ALBERT

Description: Abstract 483 The essential challenge in pharmacologic control of platelet activation during thrombus formation is to achieve inhibition of platelet mediated thrombosis without impairing hemostasis. Inhibition of platelet GPCRs has focused primarily on inhibition of orthosteric binding sites, selecting for compounds that compete with endogenous ligands. However, small molecules that act at non-orthosteric sites can stabilize alternative GPCR conformations and modify GPCR function. Such allosteric modulators demonstrate saturable binding, selective effects on affinity and efficacy, and could provide improved physiologic control of platelet function. However, cell-based screens are required for the identification of such modulators. We screened a 16,000 compound library for inhibitors of platelet activation and identified a family of alkylated quinolines that modified Par1-mediated granule secretion. Activity of these compounds was determined by the length of their alkyl tails such that a two carbon addition to the tail converted a compound that inhibited Par1 activating peptide (SFLLRN)-induced P-selectin expression into a compound that augmented Par1-mediated activation. The most potent inhibitory compound, JF5 (IC50 = 4 μM), blocked Par1 activating peptide-induced GTPase activity and GTP[γ-35S] binding but not GTPγS-induced platelet activation in permeabilized platelets, thus indicating activity at Par1. The compound failed to inhibit platelet activation induced by a Par4 activating peptide, the thromboxane analog U46619, arginine vasopressin, PMA, or Ca2+ ionophore. Studies using cultured cells overexpressing specific GPCRs demonstrated that JF5 inhibited Par1-mediated activation, but failed to inhibit activation through TP, P2Y1 or EP3 receptors. However, JF5 did demonstrate inhibition of the α2A-adrenergic receptor. Radioligand binding studies confirmed non-competitive inhibition of the α2A-adrenergic receptor by JF5. Comparative phenotyping using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to select GPCRs. Evaluation of the amino acid sequence of these receptors demonstrated that sensitive receptors possessed a constrained 8th helix, as defined by a C-terminal palmitoylation site and interactions of the 8th helix with TM7 and the i1 loop. The 8th helix of GPCRs is oriented parallel to the intracellular face of the plasma membrane and is thought to transduce signals to cognate Gα subunits. To assess the hypothesis that the 8th helix contributes to the inhibitory activity of JF5, the compound was evaluated for its ability to inhibit IP3 generation in COS7 cells overexpressing wild-type Par1 or cells expressing a Par1 mutant in which residues 376–386 had been replaced with 3 alanine residues (ΔH8 Par1). JF5 completely inhibited SFLLRN-induced IP3 generation in cells expressing wild-type Par1. Inhibition was reversed in the ΔH8 Par1 mutant, confirming the involvement of the 8th helix in JF5 inhibitory activity. Despite its potent inhibition of Par1-mediated aggregation, secretion, and IP3 generation, JF5 did not inhibit SFLLRN-induced shape change. To evaluate the premise that JF5 inhibited coupling to Gαq but not Gα12/13, we determined the effect of JF5 on SFLLRN-induced Gα12-dependent decreased barrier function by measuring changes in transepithelial resistance (TER) in MDCK cells overexpressing Gα12. JF5 demonstrated no inhibition of SFLLRN-mediated decrease in TER at concentrations up to 200 μM, whereas the orthosteric Par1 inhibitor, Sch79797, completely blocked SFLLRN-induced TER at 1 μM. These results confirmed that JF5 inhibits Par1 coupling to Gαq, but not Gα12. JF was also evaluated in a mouse model of thrombus formation following laser-induced injury of cremaster arterioles. JF5 inhibited thrombus formation with an IC50 of 1 mg/kg and delayed thrombus formation by 2.3-fold. Evaluation of the 8th helix of mouse Par4 demonstrated that, unlike the 8th helix of human Par4, it contained a palmitoylation site and is predicted to have a constrained conformation based on interactions with TM7. These results indicate a role for the 8th helix in conferring sensitivity to small molecules and show that this sensitivity can be exploited to control platelet activation during thrombus formation. JF5 will serve as a useful probe to evaluate the role of the 8th helix in coupling of GPCRs to cognate Gα subunits. