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Thrombin, a serine protease, is a potent platelet agonist that signals primarily through the protease-activated receptors (PARs). The PAR family contains four G-protein coupled receptors (GPCRs), named PARs1–4, that are activated by enzymatic cleavage of the N-terminus to expose a tethered ligand that intramolecularly activates the receptor. The expression profile of PARs on platelets is species-specific; PAR1 and PAR4 are expressed on human platelets and are the focus of this chapter. PAR1 and PAR4 interact with thrombin through distinct mechanisms. Once activated, PARs mediate signaling through overlapping pathways with distinct kinetics. PAR1 signaling is rapid and short-lived, while PAR4 initiates slower but sustained signaling. PARs have become important targets for antiplatelet therapies and the development of PAR antagonists has facilitated a greater understanding of their molecular structure and physiological roles in vivo.

Protease activated receptor 4 (PAR4) is a G protein coupled receptor (GPCR) which is activated by proteolytic cleavage of its N-terminal exodomain. This generates a tethered ligand that activates the receptor and triggers downstream signaling events. With the current focus in the development of anti-platelet therapies shifted towards PARs, new reagents are needed for expanding the field's knowledge on PAR4. Currently, there are no PAR4 reagents which are able to detect activation of the receptor.Monoclonal PAR4 antibodies were purified from hybridomas producing antibody that were generated by fusing splenocytes with NS-1 cells. Immunoblotting, immunofluorescence, and flow cytometry were utilized to detect the epitope for each antibody and to evaluate the interaction of the antibodies with cells.Here, we report the successful generation of three monoclonal antibodies to the N-terminal extracellular domain of PAR4: 14H6, 5F10, and 2D6. We mapped the epitope on PAR4 of 14H6, 5F10, and 2D6 antibodies to residues (48-53), (41-47), and (73-78), respectively. Two of the antibodies (14H6 and 5F10) interacted close to the thrombin cleavage and were sensitive to α-thrombin cleavage of PAR4. In addition, 5F10 was able to partially inhibit the cleavage of PAR4 expressed in HEK293 cells by α-thrombin.These new antibodies provide a means to monitor endogenous PAR4 expression and activation by proteases on cells.

International audience; Although situated ∼400 km from the east coast of Africa, Madagascar exhibits cultural, linguistic, and genetic traits from both Southeast Asia and Eastern Africa. The settlement history remains contentious; we therefore used a grid-based approach to sample at high resolution the genomic diversity (including maternal lineages, paternal lineages, and genome-wide data) across 257 villages and 2,704 Malagasy individuals. We find a common Bantu and Austronesian descent for all Malagasy individuals with a limited paternal contribution from Europe and the Middle East. Admixture and demographic growth happened recently, suggesting a rapid settlement of Madagascar during the last millennium. However, the distribution of African and Asian ancestry across the island reveals that the admixture was sex biased and happened heterogeneously across Madagascar, suggesting independent colonization of Madagascar from Africa and Asia rather than settlement by an already admixed population. In addition, there are geographic influences on the present genomic diversity, independent of the admixture, showing that a few centuries is sufficient to produce detectable genetic structure in human populations.

Thrombin is a potent platelet agonist that activates platelets and other cells of the cardiovascular system by cleaving its G-protein-coupled receptors, protease-activated receptor 1 (PAR1), PAR4, or both. We now show that cleaving PAR1 and PAR4 with α-thrombin induces heterodimer formation. PAR1-PAR4 heterodimers were not detected when unstimulated; however, when the cells were stimulated with 10 nm α-thrombin, we were able to detect a strong interaction between PAR1 and PAR4 by bioluminescence resonance energy transfer. In contrast, activating the receptors without cleavage using PAR1 and PAR4 agonist peptides (TFLLRN and AYPGKF, respectively) did not enhance heterodimer formation. Preventing PAR1 or PAR4 cleavage with point mutations or hirugen also prevented the induction of heterodimers. To further characterize the PAR1-PAR4 interactions, we mapped the heterodimer interface by introducing point mutations in transmembrane helix 4 of PAR1 or PAR4 that prevented heterodimer formation. Finally, we show that mutations in PAR1 or PAR4 at the heterodimer interface prevented PAR1-assisted cleavage of PAR4. These data demonstrate that PAR1 and PAR4 require allosteric changes induced via receptor cleavage by α-thrombin to mediate heterodimer formation, and we have determined the PAR1-PAR4 heterodimer interface. Our findings show that PAR1 and PAR4 have dynamic interactions on the cell surface that should be taken into account when developing and characterizing PAR antagonists.

