James Dilks

p63 Is a Prostate Basal Cell Marker and Is Required for Prostate Development

The p53 homologue p63 encodes for different isotypes able to either transactivate p53 reporter genes (TAp63) or act as p53-dominant-negatives (DeltaNp63). p63 is expressed in the basal cells of many epithelial organs and its germline inactivation in the mouse results in agenesis of organs such as skin appendages and the breast. Here, we show that prostate basal cells, but not secretory or neuroendocrine cells, express p63. In addition, prostate basal cells in culture predominantly express the DeltaNp63alpha isotype. In contrast, p63 protein is not detected in human prostate adenocarcinomas. Finally, and most importantly, p63(-/-) mice do not develop the prostate. These results indicate that p63 is required for prostate development and support the hypothesis that basal cells represent and/or include prostate stem cells. Furthermore, our results show that p63 immunohistochemistry may be a valuable tool in the differential diagnosis of benign versus malignant prostatic lesions.

Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents.

Thrombosis, or blood clot formation, and its sequelae remain a leading cause of morbidity and mortality, and recurrent thrombosis is common despite current optimal therapy. Protein disulfide isomerase (PDI) is an oxidoreductase that has recently been shown to participate in thrombus formation. While currently available antithrombotic agents inhibit either platelet aggregation or fibrin generation, inhibition of secreted PDI blocks the earliest stages of thrombus formation, suppressing both pathways. Here, we explored extracellular PDI as an alternative target of antithrombotic therapy. A high-throughput screen identified quercetin-3-rutinoside as an inhibitor of PDI reductase activity in vitro. Inhibition of PDI was selective, as quercetin-3-rutinoside failed to inhibit the reductase activity of several other thiol isomerases found in the vasculature. Cellular assays showed that quercetin-3-rutinoside inhibited aggregation of human and mouse platelets and endothelial cell-mediated fibrin generation in human endothelial cells. Using intravital microscopy in mice, we demonstrated that quercetin-3-rutinoside blocks thrombus formation in vivo by inhibiting PDI. Infusion of recombinant PDI reversed the antithrombotic effect of quercetin-3-rutinoside. Thus, PDI is a viable target for small molecule inhibition of thrombus formation, and its inhibition may prove to be a useful adjunct in refractory thrombotic diseases that are not controlled with conventional antithrombotic agents.

Endobrevin/VAMP-8-dependent dense granule release mediates thrombus formation in vivo.

Individuals whose platelets lack dense or alpha-granules suffer various degrees of abnormal bleeding, implying that granule cargo contributes to hemostasis. Despite these clinical observations, little is known regarding the effects of impaired platelet granule secretion on thrombus formation in vivo. In platelets, SNARE proteins mediate the membrane fusion events required for granule cargo release. Endobrevin/VAMP-8 is the primary vesicle-SNARE (v-SNARE) responsible for efficient release of dense and alpha-granule contents; thus, VAMP-8(-/-) mice are a useful model to evaluate the importance of platelet granule secretion in thrombus formation. Thrombus formation, after laser-induced vascular injury, in these mice is delayed and decreased, but not absent. In contrast, thrombus formation is almost completely abolished in the mouse model of Hermansky-Pudlak syndrome, ruby-eye, which lacks dense granules. Evaluation of aggregation of VAMP-8(-/-) and ruby-eye platelets indicates that defective ADP release is the primary abnormality leading to impaired aggregation. These results demonstrate the importance of dense granule release even in the earliest phases of thrombus formation and validate the distal platelet secretory machinery as a potential target for antiplatelet therapies.

Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1

G protein-coupled receptors (GPCRs) can assume multiple conformations and possess multiple binding sites. Whereas endogenous agonists acting at the orthosteric binding site stabilize the active receptor conformation, small molecules that act at nonorthosteric sites can stabilize alternative conformations. The large majority of these allosteric modulators associate with extracellular loops of GPCRs. The role of intracellular domains in mediating allosteric modulation is largely unknown. In screening a small-molecule library for inhibitors of platelet activation, we identified a family of compounds that modified PAR1-mediated granule secretion. The most potent inhibitory compound, termed JF5, also demonstrated noncompetitive inhibition of the α2A-adrenergic receptor. Aggregation studies using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to GPCRs that possessed a constrained eighth helix, as defined by a C-terminal palmitoylation site and interactions with TM7 and the i1 loop. Inhibition by JF5 was overcome in a PAR1 mutant in which the eighth helix was deleted, confirming a role for helix 8 in JF5 activity. Evaluation of downstream signaling showed that JF5 was selective with regard to G protein coupling, blocking signaling mediated by Gαq but not Gα12. The compound inhibited thrombus formation in vivo following vascular injury with an IC50 of ∼1 mg/kg. These results indicate a role for helix 8 in conferring sensitivity to small molecules, and show that this sensitivity can be exploited to control platelet activation during thrombus formation.

