Evaluation of the Efficacy and Safety of Antiplatelet Therapeutics in Rabbits.

Hemostasis is a multifactorial process that involves vasoconstriction of blood vessels, activation of the coagulation cascade, and platelet aggregation. Inappropriate activation of hemostatic processes can result in thrombosis and tissue ischemia. In patients at risk for thrombotic events, antiplatelet therapeutic agents inhibit platelet activation, thereby reducing the incidence of pathologic clot formation. Platelets are activated by several endogenous chemical mediators, including adenosine diphosphate, thrombin, and thromboxane. These activation pathways serve as attractive drug targets. The protocols described in this article are designed to evaluate the preclinical efficacy and safety of novel antiplatelet therapeutics in rabbits. Here, we provide two protocols for blood collection, two for determining platelet activation, and one for assessing bleeding safety. Together, these protocols can be used to characterize the efficacy and safety of antiplatelet agents for hemostasis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Blood collection via the central ear artery Alternative Protocol 1: Blood collection via the jugular vein Basic Protocol 2: Platelet aggregation assessment via light transmission aggregometry Alternative Protocol 2: Platelet activation assessment via flow cytometry Basic Protocol 3: Determination of tongue bleeding time.

Clopidogrel treatment inhibits P2Y2-Mediated constriction in the rabbit middle cerebral artery.

Clopidogrel is an effective purinergic 2Y12 receptor (P2Y12) antagonist used to prevent arterial thrombosis, but its use is associated with adverse bleeding. Clinical studies have demonstrated that clopidogrel users have an increased risk of cerebral microbleeds and intracerebral hemorrhage. Our previous studies suggest that non-platelet mechanisms mediate these adverse bleeding events; we hypothesize that clopidogrel or one of its metabolites interacts with blood vessels directly to cause bleeding. New Zealand white rabbits (1.9-2.7 kg) were treated orally with vehicle or clopidogrel (3 or 10 mg/kg) for three days. On the fourth day, the rabbits were anesthetized for blood collection and then euthanized. The brain was collected, and the middle cerebral arteries were isolated. We used light transmission aggregometry and pressure myography to elucidate the mechanisms of the off-target effects associated with clopidogrel treatment. We confirmed that inhibition of P2Y12 activation by clopidogrel inhibited ADP-induced platelet aggregation but had no impact on P2Y12-independent arachidonic acid- or collagen-induced platelet aggregation. Analysis of middle cerebral arteries from clopidogrel treated rabbits showed that clopidogrel did not affect P2Y4, P2Y6, and P2Y14 receptor-mediated contraction but attenuated the contractile response after P2Y2 receptor activation. Further analysis determined P2Y2-mediated constriction was endothelium-dependent. Vasoconstriction is a primary component of hemostasis, and impaired vasoconstriction can prolong bleeding. These results suggest clopidogrel inhibits the endothelial P2Y2 receptor in the middle cerebral artery, which provides a mechanistic explanation for the adverse cerebral bleeding associated with the drug.

DT-678 inhibits platelet activation with lower tendency for bleeding compared to existing P2Y12 antagonists.

The novel clopidogrel conjugate, DT-678, is an effective inhibitor of platelets and thrombosis in preclinical studies. However, a comparison of the bleeding risk with DT-678 and currently approved P2Y12 antagonists has yet to be determined. The objective of this study was to evaluate the bleeding tendency of animals treated with clopidogrel, ticagrelor, and DT-678. Ninety-one New Zealand white rabbits were randomized to one of 13 treatment groups (n = 7). Platelet activation was assessed by flow cytometry and light transmission aggregometry before and after the administration of various doses of DT-678, clopidogrel, and ticagrelor. Tongue template bleeding times were also measured before and after drug treatment. Treatment with P2Y12 receptor antagonists caused a dose-dependent reduction in markers of platelet activation (P-selectin and integrin αIIbß3) and aggregation in response to adenosine diphosphate stimulation. At the same doses required for platelet inhibition, clopidogrel and ticagrelor significantly prolonged bleeding times, while DT-678 did not. DT-678 and the FDA-approved P2Y12 antagonists clopidogrel and ticagrelor are effective inhibitors of platelet activation and aggregation. However, unlike clopidogrel and ticagrelor, DT-678 did not prolong bleeding times at equally effective antiplatelet doses. The results suggest a more favorable benefit/risk ratio for DT-678 and potential utility as part of a dual antiplatelet therapy regimen.

