57 research outputs found
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
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
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
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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]
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.
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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
Racial differences in human platelet PAR4 reactivity reflect 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. Platelet aggregation and calcium mobilization induced by the PAR4 thrombin receptor were significantly greater in black subjects. Numerous differentially expressed RNAs were associated with both race and PAR4 reactivity, including PCTP (encoding phosphatidylcholine transfer protein), and platelets from black subjects expressed higher levels of PC-TP protein. PC-TP inhibition or depletion blocked PAR4- but not PAR1-mediated activation of platelets and megakaryocytic cell lines. miR-376c levels were differentially expressed by race and PAR4 reactivity and were inversely correlated with PCTP mRNA levels, PC-TP protein levels and PAR4 reactivity. miR-376c regulated the expression of PC-TP in human megakaryocytes. A disproportionately high number of microRNAs that were differentially expressed by race and PAR4 reactivity, including miR-376c, are encoded in the DLK1-DIO3 locus and were expressed at lower levels in platelets from black subjects. These results suggest that PC-TP contributes to 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 the effects of race when developing anti-thrombotic drugs
RhoA regulation of endothelial monolayer integrity.
RhoA regulation of endothelial monolayer integrity
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