Parmodulins inhibit thrombus formation without inducing endothelial injury caused by vorapaxar

Protease-activated receptor-1 (PAR1) couples the coagulation cascade to platelet activation during myocardial infarction and to endothelial inflammation during sepsis. This receptor demonstrates marked signaling bias. Its activation by thrombin stimulates prothrombotic and proinflammatory signaling, whereas its activation by activated protein C (APC) stimulates cytoprotective and antiinflammatory signaling. A challenge in developing PAR1-targeted therapies is to inhibit detrimental signaling while sparing beneficial pathways. We now characterize a novel class of structurally unrelated small-molecule PAR1 antagonists, termed parmodulins, and compare the activity of these compounds to previously characterized compounds that act at the PAR1 ligand-binding site. We find that parmodulins target the cytoplasmic face of PAR1 without modifying the ligand-binding site, blocking signaling through Gαq but not Gα13 in vitro and thrombus formation in vivo. In endothelium, parmodulins inhibit prothrombotic and proinflammatory signaling without blocking APC-mediated pathways or inducing endothelial injury. In contrast, orthosteric PAR1 antagonists such as vorapaxar inhibit all signaling downstream of PAR1. Furthermore, exposure of endothelial cells to nanomolar concentrations of vorapaxar induces endothelial cell barrier dysfunction and apoptosis. These studies demonstrate how functionally selective antagonism can be achieved by targeting the cytoplasmic face of a G-protein-coupled receptor to selectively block pathologic signaling while preserving cytoprotective pathways.

Platelets Possess and Require an Active Protein Palmitoylation Pathway for Agonist-Mediated Activation and In Vivo Thrombus Formation

Objective—Several platelet proteins are palmitoylated, but whether protein palmitoylation functions in platelet activation is unknown. We sought to determine the role of platelet protein palmitoylation in platelet activation and thrombus formation. Methods and Results—Platelet proteins were depalmitoylated by infusing acyl-protein thioesterase 1 into permeabilized platelets. In intact platelets, platelet protein palmitoylation was blocked using the protein palmitoylation inhibitor cerulein. The effects of inhibiting platelet protein palmitoylation on platelet function and on thrombus formation in vivo were evaluated. When infused into permeabilized platelets, acyl-protein thioesterase 1 reduced total platelet protein palmitoylation and inhibited protease-activated receptor-1–mediated α-granule secretion with an IC50 of 175 nmol/L and maximal inhibition of ≥90%. Gαq and SNAP-23, membrane-associated proteins that are constitutively palmitoylated, translocated to the cytosol when permeabilized platelets were exposed to recombinant acyl-protein thioesterase 1. The protein palmitoylation inhibitor cerulein also inhibited platelet granule secretion and aggregation. Studies using intravital microscopy showed that incubation with cerulein decreased the rate of platelet accumulation into thrombi formed after laser-induced injury of mouse arterioles and inhibited maximal platelet accumulation by >60%. Conclusion—These studies show that platelets possess a protein palmitoylation machinery that is required for both platelet activation and platelet accumulation into thrombi. These studies show that inhibition of platelet protein palmitoylation blocks platelet aggregation and granule secretion. In a murine model of thrombus formation, inhibition of protein palmitoylation markedly inhibits platelet accumulation into thrombi at sites of vascular injury.

Dynamin-Related Protein-1 Controls Fusion Pore Dynamics During Platelet Granule Exocytosis

Objective—Platelet granule exocytosis serves a central role in hemostasis and thrombosis. Recently, single-cell amperometry has shown that platelet membrane fusion during granule exocytosis results in the formation of a fusion pore that subsequently expands to enable the extrusion of granule contents. However, the molecular mechanisms that control platelet fusion pore expansion and collapse are not known. Methods and Results—We identified dynamin-related protein-1 (Drp1) in platelets and found that an inhibitor of Drp1, mdivi-1, blocked exocytosis of both platelet dense and &agr;-granules. We used single-cell amperometry to monitor serotonin release from individual dense granules and, thereby, measured the effect of Drp1 inhibition on fusion pore dynamics. Inhibition of Drp1 increased spike width and decreased prespike foot events, indicating that Drp1 influences fusion pore formation and expansion. Platelet-mediated thrombus formation in vivo after laser-induced injury of mouse cremaster arterioles was impaired after infusion of mdivi-1. Conclusion—These results demonstrate that inhibition of Drp1 disrupts platelet fusion pore dynamics and indicate that Drp1 can be targeted to control thrombus formation in vivo.