The serine protease thrombin is a potent activator of platelets. It binds to two classes of receptors, the GPIb-IX complex and protease-activated receptors (PARs). PARs constitute a family of four G protein-coupled receptors named PARs 1–4 that mediate protease signaling in a wide variety of cells. In this chapter we describe the genomic organization and expression of PARs in platelets from humans and other species. Thrombin is the primary activator of PARs in platelets. We focus on the factors that determine the specificity and rate of cleavage PARs by thrombin, which are the initiating events of thrombin-induced platelet activation. Human platelets express PAR1 and PAR4, which have both overlapping and distinct signaling pathways. These differences have become increasingly important as therapeutics targeting PAR1 and PAR4 are developed. In addition to thrombin-PAR interactions, the activation and downstream signaling of PAR subtypes is influenced by dimerization with one another and other platelet GPCRs. We also discuss the recent identification of genetic variations that impact PAR4 signaling in humans. Finally, we highlight the differences in PAR expression on platelets across species that impact how animal models can be used as preclinical tools.

Macroautophagy (autopagy herein) is a cellular stress mechanism characterized by the engulfment of portions of cytoplasm, proteins and organelles in double or multimembrane vesicles. Cargo carried in these autophagic vesicles are then delivered and subsequently degraded by lysosomal/vacuolar systems. Autophagy occurs at low basal levels in all cell types (from yeast to mammals) under non-deprived conditions, performing homeostatic functions. Under conditions leading to cellular stress such as nutrient or growth factor deprivation, autophagy is activated to provide the cell with intracellular building blocks and substrates for energy generation. In addition to the ubiquitin-proteasome system, autophagy is a major degradation pathway for misfolded, mutant or abnormal proteins. Deregulations and abnormalities of autophagy are deleterious for all cell types including neurons. Consequently, autophagy abnormalities are observed in various neuronal diseases. Here, we summarize the basic autophagy machinery and its regulation, and provide a brief summary of the role of autophagy in healthy neurons and in major neurodegenerative diseases.

Protease activated receptors (PAR) are G-protein-coupled receptors (GPCR) that are activated by proteolytic cleavage of the N-terminus. Mouse platelets use PAR3 and PAR4 to respond to thrombin. PAR3 itself does not signal, but it is a cofactor that enhances cleavage and activation of PAR4 by thrombin ∼ 10-fold. PAR4 is the signaling thrombin receptor and is sufficient to induce platelet activation at >10 nM thrombin. The aim of this study is to determine how PAR3 regulates PAR4 signaling in mouse platelets. In this study we show that platelets from PAR3 knockout mice (PAR3-/-) had a 1.6-fold increase in the maximum calcium mobilization at thrombin concentrations above 10 nM, compared to wild type platelets. The PAR3+/- had an intermediate increase (∼ 1.2-fold) in the maximum calcium mobilization. PAR3 -/- platelets also had an increase in PKC activation, as measured by serine phosphorylation of PKC substrates in response to thrombin (10-100nM). The PAR4 agonist peptide (AYPGKF) also induced an increase in maximum calcium mobilization and PKC activation in PAR3 -/- platelets, indicating that PAR3 may be influencing PAR4 at the level of the receptor. PAR4 is coupled to Gq and G12/13. PAR3 influences PAR4-mediated calcium mobilization and PKC activation which are both downstream of Gq. We next, determined the influence of PAR3 on PAR4 mediated G12/13 signaling by measuring the activation of small GTPase RhoA protein in response to thrombin (1-100nM). The level of RhoA activation measured was not different in PAR3 -/- compared to wild-type platelets in response to thrombin. In conclusion, PAR3 negatively regulates PAR4 signaling through the Gq pathway without affecting the G12/13 pathway. Since the increase of Gq signaling is independent of PAR4 agonist, PAR3 is influencing PAR4 signaling at the level of the receptors, perhaps by forming heterodimers. The crosstalk between platelet GPCRs may be a general mechanism for regulating platelet signaling and uncovering the details of these interactions may lead to novel insights for developing therapeutics.

