Newer agents in antiplatelet therapy: a review.

Antiplatelet therapy remains the mainstay in preventing aberrant platelet activation in pathophysiological conditions such as myocardial infarction, ischemia, and stroke. Although there has been significant advancement in antiplatelet therapeutic approaches, aspirin still remains the gold standard treatment in the clinical setting. Limitations in safety, efficacy, and tolerability have precluded many of the antiplatelet inhibitors from use in patients. Unforeseen incidences of increased bleeding risk and recurrent arterial thrombosis observed in patients have hampered the development of superior next generation antiplatelet therapies. The pharmacokinetic and pharmacodynamic profiles have also limited the effectiveness of a number of antiplatelet inhibitors currently in use due to variability in metabolism, time to onset, and reversibility. A focused effort in the development of newer antiplatelet therapies to address some of these shortcomings has resulted in a significant number of potential antiplatelet drugs which target enzymes (phosphodiesterase, cyclooxygenase), receptors (purinergic, prostaglandins, protease-activated receptors, thromboxane), and glycoproteins (αIIbβ3, GPVI, vWF, GPIb) in the platelet. The validation and search for newer antiplatelet therapeutic approaches proven to be superior to aspirin is still ongoing and should yield a better pharmacodynamic profile with fewer untoward side-effects to what is currently in use today

The emerging role of oxylipins in thrombosis and diabetes.

The prevalence of cardiovascular disease (CVD), the leading cause of death in the US, is predicted to increase due to the shift in age of the general population and increase in CVD risk factors such as obesity and diabetes. New therapies are required to decrease the prevalence of CVD risk factors (obesity and diabetes) as well as reduce atherothrombosis, the major cause of CVD related mortality. Oxylipins, bioactive metabolites derived from the oxygenation of polyunsaturated fatty acids, play a role in the progression of CVD risk factors and thrombosis. Aspirin, a cyclooxygenase-1 inhibitor, decreases atherothrombotic associated mortality by 25%. These potent effects of aspirin have shown the utility of modulating oxylipin signaling pathways to decrease CVD mortality. The role of many oxylipins in the progression of CVD, however, is still uncertain or controversial. An increased understanding of the role oxylipins play in CVD risk factors and thrombosis could lead to new therapies to decrease the prevalence of CVD and its associated mortality

5 S,15 S-Dihydroperoxyeicosatetraenoic Acid (5,15-diHpETE) as a Lipoxin Intermediate: Reactivity and Kinetics with Human Leukocyte 5-Lipoxygenase, Platelet 12-Lipoxygenase, and Reticulocyte 15-Lipoxygenase-1.

The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA4 when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB4. For 12-LOX and 5,15-diHpETE, the kinetic parameters are kcat = 0.17 s-1 and kcat/ KM = 0.011 μM-1 s-1 [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are kcat = 4.6 s-1 and kcat/ KM = 0.21 μM-1 s-1 (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB4 from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC50 of 1.3 μM; however, LXB4 did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB4 is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA4 is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA4 and LXB4 are produced by 12-LOX but only if LTA4 is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234]

Identification of a functional genetic variant driving racially dimorphic platelet gene expression of the thrombin receptor regulator, PCTP.

Platelet activation in response to stimulation of the Protease Activated Receptor 4 (PAR4) receptor differs by race. One factor that contributes to this difference is the expression level of Phosphatidylcholine Transfer Protein (PCTP), a regulator of platelet PAR4 function. We have conducted an expression Quantitative Trait Locus (eQTL) analysis that identifies single nucleotide polymorphisms (SNPs) linked to the expression level of platelet genes. This analysis revealed 26 SNPs associated with the expression level of PCTP at genome-wide significance (p \u3c 5×10(-8)). Using annotation from ENCODE and other public data we prioritised one of these SNPs, rs2912553, for functional testing. The allelic frequency of rs2912553 is racially-dimorphic, in concordance with the racially differential expression of PCTP. Reporter gene assays confirmed that the single nucleotide change caused by rs2912553 altered the transcriptional potency of the surrounding genomic locus. Electromobility shift assays, luciferase assays, and overexpression studies indicated a role for the megakaryocytic transcription factor GATA1. In summary, we have integrated multi-omic data to identify and functionalise an eQTL. This, along with the previously described relationship between PCTP and PAR4 function, allows us to characterise a genotype-phenotype relationship through the mechanism of gene expression

Benchside-to-Bedside translation of novel targets for regulating blood clots in man

Please click Download on the upper right corner to see the full descriptio

Lipid targets in prevention of clotting: Translating in vitro concepts to in vivo application

