PAR4 (Protease-Activated Receptor 4) Antagonism with BMS-986120 Inhibits Human Ex Vivo Thrombus Formation

Objective-BMS-986120 is a novel first-in-class oral PAR4 (protease-Activated receptor 4) antagonist with potent and selective antiplatelet effects. We sought to determine for the first time, the effect of BMS-986120 on human ex vivo thrombus formation. Approach and Results-Forty healthy volunteers completed a phase 1 parallel-group PROBE trial (Prospective Randomized Open-Label Blinded End Point). Ex vivo platelet activation, platelet aggregation, and thrombus formation were measured at 0, 2, and 24 hours after (1) oral BMS-986120 (60 mg) or (2) oral aspirin (600 mg) followed at 18 hours with oral aspirin (600 mg) and oral clopidogrel (600 mg). BMS-986120 demonstrated highly selective and reversible inhibition of PAR4 agonist peptide (100 μM)-stimulated P-selectin expression, platelet-monocyte aggregates, and platelet aggregation (P<0.001 for all). Compared with pretreatment, total thrombus area (μm2/mm) at high shear was reduced by 29.2% (95% confidence interval, 18.3%-38.7%; P<0.001) at 2 hours and by 21.4% (9.3%-32.0%; P=0.002) at 24 hours. Reductions in thrombus formation were driven by a decrease in platelet-rich thrombus deposition: 34.8% (19.3%-47.3%; P<0.001) at 2 hours and 23.3% (5.1%-38.0%; P=0.016) at 24 hours. In contrast to aspirin alone, or in combination with clopidogrel, BMS-986120 had no effect on thrombus formation at low shear (P=nonsignificant). BMS-986120 administration was not associated with an increase in coagulation times or serious adverse events. Conclusions-BMS-986120 is a highly selective and reversible oral PAR4 antagonist that substantially reduces platelet-rich thrombus formation under conditions of high shear stress. Our results suggest PAR4 antagonism has major potential as a therapeutic antiplatelet strategy. Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT02439190

The neurofibromin GAP-related domain rescues endothelial but not neural crest development in Nf1(–/–) mice

Neurofibromatosis type I (NF1; also known as von Recklinghausen’s disease) is a common autosomal-dominant condition primarily affecting neural crest–derived tissues. The disease gene, NF1, encodes neurofibromin, a protein of over 2,800 amino acids that contains a 216–amino acid domain with Ras–GTPase-activating protein (Ras-GAP) activity. Potential therapies for NF1 currently in development and being tested in clinical trials are designed to modify NF1 Ras-GAP activity or target downstream effectors of Ras signaling. Mice lacking the murine homolog (Nf1) have mid-gestation lethal cardiovascular defects due to a requirement for neurofibromin in embryonic endothelium. We sought to determine whether the GAP activity of neurofibromin is sufficient to rescue complete loss of function or whether other as yet unidentified functions of neurofibromin might also exist. Using cre-inducible ubiquitous and tissue-specific expression, we demonstrate that the isolated GAP-related domain (GRD) rescued cardiovascular development in Nf1(–/–) embryos, but overgrowth of neural crest–derived tissues persisted, leading to perinatal lethality. These results suggest that neurofibromin may possess activities outside of the GRD that modulate neural crest homeostasis and that therapeutic approaches solely aimed at targeting Ras activity may not be sufficient to treat tumors of neural crest origin in NF1

Jun Is Required in <em>Isl1</em>-Expressing Progenitor Cells for Cardiovascular Development

<div><p>Jun is a highly conserved member of the multimeric activator protein 1 transcription factor complex and plays an important role in human cancer where it is known to be critical for proliferation, cell cycle regulation, differentiation, and cell death. All of these biological functions are also crucial for embryonic development. Although all <i>Jun</i> null mouse embryos die at mid-gestation with persistent truncus arteriosus, a severe cardiac outflow tract defect also seen in human congenital heart disease, the developmental mechanisms are poorly understood. Here we show that murine Jun is expressed in a restricted pattern in several cell populations important for cardiovascular development, including the second heart field, pharyngeal endoderm, outflow tract and atrioventricular endocardial cushions and post-migratory neural crest derivatives. Several genes, including <i>Isl1</i>, molecularly mark the second heart field. <i>Isl1</i> lineages include myocardium, smooth muscle, neural crest, endocardium, and endothelium. We demonstrate that conditional knockout mouse embryos lacking Jun in <i>Isl1</i>-expressing progenitors display ventricular septal defects, double outlet right ventricle, semilunar valve hyperplasia and aortic arch artery patterning defects. In contrast, we show that conditional deletion of Jun in <i>Tie2-</i>expressing endothelial and endocardial precursors does not result in aortic arch artery patterning defects or embryonic death, but does result in ventricular septal defects and a low incidence of semilunar valve defects, atrioventricular valve defects and double outlet right ventricle. Our results demonstrate that Jun is required in <i>Isl1</i>-expressing progenitors and, to a lesser extent, in endothelial cells and endothelial-derived endocardium for cardiovascular development but is dispensable in both cell types for embryonic survival. These data provide a cellular framework for understanding the role of Jun in the pathogenesis of congenital heart disease.</p> </div

Cardiac and vascular functions of the zebrafish orthologues of the type I neurofibromatosis gene NFI