Elevated hepatic fatty acid elongase-5 activity affects multiple pathways controlling hepatic lipid and carbohydrate composition.

Hepatic fatty acid elongase-5 (Elovl-5) plays an important role in long chain monounsaturated and polyunsaturated fatty acid synthesis. Elovl-5 activity is regulated during development, by diet, hormones, and drugs, and in chronic disease. This report examines the impact of elevated Elovl-5 activity on hepatic function. Adenovirus-mediated induction of Elovl5 activity in livers of C57BL/6 mice increased hepatic and plasma levels of dihomo-gamma-linolenic acid (20:3,n-6) while suppressing hepatic arachidonic acid (20:4,n-6) and docosahexaenoic acid (22:6,n-3) content. The fasting-refeeding response of peroxisome proliferator-activated receptor alpha-regulated genes was attenuated in mice with elevated Elovl5 activity. In contrast, the fasting-refeeding response of hepatic sterol-regulatory element binding protein-1 (SREBP-1)-regulated and carbohydrate-regulatory element binding protein/Max-like factor X-regulated genes, Akt and glycogen synthase kinase (Gsk)-3beta phosphorylation, and the accumulation of hepatic glycogen content and nuclear SREBP-1 were not impaired by elevated Elovl5 activity. Hepatic triglyceride content and the phosphorylation of AMP-activated kinase alpha and Jun kinase 1/2 were reduced by elevated Elovl5 activity. Hepatic phosphoenolpyruvate carboxykinase expression was suppressed, while hepatic glycogen content and phosphorylated Gsk-3beta were significantly increased, in livers of fasted mice with increased Elovl5 activity. As such, hepatic Elovl5 activity may affect hepatic glucose production during fasting. In summary, Elovl5-induced changes in hepatic fatty acid content affect multiple pathways regulating hepatic lipid and carbohydrate composition.

Docosahexaenoic acid (DHA) and hepatic gene transcription.

The type and quantity of dietary fat ingested contributes to the onset and progression of chronic diseases, like diabetes and atherosclerosis. The liver plays a central role in whole body lipid metabolism and responds rapidly to changes in dietary fat composition. Polyunsaturated fatty acids (PUFA) play a key role in membrane composition and function, metabolism and the control of gene expression. Certain PUFA, like the n-3 PUFA, enhance hepatic fatty acid oxidation and inhibit fatty acid synthesis and VLDL secretion, in part, by regulating gene expression. Our studies have established that key transcription factors, like PPARalpha, SREBP-1, ChREBP and MLX, are regulated by n-3 PUFA, which in turn control levels of proteins involved in lipid and carbohydrate metabolism. Of the n-3 PUFA, 22:6,n-3 has recently been established as a key controller of hepatic lipid synthesis. 22:6,n-3 controls the 26S proteasomal degradation of the nuclear form of SREBP-1. SREBP-1 is a major transcription factor that controls the expression of multiple genes involved fatty acid synthesis and desaturation. 22:6,n-3 suppresses nuclear SREBP-1, which in turn suppresses lipogenesis. This mechanism is achieved, in part, through control of the phosphorylation status of protein kinases. This review will examine both the general features of PUFA-regulated hepatic gene transcription and highlight the unique mechanisms by which 22:6,n-3 impacts gene expression. The outcome of this analysis will reveal that changes in hepatic 22:6,n-3 content has a major impact on hepatic lipid and carbohydrate metabolism. Moreover, the mechanisms involve 22:6,n-3 control of several well-known signaling pathways, such as Akt, Erk1/2, Gsk3beta and PKC (novel or atypical). 22:6,n-3 control of these same signaling pathways in non-hepatic tissues may help to explain the diverse actions of n-3 PUFA on such complex physiological processes as visual acuity and learning.

Regulation of hepatic fatty acid elongase and desaturase expression in diabetes and obesity.