Bioactive lipids have been shown to play both pro- and anti-clotting regulatory roles in platelet function resulting in modulation of hemostasis and thrombosis. While much is known about COX-1 regulation and the role of its free fatty acid metabolites in regulation of the platelet, less is known about how 12-LOX and its fatty acid eicosanoids mediate these essential functions. Nearly 33% of deaths annually are associated with cardiovascular disease and platelet activation is essential to arteriothrombotic clots leading to myocardial infarction and stroke. Therefore a greater understanding of the role of 12-LOX in this process is needed and may represent a novel target for prevention of thrombosis. Our group has developed a highly selective 12-LOX inhibitor to target 12-LOX in the platelet and determine its potential role in platelet activation and thrombotic risk. Here, we show for the first time the in vivo utility of inhibiting 12-LOX. In human platelets run through a microfluidics system at arterial shear, treatment with the 12-LOX inhibitor ML355 was shown to be more effective at decreasing platelet adhesion to collagen compared to aspirin. In vivo, platelet accumulation at the site of injury in a number of thrombotic models in the mouse was prevented in the presence of ML355. Importantly, bleeding, a common side effect of platelet inhibition, was not affected, supporting 12-LOX as an important enzyme in regulation of hemostasis and thrombosis in vivo (Adili et al. Arterioscler Thromb Vasc Biol 2017,). These observations, coupled to the earlier observation by our group that inhibition or ablation of 12-LOX was effective in preventing immune-meditated thrombosis in human platelets and mouse models (Yeung et al. Blood 2014), raised the question of whether inhibition of 12-LOX might be a viable treatment of immune-mediated thrombocytopenia and thrombosis (ITTs). To address this question, transgenic mice expressing human immune receptor FcgRIIa but not ALOX12, were retro-orbitally injected with a fluorescent antibody for the platelet receptor -GPIX to induce ITT-like symptoms. Blood was collected at several time points to assess platelet count and the mice were sacrificed after 4 hours to determine the degree of thrombosis in vascular beds such as the lungs. While induction of ITT resulted in over 80% platelet loss within an hour and significant thrombosis in the lungs within 4 hours, animals lacking 12-LOX showed protection from both of these pathologies. Hence, targeting 12-LOX with ML355 demonstrates that 12-LOX is a viable antiplatelet target for arteriothrombotic events while exhibiting limited bleeding

12-Hepe Regulates the Antiplatelet Effects of Epa, the ω-3 Fatty Acid

Fish oil supplements containing omega-3 polyunsaturated fatty acids (PUFAs) are popular over-the-counter supplements due to their ability to reduce the risk for cardiovascular disease. Fish oil supplements are enriched with the omega-3 fatty acid, eicosapentaenoic acid (EPA). Supplementation with EPA alone provides cardiovascular protection, and the recent REDUCE-IT trial demonstrated the effectiveness of icosapent ethyl, a modified form of EPA, at reducing the risk of major cardiovascular events in at risk patients. Despite the wide use of dietary supplements containing high levels of EPA, the mechanisms regulating the cardiovascular protective effects of EPA remain unclear. The previous hypothesis was that high levels of EPA reduced the production of arachidonic acid (AA) metabolites, the most abundant proplatelet fatty acid, by competing at the oxygenase enzymes, however our group has shown the antiplatelet effects of several PUFAs are regulated through their 12-lipoxygease (12-LOX) metabolites. Therefore, we set out to determine whether the cardiovascular protective effects observed in individuals taking dietary supplements with EPA are realized by altering platelet function, and if these effects are mediated through the 12-lipoxygenase derived metabolite, 12-hydroxyeicosapentaenoic acid (12-HEPE). To determine if the presence of EPA alters the production of 12-LOX and cyxlooxygnease-1 (COX-1) metabolites, the lipid releasate from platelets treated with EPA and stimulated with collagen was analyzed via mass spectrometry. Production of 12-HEPE, the 12-LOX metabolite of EPA, is significantly increased in the presence of EPA, while COX-1 derived metabolites of EPA remain undetected. The presence of EPA does not alter levels of the 12-LOX and COX-1 derived metabolites of arachidonic acid. To assess the ability of EPA and 12-HEPE to alter platelet activation, isolated platelets from healthy human donors were treated with EPA or 12-HEPE and stimulated with various agonists targeting different steps of the hemostatic response to vascular injury. Both EPA and 12-HEPE dose-dependently inhibit collagen and thrombin-induced platelet aggregation with 12-HEPE having increased potency compared to EPA. Furthermore, 12-HEPE is a more potent inhibitor of surface expression of platelet integrin αIIbβ3 activation and surface exposure of P-selectin analyzed via flow cytometry in comparison to EPA. Additionally, EPA fully inhibits thrombus formation in whole blood under arterial shear via Total Thrombus formation Analysis System (T-TAS), while 12-HEPE only partially inhibits thrombus formation. Similarly, only EPA attenuates platelet adhesion to collagen in whole blood under arterial shear, while 12-HEPE has no effect. Our findings show for the first time 12-HEPE, the 12-LOX metabolite of EPA, is the most abundant metabolite produced by platelets when activated in the presence of EPA, suggesting the effects of EPA on platelets are regulated by 12-HEPE and are not by reducing the production of proplatelet arachidonic acid derived metabolites. We also show both EPA and 12-HEPE directly inhibit platelet activation, and 12-HEPE has more potent antiplatelet effects in isolated platelets. Interestingly, in whole blood, 12-HEPE has a reduced effect compared to EPA, suggesting 12-HEPE may be unstable or bound by circulating proteins in whole blood, making the local production of 12-HEPE within the platelet critical to its antiplatelet effect. These findings provide further insight into the mechanisms underlying the cardioprotective effects of EPA. A better understanding of current PUFA supplements containing EPA can inform treatment and prevention of cardiovascular diseases