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1062 Protein palmitoylation is a reversible post-translational modification that regulates both lipid-protein and protein-protein interactions. During the palmitoylation cycle, palmitoylation occurs through a thioester linkage to a cysteine residue. Depalmitoylation occurs primarily through cleavage of this bond by acyl-protein thioesterase 1 (APT1). We have previously demonstrated the presence of APT1 in platelets and showed that APT1 translocates to membranes in an activation-dependent manner. However, the function of APT1 in platelet activation is not known. To determine whether APT1 functions in platelet signal transduction we evaluated the effect of palmostatins, novel small molecule inhibitors of APT1, on platelet function. Palmostatins B and M both inhibited platelet aggregation and α -granule secretion induced through protease-activated receptor (PAR) 1 with an IC50 of 15 μM. To assess which signaling pathways were affected by APT1 inhibition, we screened palmostatins for their ability to inhibit activation induced by several agonists. Palmostatins blocked platelet aggregation induced by a PAR1 agonist, a PAR4 agonist, TxA2, or epinephrine. In contrast, palmostatins failed to inhibit aggregation induced by collagen, PMA, or ionophore. Palmostatins also inhibited α -granule exocytosis induced by a PAR1 agonist or TxA2, but not exocytosis induced by PMA or ionophore. These results suggested that palmostatins blocked proximal signaling events mediated through G protein coupled receptors (GPCRs). To evaluate this supposition, we tested the effect of palmostatin B on PAR1-mediated [Ca2+]i flux. Palmostatin B inhibited PAR1-induced Ca2+ signaling with and IC50 of 15 μM, the same concentration required for inhibition of platelet aggregation and α -granule secretion. We have recently described the platelet palmitoylome (Dowal et al., Blood, 118:e62-73) and found several components of the proximal G protein signaling pathway that are palmitoylated, including many Gα subunits. To directly assess the effect of APT1 inhibition on palmitoylation/depalmitoylation cycles on a target Gα subunit, we evaluated Gα q palmitoylation using acyl biotin exchange chemistry. Total Gα q palmitoylation decreased substantially with activation of platelets through PAR1. In the presence of palmostatin B, however, Gα q palmitoylation increased following PAR1 activation. These results demonstrate that Gα q is a substrate for APT1. Our studies demonstrate a role for palmitoylation/depalmitoylation cycles in proximal signaling pathways downstream of GPCRs and implicate APT1 as an essential regulator of G protein signaling in platelets. Disclosures: No relevant conflicts of interest to declare.

Description: Protein palmitoylation is a dynamic process that regulates membrane targeting of proteins and protein-protein interactions. We have previously demonstrated a critical role for protein palmitoylation in platelet activation and have identified palmitoylation machinery in platelets. Using a novel proteomic approach, Palmitoyl Protein Identification and Site Characterization, we have begun to characterize the human platelet palmitoylome. Palmitoylated proteins were enriched from membranes isolated from resting platelets using acyl-biotinyl exchange chemistry, followed by identification using liquid chromatography-tandem mass spectrometry. This global analysis identified 〉 1300 proteins, of which 215 met criteria for significance and represent the platelet palmitoylome. This collection includes 51 known palmitoylated proteins, 61 putative palmitoylated proteins identified in other palmitoylation-specific proteomic studies, and 103 new putative palmitoylated proteins. Of these candidates, we chose to validate the palmitoylation of triggering receptors expressed on myeloid cell (TREM)–like transcript-1 (TLT-1) as its expression is restricted to platelets and megakaryocytes. We determined that TLT-1 is a palmitoylated protein using metabolic labeling with [3H]palmitate and identified the site of TLT-1 palmitoylation as cysteine 196. The discovery of new platelet palmitoyl protein candidates will provide a resource for subsequent investigations to validate the palmitoylation of these proteins and to determine the role palmitoylation plays in their function.