R-lineage mitochondrial DNA represents over 90% of the European population and is significantly present all around the planet (North Africa, Asia, Oceania, and America). This lineage played a major role in migration "out of Africa" and colonization in Europe. In order to determine an accurate dating of the R lineage and its sublineages, we analyzed 1173 individuals and complete mtDNA sequences from Mitomap. This analysis revealed a new coalescence age for R at 54.500 years, as well as several limitations of standard dating methods, likely to lead to false interpretations. These findings highlight the association of a striking under-accumulation of synonymous mutations, an over-accumulation of non-synonymous mutations, and the phenotypic effect on haplogroup J. Consequently, haplogroup J is apparently not a Neolithic group but an older haplogroup (Paleolithic) that was subjected to an underestimated selective force. These findings also indicated an under-accumulation of synonymous and non-synonymous mutations localized on coding and non-coding (HVS1) sequences for haplogroup R0, which contains the major haplogroups H and V. These new dates are likely to impact the present colonization model for Europe and confirm the late glacial resettlement scenario.

Objective— Relationships between intracellular Ca 2+ concentration ([Ca 2+ ] cyt ) and apoptotic events, such as mitochondrial depolarization (ΔΨm loss) and Bcl-2 and Bad phosphorylation, were analyzed in platelets and Jurkat cells in relation to rapid procoagulant phosphatidylserine (PS) exposure. Methods and Results— Platelets were stimulated with A23187, thapsigargin (TG) and thrombin plus convulxin (Thr/Cvx), and Jurkat cells with ionomycin, in the presence or absence of cyclosporin A (CsA), a mitochondrial permeability transition pore inhibitor. ΔΨm loss occurred when platelets were stimulated in Ca 2+ medium in conditions exposing PS, but also in EGTA medium. CsA inhibited PS exposure, [Ca 2+ ] cyt increase, and ΔΨm loss in platelets stimulated with TG and Thr/Cvx, but had no inhibitory effect on A23187 stimulation. CsA reduced TG-induced Ca 2+ release from the endoplasmic reticulum and, consequently, external Ca 2+ influx. In ionomycin-stimulated Jurkat cells, rapid PS exposure was evidenced but not ΔΨm loss, and CsA did not inhibit the process. The status of phosphorylated Bad and Bcl-2 in both cell types remained unchanged on stimulation. Conclusions— Whether ΔΨm loss occurs or not, PS exposure is triggered by a high [Ca 2+ ] cyt increase. Data further demonstrate that CsA prevents membrane scrambling by inhibiting the high [Ca 2+ ] cyt increase, independently of its effect on mitochondrial permeability transition pore.

An immunodetection study of protein tyrosine phosphatase 1B (PTP-1B), SHP-2, and Src in isolated mitochondria from different rat tissues (brain, muscle, heart, liver, and kidney) revealed their exclusive localization in the brain. Given this result, we sought whether mitochondria respond to ATP and to the general tyrosine phosphatase inhibitor orthovanadate and found little or no change in the tyrosine phosphorylation profile of mitochondria from muscle, heart, liver, and kidney. In contrast, ATP induced an enhancement in the tyrosine-phosphorylated protein profile of brain mitochondria, which was further greatly enhanced with orthovanadate and which disappeared when Src was inhibited with two inhibitors: PP2 and PP1. Importantly, we found that in brain mitochondria, ATP addition induced Src autophosphorylation at Tyr-416 in its catalytic site, leading to its activation, whereas the regulatory Tyr-527 site remained unphosphorylated. Functional implications were addressed by measurements of the enzymatic activity of each of the oxidative phosphorylation complexes in brain mitochondria in the presence of ATP. We found an increase in complex I, III, and IV activity and a decrease in complex V activity, partially reversed by Src inhibition, demonstrating that the complexes are Src substrates. These results complemented and reinforced our initial study showing that respiration of brain mitochondria was partially dependent on tyrosine phosphorylation. Therefore, the present data suggest a possible control point in the regulation of respiration by tyrosine phosphorylation of the complexes mediated by Src auto-activation.