Fatty acid elongases and desaturases play an important role in hepatic and whole body lipid composition. We examined the role that key transcription factors played in the control of hepatic elongase and desaturase expression. Studies with peroxisome proliferator-activated receptor alpha (PPARalpha)-deficient mice establish that PPARalpha was required for WY14643-mediated induction of fatty acid elongase-5 (Elovl-5), Elovl-6, and all three desaturases [Delta(5) desaturase (Delta(5)D), Delta(6)D, and Delta(9)D]. Increased nuclear sterol-regulatory element binding protein-1 (SREBP-1) correlated with enhanced expression of Elovl-6, Delta(5)D, Delta(6)D, and Delta(9)D. Only Delta(9)D was also regulated independently by liver X receptor (LXR) agonist. Glucose induction of l-type pyruvate kinase, Delta(9)D, and Elovl-6 expression required the carbohydrate-regulatory element binding protein/MAX-like factor X (ChREBP/MLX) heterodimer. Suppression of Elovl-6 and Delta(9)D expression in livers of streptozotocin-induced diabetic rats and high fat-fed glucose-intolerant mice correlated with low levels of nuclear SREBP-1. In leptin-deficient obese mice (Lep(ob/ob)), increased SREBP-1 and MLX nuclear content correlated with the induction of Elovl-5, Elovl-6, and Delta(9)D expression and the massive accumulation of monounsaturated fatty acids (18:1,n-7 and 18:1,n-9) in neutral lipids. Diabetes- and obesity-induced changes in hepatic lipid composition correlated with changes in elongase and desaturase expression. In conclusion, these studies establish a role for PPARalpha, LXR, SREBP-1, ChREBP, and MLX in the control of hepatic fatty acid elongase and desaturase expression and lipid composition.

Regulation of rat hepatic L-pyruvate kinase promoter composition and activity by glucose, n-3 polyunsaturated fatty acids, and peroxisome proliferator-activated receptor-alpha agonist.

Carbohydrate regulatory element-binding protein (ChREBP), MAX-like factor X (MLX), and hepatic nuclear factor-4alpha (HNF-4alpha) are key transcription factors involved in the glucose-mediated induction of hepatic L-type pyruvate kinase (L-PK) gene transcription. n-3 polyunsaturated fatty acids (PUFA) and WY14643 (peroxisome proliferator-activated receptor alpha (PPARalpha) agonist) interfere with glucose-stimulated L-PK gene transcription in vivo and in rat primary hepatocytes. Feeding rats a diet containing n-3 PUFA or WY14643 suppressed hepatic mRNA(L-PK) but did not suppress hepatic ChREBP or HNF-4alpha nuclear abundance. Hepatic MLX nuclear abundance, however, was suppressed by n-3 PUFA but not WY14643. In rat primary hepatocytes, glucose-stimulated accumulation of mRNA(LPK) and L-PK promoter activity correlated with increased ChREBP nuclear abundance. This treatment also increased L-PK promoter occupancy by RNA polymerase II (RNA pol II), acetylated histone H3 (Ac-H3), and acetylated histone H4 (Ac-H4) but did not significantly impact L-PK promoter occupancy by ChREBP or HNF-4alpha. Inhibition of L-PK promoter activity by n-3 PUFA correlated with suppressed RNA pol II, Ac-H3, and Ac-H4 occupancy on the L-PK promoter. Although n-3 PUFA transiently suppressed ChREBP and MLX nuclear abundance, this treatment did not impact ChREBP-LPK promoter interaction. HNF4alpha-LPK promoter interaction was transiently suppressed by n-3 PUFA. Inhibition of L-PK promoter activity by WY14643 correlated with a transient decline in ChREBP nuclear abundance and decreased Ac-H4 interaction with the L-PK promoter. WY14643, however, had no impact on MLX nuclear abundance or HNF4alpha-LPK promoter interaction. Although overexpressed ChREBP or HNF-4alpha did not relieve n-3 PUFA suppression of L-PK gene expression, overexpressed MLX fully abrogated n-3 PUFA suppression of L-PK promoter activity and mRNA(L-PK) abundance. Overexpressed ChREBP, but not MLX, relieved the WY14643 inhibition of L-PK. In conclusion, n-3 PUFA and WY14643/PPARalpha target different transcription factors to control L-PK gene transcription. MLX, the heterodimer partner for ChREBP, has emerged as a novel target for n-3 PUFA regulation.

Docosahexaneoic acid (22:6,n-3) regulates rat hepatocyte SREBP-1 nuclear abundance by Erk- and 26S proteasome-dependent pathways.