Description: Abstract 2017 Protein palmitoylation is a dynamic process that regulates membrane targeting of proteins and protein-protein interactions. It is unique among the fatty acid modifications as it is reversible, and its reversibility suggests that it can participate in the regulation of cell signaling. We have previously demonstrated a critical role for protein palmitoylation in platelet activation and have begun to characterize the palmitoylation machinery in platelets. We have now employed a novel proteomic approach termed Palmitoyl Protein Identification and Site Characterization (PalmPISC) to define the platelet “palmitoylome.” Using acyl biotin exchange (ABE) chemistry, we have purified palmitoylated proteins from membranes of resting platelets and identified them using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Spectral counting analysis identified 131 putative palmitoylated proteins including 58 novel palmitoylated proteins. Components of the G protein signal transduction pathways (15% of palmitoylated proteins) and membrane fusion proteins (10% of palmitoylated proteins) were highly represented. Platelets undergo a dramatic phenotypic change upon activation and platelet proteins are known to undergo activation-dependent palmitoylation. Changes in the palmitoylation state of proteins during platelet signaling may be reflective of the activation process. We have compared changes in protein palmitoylation in resting and thrombin-activated platelets to identify proteins that undergo activation-dependent palmitoylation or depalmitoylation. To quantify these changes by mass spectrometry, we employed iTRAQ labeling and identified 32 proteins that increase or decrease their palmitoylation upon activation. We have focused our initial efforts on one of these proteins, Triggering Receptor Expressed on Myloid cells (TREM)-like transcript-1 (TLT-1), an immunoglobulin domain-containing receptor expressed exclusively in platelets and megakaryocytes. We have validated that platelet TLT-1 is palmitoylated using [3H]palmitate labeling and have identified the site of TLT-1 palmitoylation as juxtamembrane Cys196, which is adjacent to an ITIM domain. Our iTRAQ results reveal that TLT-1 exhibits a 2-fold decrease in palmitoylation upon activation. A decrease in TLT-1 palmitoylation upon Par1-mediated activation was confirmed using an ABE strategy, which detects total protein palmitoylation. In contrast, there is a 2.5-fold increase in [3H]palmitate labeling of TLT-1 upon activation of platelets, indicating increased turnover of palmitate with activation. These observations suggest that activation-dependent depalmitoylation of TLT-1 occurs more rapidly than activation-dependent palmitoylation and underscores the importance of measuring both total palmitoylation and palmitate turnover in assessing activation-dependent palmitoylation. This global analysis of platelet protein palmitoylation provides a platform to inform future investigations identifying the role of palmitoylation in the function of specific platelet proteins. Identification of proteins that undergo activation-dependent palmitoylation or depalmitoylation will enable studies of the platelet protein palmitoylation machinery. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1138 Protease-activated receptor-1 (PAR1) is a widely expressed G protein-coupled receptor (GPCR) that functions in thrombus formation, inflammation, and mitogenesis. Like other GPCRs, PAR1 can assume multiple conformations and possess multiple binding sites. We hypothesized that the binding of small molecules to sites outside of the tethered ligand binding pocket can stabilize these alternative conformations, resulting in novel signaling properties. To identify allosteric modulators of PAR1, we screened 〉300,000 compounds for the ability to inhibit PAR1-mediated dense granule release. Following the identification of potent compounds with a 1,3-diaminophenyl scaffold, 81 analogs were synthesized or procured and tested for inhibition of PAR1-mediated granule secretion. Structural features that were required for optimal inhibition included a 4-carbon chain at the “western end” and a 2'-substituted benzamide at the “eastern end” of the molecule. The most potent compound, ML161, inhibited PAR1-mediated platelet activation with an IC50 of 300 nM, 〉10-fold more potently than JF5, a previously identified allosteric modulator of PAR1. ML161 inhibited platelet activation induced by the PAR1 agonists SFLLRN and thrombin, but not other platelet agonists including the PAR4 agonist AYPGKF, PMA, ionophore, collagen, or ADP. ML161 inhibited SFLLRN-induced Ca2+ flux in platelets and HEK293 cells overexpressing PAR1, confirming activity at PAR1. Modeling of multiple Ca2+ flux curves at different ML161 concentrations over a range of SFLLRN doses indicated an allosteric mode of inhibition. A similar analysis of the effect of ML161 on PAR1-mediated P-selectin expression confirmed an allosteric mechanism. In platelet aggregation studies, ML161 inhibited SFLLRN-induced aggregation, but not shape change, raising the possibility that it inhibits Gαq-mediated, but not Gα12/13-mediated pathways. Shape change in the presence of ML161 was sensitive to the Rho kinase inhibitor Y27632, indicating involvement of Rho kinase in ML161-resistant signaling. ML161 failed to inhibit PAR1-mediated decreases in transepithelial resistance (TER) in MDCK cells overexpressing Gα12. In contrast, orthosteric inhibitors of PAR1, 3,5-difluoro aminoisoxazole and SCH79797, blocked PAR1-mediated shape change in platelets and PAR1-mediated decrease in TER in MDCK cells. These results confirmed that PAR1 modified by ML161 couples to Gα12/13, but loses coupling to Gαq. Although ML161 did not inhibit activation through human PAR4, whose 8th helix lacks a palmitoylation site and contains amino acids that disrupt helices, it inhibited activation through mouse PAR4, which like human PAR1 possesses a constrained 8th helix with a C-terminal palmitoylation site. Consistent with its ability to inhibit mouse PAR4-mediated activation of mouse platelets, 5 mg/kg ML161 inhibited by 〉90% platelet accumulation during thrombus formation following laser-induced injury of the cremaster arteriole in mice. These results demonstrate that ML161 acts as an allosteric modulator of PAR1 that blocks coupling to Gαq, but not Gα12/13. ML161 demonstrates saturable inhibition as well as selective blockade of Gα subunits coupling and could provide improved control of PAR1 function in the setting of thrombotic disease. Disclosures: No relevant conflicts of interest to declare.