Platelet activation by thrombin plays an important role in hemostasis and thrombosis. Protease activated receptor 1 (PAR1) and PAR4 are G- protein coupled receptors expressed in human platelets and activated by the proteolytic cleavage of their N-terminus with thrombin. We have previously shown by Bioluminescence Resonance Energy Transfer (BRET) that thrombin induces the formation of PAR1-PAR4 heterodimers in HEK293 cells. In contrast, simultaneous stimulation with the PAR1 and PAR4 agonist peptides, TFLLRN and AYPGKF, respectively, does not induce the formation of heterodimers. Signaling events that are specific to PAR1-PAR4 heterodimers in platelets are not known. In the current study we compared signaling in human platelets activated with thrombin or a mixture of TFLLRN and AYPGKF agonist peptides by examining the phosphoproteome using mass spectrometry. A global analysis of proteins phosphorylated on tyrosine, serine, or threonine residues was performed using nanoflow liquid chromatography-tandem mass spectrometry (MS/MS). Human platelets were pretreated with tirofiban, a GPIIb/IIIa integrin inhibitor, in order to block the outside in signaling and limit the analysis to the initial signaling events prior to integrin activation. Platelets were then activated with 3 nM thrombin or both TFLLRN (50 μM) and AYPGKF (500 μM) for 3 min and compared to unstimulated platelets. The results showed the expected signaling pathways activated in both thrombin-stimulated and agonist peptide-stimulated platelets. Importantly we identified 51 proteins that were specifically phosphorylated in thrombin-stimulated platelets. Next we wanted to know if the specific phosphorylation sites identified play a role in the function of these proteins. We identified a single phosphorylation residue on Ser552 of β-catenin, a signaling component of the Wnt pathway, after stimulation with thrombin but not with TFLLRN+ AYPGKF. This phosphorylation is important for the stabilization of β-catenin. We also identified that Disabled-2 (Dab2) and protein kinase C beta (PKC-β) are phosphorylated after stimulation with TFLLRN+ AYPGKF, but not thrombin. Dab2 is a cargo-specific adaptor protein involved in endocytosis and cell signaling. The phosphorylation on Ser401 regulates function and localization of Dab2 whereas the phosphorylation on Thr500 and Thr642 regulates PKC-β function. Taken together, we have identified specific signaling pathways initiated by thrombin stimulation, since thrombin induces the formation of PAR1-PAR4 heterodimers, the signaling events identified in our study may be specific to PAR1-PAR4 heterodimers. In conclusion, the understanding of the molecular arrangement of PAR1 and PAR4 in platelets will provide insight for the development of specific antiplatelet therapies. Disclosures: No relevant conflicts of interest to declare.

Abstract 2203 Thrombin is a potent platelet agonist. Thrombin activates platelets and other cells of the cardiovascular system by cleaving its receptors, protease activated receptor 1 (PAR1), PAR4 or both. PARs are G-protein coupled receptors that activate cellular signaling through Gq and G12/13. There is structural evidence that GPCRs, as a class, function as dimers and that dimerization can alter signaling specificity. Our previous studies have determined that PAR4 forms homodimers and have mapped the homodimer interface to transmembrane helix 4 (TM4). We have also shown that coexpression of PAR1 with PAR4 lowers the threshold for PAR4 activation by thrombin ∼10-fold. The purpose of the current study is to examine the physical interaction between PAR1 and PAR4 and how these interactions influence PAR1's ability to enhance PAR4 activation. The PAR1-PAR4 heterodimers were examined by bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC). Similar to our previous studies with PAR4 homodimers, PAR1 homodimers were constitutive and did not require receptor activation. In contrast, PAR1-PAR4 heterodimers were not detected under basal conditions. However, when the cells were stimulated with 10 nM thrombin, we were able to detect a strong interaction between PAR1 and PAR4. We next examined if PAR1-PAR4 heterodimers would be induced by stimulating PAR1 or PAR4 individually with their agonist peptides TFLLRN (100 μM) or AYPGKF (500 μM), respectively. The agonist peptides were unable to induce heterodimers when added to the cells individually or simultaneously. These data demonstrate that PAR1 and PAR4 require allosteric changes induced by receptor cleavage by thrombin to mediate heterodimer formation. To examine this further, we removed 37 amino acids from the C-terminus of PAR1, which disrupts the eighth helix. The truncated PAR1 was able to form constitutive heterodimers with PAR4 and these heterodimers were unaffected by thrombin. These data suggest that PAR1 is the allosteric modulator of the PAR1-PAR4 heterodimers. Finally, the stability of the constitutive PAR1 and PAR4 homodimers was unchanged in response to thrombin or the agonist peptides. Taken together, these data suggest that PAR1 and PAR4 have a dynamic interaction depending on the context of their expression. Since PAR1 is an attractive antiplatelet target, the molecular interactions of this receptor on the cells surface must be taken into account when developing and characterizing these antagonists. Disclosures: No relevant conflicts of interest to declare.