kinases play an important role in thromboxane generation. Until now, the role of specific SFKs downstream of G protein signaling in platelets is not well understood. In the present study we characterized functional roles of specific SFK members Fyn, Lyn, Lck and Src Kinases downstream of ADP receptors. Src, Lyn and Fyn are activated downstream of ADP receptors (P2Y) receptors in a time and concentration dependent manner. The presence of fibrinogen receptor antagonist, GR144053, did not affect the activation of Src and Fyn; however, Lyn is not activated in the presence of GR144053, suggesting that Lyn requires outside-in signaling for it's activation. On the other hand, Lck kinase is not activated downstream of ADP receptors. ADP activates P2Y1 and P2Y12 receptors which in turn couple to Gq and Gi, respectively. In order to further delineate the Src activation pathways we used P2Y1 and P2Y12 receptor antagonists MRS 2179 and AR-C69931MX respectively. Src activation is not inhibited by either P2Y1 or P2Y12 receptor antagonists, suggesting that both receptors independently contribute to the activation of Src kinase. Platelets from mice deficient in Src, Fyn or Lyn were analyzed for thromboxane generation upon stimulation with ADP. Lyn and Fyn KO mice had reduced levels of thromboxane A2 compared to wild type littermates. However, thromboxane generation from platelets lacking Src was unaffected. Hence, we conclude Lyn and Fyn kinases, but not Src, positively regulate thromboxane generation downstream of ADP receptors. We also conclude that Lyn requires outside-in signaling for its activation.

Regulation and functional consequences of ADP receptor-mediated ERK2 activation in platelets

We have previously shown that ADP-induced thromboxane generation in platelets requires signalling events from the G(q)-coupled P2Y1 receptor (platelet ADP receptor coupled to stimulation of phospholipase C) and the G(i)-coupled P2Y12 receptor (platelet ADP receptor coupled to inhibition of adenylate cyclase) in addition to outside-in signalling. While it is also known that extracellular calcium negatively regulates ADP-induced thromboxane A2 generation, the underlying mechanism remains unclear. In the present study we sought to elucidate the signalling mechanisms and regulation by extracellular calcium of ADP-induced thromboxane A2 generation in platelets. ERK (extracllular-signal-regulated kinase) 2 activation occurred when outside-in signalling was blocked, indicating that it is a downstream event from the P2Y receptors. However, blockade of either P2Y1 or the P2Y12 receptors with corresponding antagonists completely abolished ERK phosphorylation, indicating that both P2Y receptors are required for ADP-induced ERK activation. Inhibitors of Src family kinases or the ERK upstream kinase MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] abrogated ADP-induced ERK phosphorylation and thromboxane A2 generation. Finally ADP- or G(i)+G(z)-induced ERK phosphorylation was blocked in the presence of extracellular calcium. The present studies show that ERK2 is activated downstream of P2Y receptors through a complex mechanism involving Src kinases and this plays an important role in ADP-induced thromboxane A2 generation. We also conclude that extracellular calcium blocks ADP-induced thromboxane A2 generation through the inhibition of ERK activation.

Reduced Blood Platelet Sensitivity to Aspirin in Coronary Artery Disease: Are Dyslipidaemia and Inflammatory States Possible Factors Predisposing to Sub‐optimal Platelet Response to Aspirin?

The study was designed to assess blood platelet sensitivity to acetylsalicylic acid and its associations with dyslipidaemia and inflammation in coronary artery disease patients. Platelet non-responsiveness to aspirin is associated with an increased risk of serious cardiovascular events. Several environmental and hereditary factors are reportedly involved in sub-optimal acetylsalicylic acid response. Forty-five coronary artery disease patients and 45 non-coronary artery disease controls received acetylsalicylic acid at a daily dose of 75-150 mg. Controls were examined twice: on the day of entering the study and 10 days later. Urinary 11-dehydrothromboxane B2 was assessed as the marker of platelet thromboxane generation. Aggregation was studied in platelet-rich plasma using turbidimetric aggregometry with collagen and arachidonic acid. Fifty to seventy percent of coronary artery disease patients showed an extent of collagen-induced aggregation above the upper quartile of the reference range compared with 8-15% in controls (P<0.003). For arachidonic acid-activated aggregation these proportions were 45-50% in coronary artery disease versus 7% in controls (P<0.007). In coronary artery disease patients, the acetylsalicylic acid-mediated platelet inhibition positively correlated with increased triglycerides (in arachidonic acid-stimulated platelets, r=0.30, P=0.0018), total cholesterol (r=0.33, P<0.0001 in coll and arachidonic acid-activated platelets) and elevated serum C-reactive protein (CRP) (r=0.27, P=0.0024). In coronary artery disease patients urine 11-dehydrothromboxane B2 concentrations were significantly increased compared to controls after 10 day acetylsalicylic acid intake (563; 313-728 pg/mg creatinine versus 321; 246-488 pg/mg creatinine, P=0.04). The incidence of suboptimal acetylsalicylic acid response incidence was more common in patients with coronary artery disease. Acetylsalicylic acid inhibition of blood platelet reactivity and thromboxane generation was less effective in these patients. Dyslipidaemia and chronic inflammatory states may promote suboptimal acetylsalicylic acid response in coronary artery disease patients.