Von Recklinghausen neurofibromatosis is a common autosomal dominant genetic disorder characterized by benign and malignant tumors of neural crest origin. Significant progress in understanding the pathophysiology of this disease has occurred in recent years, largely aided by the development of relevant animal models. Von Recklinghausen neurofibromatosis is caused by mutations in the NF1 gene, which encodes neurofibromin, a large protein that modulates the activity of Ras. Here, we describe the identification and characterization of zebrafish nf1a and nf1b, orthologues of NF1, and show neural crest and cardiovascular defects resulting from morpholino knockdown, including vascular and cardiac valvular abnormalities. Development of a zebrafish model of von Recklinghausen neurofibromatosis will allow for structure-function analysis and genetic screens in this tractable vertebrate system

<i>Tie2</i>-specific deletion of <i>Jun</i> results in cardiovascular defects.

<p>Compared to control embryos (<b>B, D</b>), the loss of <i>Jun</i> in <i>Tie2</i>-expressing precursors results in VSD (open arrowhead, <b>A</b>), thinned RV (arrows, <b>A</b>) and thickened and hyperplastic mitral valve (closed arrowhead, <b>A</b>) and pulmonary valve leaflets (open arrowhead, <b>C</b>). Ao, aorta; PA, pulmonary artery; RV, right ventricle.</p

Effect of the loss of Jun in <i>Isl1</i>-expressing progenitors on proliferation, apoptosis, cardiac neural crest cells and the endocardium.

<p>Sagittal sections of E10.5 mutant (<b>B, D, G</b>) and control (<b>A, C, F</b>) embryos analyzed by pHH3 and NFATc1 immunostaining and by TUNEL assay. pHH3 immunostaining (pink) reveals similar proliferation rates in mutant and control OFT cells (<b>A, B</b>). Dotted lines show representative areas used for cell counting with ImageJ software. TUNEL assay reveals similar numbers of TUNEL-positive (green) OFT cells in mutant and control embryos (<b>C, D</b>). (<b>E</b>) Quantitative analysis of the percentage of pHH3- and TUNEL-positive OFT cells in serial sections showing a statistically insignificant trend toward less proliferation and more apoptosis in conditionally deleted embryos at E10.5. Results are expressed as mean ± SEM of the percent positive nuclei. The statistical significance of differences between groups was analyzed by the Student’s t-test. (<b>F, G</b>) Expression of the endocardial marker NFATc1 (pink), is not significantly different between conditional mutant and control embryos. Saggital sections of anti-Tfap2a immunostained <i>Jun</i>^flox/− (<b>H</b>) and <i>Isl1</i>^Cre/+<i>; Jun</i>^flox/− (<b>I</b>) embryos showing no significant difference in Tfap2a-expressing neural crest cells (arrowheads) at E10.5. Sections were co-stained with DAPI to illustrate nuclei. A, atrium; AVC, atrioventricular cushion; LV, left ventricle; OFT, outflow tract; OFTC, outflow tract cushion; SMA, smooth muscle actin.</p

<i>Isl1</i>-specific deletion of <i>Jun</i> results in cardiovascular defects.

<p>Compared to control embryos (<b>A, A', C–E, I–L</b>), the loss of <i>Jun</i> in <i>Isl1</i>-expressing precursors results in IAA (<b>B, B'</b>), VSD (arrowhead, <b>E</b>), and enlarged and hyperplastic pulmonary valve leaflets (arrowhead, <b>O</b>). The atrioventricular valves are unaffected (<b>C, D, F, G</b>). Ao, aorta; CA, carotid artery; DA, ductus arteriosus; DAo, descending aorta; LA, left atrium; lv, left ventricle; MV, mitral valve; PA, pulmonary artery; RA, right atrium; rv, right ventricle; SCA, subclavian artery; TV, tricuspid valve.</p

Loss of <i>Jun</i> does not alter the fate of <i>Tie2</i>-expressing endothelial derivatives or cardiac neural crest cells in the developing OFT.

<p>Transverse sections of X-gal stained <i>Jun</i>^+/−<i>; Tie2-Cre;R26R</i> (<b>A, B</b>) and <i>Jun</i>^−/−<i>; Tie2-Cre; R26R</i> (<b>D, E</b>) embryos showing no significant difference in endothelial derivatives (blue) populating the OFT endocardial cushion mesenchyme (arrowheads) at E10.5. Transverse sections of anti-GFP immunostained <i>Tie2-Cre; Jun</i>^flox/+<i>; mT/mG</i> (<b>C</b>) and <i>Tie2-Cre; Jun</i>^flox/flox<i>; mT/mG</i> (<b>F</b>) embryos showing no significant difference in endothelial derivatives (green) populating the semilunar valves, heart and blood vessels at E15.5. Transverse sections of anti-Tfap2a immunostained <i>Tie2-Cre; Jun</i>^flox/+ (<b>G, H</b>) and <i>Tie2-Cre; Jun</i>^flox/− (<b>I, J</b>) embryos showing no significant difference in Tfap2a-expressing neural crest cells at E10.5. Sections were co-stained with DAPI to illustrate nuclei. Ao, aorta; AoS, aortic sac; LA, left atrium; PA, pulmonary artery; RA, right atrium; TA, truncus arteriosus.</p