Insulin induces and dietary n-3 PUFAs suppress hepatic de novo lipogenesis by controlling sterol-regulatory element binding protein-1 nuclear abundance (nSREBP-1). Our goal was to define the mechanisms involved in this regulatory process. Insulin treatment of rat primary hepatocytes rapidly augments nSREBP-1 and mRNA(SREBP-1c) while suppressing mRNA(Insig-2) but not mRNA(Insig-1). These events are preceded by rapid but transient increases in Akt and Erk phosphorylation. Removal of insulin from hepatocytes leads to a rapid decline in nSREBP-1 [half-time (T1/2) approximately 10 h] that is abrogated by inhibitors of 26S proteasomal degradation. 22:6,n-3, the major n-3 PUFA accumulating in livers of fish oil-fed rats, suppresses hepatocyte levels of nSREBP-1, mRNA(SREBP-1c), and mRNA(Insig-2) but modestly and transiently induces mRNA(Insig-1). More importantly, 22:6,n-3 accelerates the disappearance of hepatocyte nSREBP-1 (T1/2 approximately 4 h) through a 26S proteasome-dependent process. 22:6,n-3 has minimal effects on microsomal SREBP-1 and sterol-regulatory element binding protein cleavage-activating protein or nuclear SREBP-2. 22:6,n-3 transiently inhibits insulin-induced Akt phosphorylation but induces Erk phosphorylation. Inhibitors of Erk phosphorylation, but not overexpressed constitutively active Akt, rapidly attenuate 22:6,n-3 suppression of nSREBP-1. Thus, 22:6,n-3 suppresses hepatocyte nSREBP-1 through 26S proteasome- and Erk-dependent pathways. These studies reveal a novel mechanism for n-3 PUFA regulation of hepatocyte nSREBP-1 and lipid metabolism.

Fatty acid regulation of hepatic gene transcription.

Dietary fat regulates gene expression by controlling the activity or abundance of key transcription factors. In vitro binding and cell culture studies have identified many transcription factors as prospective targets for fatty acid regulation, including peroxisome proliferator-activated receptors (PPARalpha, beta, gamma1, and gamma2), sterol regulatory element binding protein-1c (SREBP-1c), hepatic nuclear factors (HNF-4alpha and gamma), retinoid X receptor (RXRalpha), liver X receptor (LXRalpha), and others. In vivo studies established that PPARalpha- and SREBP-1c-regulated genes are key targets for PUFA control of hepatic gene expression. PUFA activate PPARalpha by direct binding, leading to the induction of hepatic fatty acid oxidation. PUFA inhibit hepatic fatty acid synthesis by suppressing SREBP-1c nuclear abundance through several mechanisms, including suppression of SREBP-1c gene transcription and enhancement of proteasomal degradation and mRNA(SREBP1c) decay. Changes in intracellular nonesterified fatty acids (NEFA) correlate well with changes in PPARalpha activity and mRNA(SREBP-1c) abundance. Several mechanisms regulate intracellular NEFA composition, including fatty acid transport, acyl CoA synthetases and thioesterases, fatty acid elongases and desaturases, neutral and polar lipid lipases, and fatty acid oxidation. Many of these mechanisms are regulated by PPARalpha or SREBP-1c. Together, these mechanisms control hepatic lipid composition and affect whole-body lipid composition.

Tissue-specific, nutritional, and developmental regulation of rat fatty acid elongases.

Of the six fatty acid elongase (Elovl) subtypes expressed in mammals, adult rat liver expresses four subtypes: Elovl-5 > Elovl-1 = Elovl-2 = Elovl-6. Overnight starvation and fish oil-enriched diets repressed hepatic elongase activity in livers of adult male rats. Diet-induced changes in elongase activity correlate with Elovl-5 and Elovl-6 mRNA abundance. Adult rats fed the peroxisome proliferator-activated receptor alpha (PPARalpha) agonist WY14,643 have increased hepatic elongase activity, Elovl-1, Elovl-5, Elovl-6, Delta5, Delta6, and Delta9 desaturase mRNA abundance, and mead acid (20:3,n-9) content. PPARalpha agonists affect both fatty acid elongation and desaturation pathways leading to changes in hepatic lipid composition. Elovl activity is low in fetal liver but increases significantly after birth. Developmental changes in hepatic elongase activity paralleled the postnatal induction of Elovl-5 mRNA and mRNAs encoding the PPARalpha-regulated transcripts, Delta5 and Delta6 desaturase, and cytochrome P450 4A. In contrast, Elovl-6, Delta9 desaturase, and FAS mRNA abundance paralleled changes in hepatic sterol regulatory element binding protein 1c (SREBP-1c) nuclear content. SREBP-1c is present in fetal liver nuclei, absent from nuclei immediately after birth, and reappears in nuclei at weaning, 21 days postpartum. In conclusion, changes in Elovl-5 expression may account for much of the nutritional and developmental control of fatty acid elongation activity in the rat liver.