Fluoxetine (Prozac) augments platelet activation mediated through protease-activated receptors.

Women s Health Initiative. Circulation 2006; 113: 2425–34. 2 Grady D, Wenger NK, Herrington D, Khan S, Furberg C, Hunninghake D, Vittinghoff E, Hulley S. Postmenopausal hormone therapy increases risk for venous thromboembolic disease. Ann Intern Med 2000; 132: 689–96. 3 Rosendaal FR, van Hylckama Vlieg A, Tanis BC, Helmerhorst FM. Estrogens, progestogens and thrombosis. J Thromb Haemost 2003; 1: 1371–80. 4 Khetawath G, Faraday N, Nealen ML, Vijayan KV, Bolton E, Noga SJ, Bray PF. Human megakaryocytes and platelets contain the estrogen receptor b and androgen receptor (AR): testosterone regulates AR expression. Blood 2000; 95: 2289–96. 5 Moro L, Reineri S, Piranda D, Pietrapiana D, Lova P, Bertoni A, Graziani A, Defilippi P, Canobbio I, Torti M, Sinigaglia F. Nongenomic effects of 17b-estradiol in human platelets: potentiation of thrombin-induced aggregation through estrogen receptor b and Src kinase. Blood 2005; 105: 115–21. 6 Akarasereenont P, Tripatara P, Chotewuttakorn S, Palo T, Thaworn A. The effects of estrone, estradiol and estriol on platelet aggregation induced by adrenaline and adenosine diphosphate. Platelets 2006; 17: 441–7. 7 Bar J, Lahav J, Hod M, Ben-Rafael Z, Weinberger I, Brosens J. Regulation of platelet aggregation and adenosine triphosphate release in vitro by 17b-estradiol and medroxyprogesterone acetate in postmenopausal women. Thromb Haemost 2000; 84: 695–700. 8 Boudoulas KD, Cooke GE, Roos CM, Bray PF, Goldschmidt-Clermont PJ. The PI Polymorphism of glycoprotein IIIa functions as a modifier for the effect of estrogen on platelet aggregation. Arch Pathol Lab Med 2001; 125: 112–5. 9 NakanoY, Oshima T,MatsuuraH,KajiyamaG,KambeM. Effect of 17b-estradiol on inhibition of platelet aggregation in vitro is medicated by an increase in NO synthesis. Arterioscler Thromb Vasc Biol 1998; 18: 961–7. 10 Leng X-H, Bray PF. Hormone therapy and platelet function. Drug Discov Today 2005; 2: 85–91. 11 Fedeniuk RW, Boison JO, MacNeil JD. Validation of a gas-chromatography-mass spectrometry method for the determination of pg ml levels of 17b-estradiol and 17b-trenbolone in bovine serum. J Chromatogr B 2004; 802: 307–15. 12 de Ronde W, Hofman A, Pols HAP, de Jong FH. A direct approach to the estimation of the origins of oestrogens and androgens in elderly men by comparison with hormone levels in postmenopausal women. Eur J Endocrinol 2005; 152: 261–8. 13 Pasqualini JR, Chetrite G, Blacker C, Feinstein MC, Delalonde L, TalbiM,Maloche C. Concentrations of estrone, estradiol, and estrone sulfate and evaluation of sulfatase and aromatase activities in preand postmenopausal breast cancer. J Clin Endocrinol Metab 1996; 81: 1460–4. 14 Lukanova A, Lundin E, Zeleniuch-Jacquotte A, Muti P, Mure A, Rinaldi S, Dossus L,Micheli A, ArslanA, Lenner P, ShoreRE,Krogh V, Koenig KL, Riboli E, Berrino F, HallmansG, Stattin P, Toniolo P, Kaaks R. Body mass index, circulating levels of sex-steroid hormones, IGF-1 and IGF-binding protein-3: a cross-sectional study in healthy women. Eur J Endocrinol 2004; 150: 161–71.