Differential actions of naftopidil, doxazosin and nifedipine on platelet thromboxane generation and platelet-derived growth factor efflux in vitro

We examined the effects of the f 1 -adrenoreceptor blockers naftopidil and doxazosin and the Ca 2+ antagonist nifedipine on platelet function with reference to stimulus-induced thromboxane (TxB 2 ) generation and platelet-derived growth factor (PDGF) efflux. Collagen (5 w g/ml) caused a 12.5-fold increase in TxB 2 generation, from a basal level of 7.69 - 1.28 ng/10 8 platelets to 96.34 - 13.37 ng/10 8 platelets ( P <0.001). Adrenaline (16 w M) increased TxB 2 production 3-fold from 2.44 - 0.61 to 8.02 - 1.08 ng/10 8 platelets ( P <0.01). Adrenaline-induced TxB 2 generation was inhibited 42.5 - 10.3% ( P <0.05) and 81.8 - 7.5% ( P <0.05) by 10 and 40 w M naftopidil, respectively. Collagen-stimulated TxB 2 generation was inhibited 59.5 - 9.2% ( P <0.01) by 40 w M naftopidil and 53.7 - 11.3% ( P <0.01) by 28 w M nifedipine. Doxazosin (7.5 and 30 w M) did not influence adrenaline- or collagen-induced TxB 2 synthesis. Collagen increased PDGF efflux from 1.17 - 0.39 to 4.25 - 0.51 ng/10 8 platelets ( P <0.01), whilst adrenaline raised concentrations from 1.08 - 0.19 to 5.37 + 1.02 ng/10 8 platelets ( P <0.01). Naftopidil had no effect on collagen-induced PDGF release. Adrenaline-stimulated PDGF efflux was, however, inhibited 82.9 - 13.7% ( P <0.001) and 125.7 - 16.3% ( P <0.001) by 10 and 40 w M naftopidil, respectively. Doxazosin (30 w M) inhibited adrenaline-induced PDGF release by 70.3 - 31.5% ( P <0.05), whilst nifedipine (28 w M) had no effect on collagen-stimulated release. We conclude that naftopidil, like nifedipine, may block stimulated TxB 2 generation via inhibition of phospholipase A 2 , the Ca 2+ -dependent, rate-limiting enzyme in thromboxane synthesis. Although adrenaline-induced PDGF release was inhibited by naftopidil and doxazosin, collagen-induced release was unaffected by either f 1 -adrenoreceptor blocker or nifedipine, indicating that platelet f -granular release is not dependent on Ca 2+ mobilisation or thromboxane generation. Thus, the effects of these drugs on PDGF release may be mediated through alternative cellular signalling mechanisms.

Increased Formation of ThromboxaneIn Vivo in Humans with Mastocytosis

Clinical manifestations of mastocytosis are mediated, at least in part, by release of the mast cell mediators histamine and prostaglandin D2. It has been previously reported that in addition to prostaglandin D2, mast cells produce other eicosanoids, including thromboxane. Nonetheless, little information exists regarding the formation of other prostanoids in vivo. The most accurate method to examine the systemic production of eicosanoids in vivo is the quantitation of urinary metabolites. We previously developed a highly accurate assay employing mass spectrometry to measure a major urinary metabolite of thromboxane, 11-dehydro-thromboxane B2, in humans. We utilized this assay to quantitate thromboxane production in 17 patients with histologically proven mastocytosis. We report that thromboxane formation was significantly increased (>2 SD above the mean) in at least one urine sample from 65% of patients studied. Of these, 91% of patients with documented systemic involvement had elevated thromboxane generation. In addition, endogenous formation of thromboxane was highly correlated with the urinary excretion of the major urinary metabolite of prostaglandin D2 (r = 0.98) and Ntau-methylhistamine (r = 0.91), suggesting that the cellular source of increased thromboxane in vivo could be the mastocyte. Enhanced thromboxane formation in patients with this disorder is unlikely to be of platelet origin as other markers of platelet activation, platelet factor 4 and beta-thromboglobulin, were not increased in three patients with marked overproduction of thromboxane. Furthermore, the recovery of 11-dehydro-thromboxane B2 excretion in two patients after the administration of aspirin occurred significantly more rapidly than the recovery of platelet thromboxane generation. These studies, therefore, report that thromboxane production is significantly increased in the majority of patients with mastocytosis that we examined and provide the basis to elucidate the role of this eicosanoid in disorders of mast cell activation.