Racial Difference in Human Platelet PAR4 Reactivity Reflects Expression of PCTP and miR-376c

Racial differences in the pathophysiology of atherothrombosis are poorly understood. We explored the function and transcriptome of platelets in healthy black (n = 70) and white (n = 84) subjects. PAR4 thrombin receptor induced platelet aggregation and calcium mobilization were significantly greater in black subjects. Numerous differentially expressed (DE) RNAs were associated with both race and PAR4 reactivity, including phosphatidylcholine transfer protein (PCTP), and platelets from blacks expressed higher levels of PC-TP protein. PC-TP inhibition or depletion blocked activation of platelets or megakaryocytic cell lines through PAR4 but not PAR1. MiR-376c levels were DE by race and PAR4 reactivity, and were inversely correlated with PCTP mRNA levels, PC-TP protein levels and PAR4 reactivity. MiR-376c regulated expression of PC-TP in human megakaryocytes. A disproportionately high number of miRNAs DE by race and PAR4 reactivity, including miR-376c, are encoded in the DLK1-DIO3 locus, and were lower in platelets from blacks. These results support PC-TP as a regulator of the racial difference in PAR4-mediated platelet activation, indicate a genomic contribution to platelet function that differs by race, and emphasize a need to consider race effects when developing anti-thrombotic drugs

Experimental evaluation of receptor-ligand interactions of dual-targeted particles to inflamed endothelium

Vascular-targeted carriers (VTCs) are often designed as leukocyte mimics, conjugated with ligands that target leukocyte adhesion molecules (LAMs) to facilitate specific adhesion to diseased endothelium. VTCs must adhere in regions with dynamic blood flow, frequently requiring multiple ligand-receptor (LR) pairs to provide particle adhesion and high disease specificity. To study LR kinetics under flow, multiple research groups have used protein-coated plates to study the adhesion and rolling of dual-targeted particles in vitro.1-4 While important knowledge is contributed by these studies, they lack the complexity of a diseased physiologic endothelium, as spatiotemporal LAM expression varies widely. Despite decades of research with the ambition of mimicking leukocytes, the specificity of multiple LAM-targeted VTCs remains poorly understood, especially in physiological environments. More specifically, there is a lack of mechanistic understanding of how multiple ligands interact with biologically complex endothelial surfaces under dynamic in vivo environments. Please click Additional Files below to see the full abstract

Evaluation of receptorâ ligand mechanisms of dualâ targeted particles to an inflamed endothelium

Vascularâ targeted carriers (VTCs) are designed as leukocyte mimics, decorated with ligands that target leukocyte adhesion molecules (LAMs) and facilitate adhesion to diseased endothelium. VTCs require different design considerations than other targeted particle therapies; adhesion of VTCs in regions with dynamic blood flow requires multiple ligandâ receptor (LR) pairs that provide particle adhesion and disease specificity. Despite the ultimate goal of leukocyte mimicry, the specificity of multiple LAMâ targeted VTCs remains poorly understood, especially in physiological environments. Here, we investigate particle binding to an inflamed mesentery via intravital microscopy using a series of particles with wellâ controlled ligand properties. We find that the total number of sites of a single ligand can drive particle adhesion to the endothelium, however, combining ligands that target multiple LR pairs provides a more effective approach. Combining sites of sialyl Lewis A (sLeA) and antiâ intercellular adhesion moleculeâ 1 (aICAM), two adhesive molecules, resulted in â ¼3â 7â fold increase of adherent particles at the endothelium over singleâ ligand particles. At a constant total ligand density, a particle with a ratio of 75% sLeA: 25% aICAM resulted in more than 3â fold increase over all over other ligand ratios tested in our in vivo model. Combined with in vivo and in silico data, we find the best dualâ ligand design of a particle is heavily dependent on the surface expression of the endothelial cells, producing superior adhesion with more particle ligand for the lesserâ expressed receptor. These results establish the importance of considering LRâ kinetics in intelligent VTC ligand design for future therapeutics.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/133573/1/btm210008-sup-0007-suppinfo07.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/133573/2/btm210008_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/133573/3/btm210008.pd