Proteinase-activated receptor 4 (PAR4): activation and inhibition of rat platelet aggregation by PAR4-derived peptides

We studied the actions of receptor-activating peptide analogues (PAR4APs), modeled on the proteolytically-revealed tethered ligand sequence of murine proteinase-activated receptor-4 (PAR4), in a rat platelet aggregation assay. The PAR4APs GYPGKF-NH2 (GY-NH2) and AYPGKF-NH2 (AY-NH2) were able to cause aggregation with EC50 values of about 40 microM and 15 microM, respectively. The reverse human PAR4 sequence (VQGPYG-NH2, YG-NH2) and the PAR1AP SFLLR-NH2, did not cause aggregation. In contrast, trans-cinnamoyl-YPGKF-NH2 (tcY-NH2) did not cause aggregation but blocked aggregation caused by GY-NH2, AY-NH2, and thrombin without affecting ADP-mediated aggregation. We conclude that in contrast to the PAR1AP, the PAR4APs GY-NH2 and AY-NH2 activate rat platelets via a PAR4-related receptor and that peptide analogues modeled on the PAR4 tethered activating sequence can serve as useful agonist and antagonist probes for assessing the consequence of activating PAR4 either by PAR4APs or thrombin in rat tissue preparations.     

Discovery of a nonpeptidic small molecule antagonist of the human platelet thrombin receptor (PAR-1)

The synthesis and biological evaluation of a series of nonpeptidic small molecule antagonists of the human platelet thrombin receptor (PAR-1) are described. Optimization of the 5-amino-3-arylisoxazole lead resulted in an approximate 100-fold increase in potency. The most potent of these compounds (54) inhibits platelet activation with IC(50)s of 90 nM against the thrombin receptor agonist peptide (TRAP) and 510 nM against thrombin as the agonist. Further, antagonist 54 fully blocks platelet aggregation stimulated by 1 nM thrombin for 10 min.     

A comparative SAR study of thrombin receptor derived non peptide mimetics: Importance of phenyl/guanidino proximity for activity

Summary Thrombin, the most potent physiological platelet agonist interacts with cells through a specific G protein-coupled receptor which has been cloned and sequenced. Synthetic thrombin receptor peptides (TRAPS) comprising the first 5 amino acids (SFLLR and SFLLR-NH₂) of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombin's proteolytic activity were found to cause full platelet aggregation. During the screening of novel thrombin receptor derived non-peptide mimetics in the platelet aggregation assay we found that 1-phenylacetyl-4-(6-guanidohexanoyl)-piperazine (1) and 1-(6-guanidohexanoyl)-4-(phenylacetylamidomethyl)-piperidine (2) exertedin vitro antagonist activities (56% and 40% correspondingly) as it is depicted by the platelet aggregation assay. Using Molecular Modeling, the synthetic compounds were overlayed with SFFLR. All three superimposed low energy structures had Phe and Arg aminoacids in spatial close proximity. The superimposition results revealed that1 resembled more the stereoelectronic environment of SFLLR than2. This difference may be related to their different antagonist efficacy.     

Tethered ligand agonist peptides. Structural requirements for thrombin receptor activation reveal mechanism of proteolytic unmasking of agonist function.

The human platelet thrombin receptor is activated when thrombin cleaves its receptor's amino-terminal extension to reveal a new amino terminus that functions as a tethered peptide ligand. Exactly how this "agonist peptide domain" remains cryptic within the uncleaved receptor and becomes functional after receptor cleavage is unknown. In this report we define the structural features of the thrombin receptor's agonist peptide domain important for receptor activation. Studies with mutant thrombin receptors have suggested that agonist peptide domain residues 2-6 contained determinants critical for receptor activation, and the synthetic peptide SFLLR-NH2 representing the 1st 5 amino-terminal residues of the agonist peptide domain was sufficient to specify agonist activity. Acetylating or removing the agonist peptide's amino-terminal ammonium group greatly attenuated agonist activity. Agonist peptide residue Phe2 was vital for agonist function; residues Leu4 and Arg5 individually played less important roles. These structure-function relationships held for both platelet activation and activation of the cloned receptor expressed in transfected mammalian cells. Our studies suggest that structures at the extreme amino terminus of the thrombin receptor's agonist peptide domain, in particular the free ammonium group of Ser1 and the phenyl ring of Phe2, are critical for receptor activation and that the agonist function of this domain is expressed when receptor proteolysis unmasks such determinants. In addition to revealing details of the thrombin receptor's proteolytic triggering mechanism, these studies open avenues to the development of drugs targeting the thrombin receptor and to further definition for the role of the thrombin receptor in cellular regulation.     

PAR4, but not PAR1, signals human platelet aggregation via Ca2+ mobilization and synergistic P2Y12 receptor activation

Regulation of platelet activation plays a central role in hemostasis and pathophysiological processes such as coronary artery disease. Thrombin is the most potent activator of platelets. Human platelets express two thrombin receptors, PAR1 and PAR4, both of which signal platelet activation. Evidence is lacking on the mechanism by which PAR1 and PAR4 may differentially signal platelet aggregation. Here we show that at the relatively high concentration of agonist most likely found at the site of a local thrombus, dual inhibition of the P2Y12 receptor and calcium mobilization result in a complete inhibition of PAR4-induced aggregation, while having no effect on either thrombin or PAR1-mediated platelet aggregation. Both PAR1- and PAR4mediated aggregation are independent of calcium mobilization. Furthermore, we show that P2Y12 receptor activation is not required for protease-activated receptor-mediated aggregation at higher agonist concentrations and is only partially required for Rap1 as well as GPIIbIIIa activation. P2Y12 receptor inhibitors clinically in use such as clopidogrel are postulated to decrease platelet aggregation through partial inhibition of PAR1 signaling. Our data, however, indicate that at high local concentrations of thrombin, it is the signaling through PAR4 rather than PAR1 that may be regulated through purinergic feedback. Thus, our data identify an intra-platelet mechanism that may function as a future site for therapeutic intervention.     

Thrombin-induced events in non-platelet cells are mediated by the unique proteolytic mechanism established for the cloned platelet thrombin receptor

We recently isolated a cDNA clone encoding a functional platelet thrombin receptor that defined a unique mechanism of receptor activation. Thrombin cleaves its receptor''s extracellular amino terminal extension, unmasking a new amino terminus that functions as a tethered peptide ligand and activates the receptor. A novel peptide mimicking this new amino terminus was a full agonist for platelet secretion and aggregation, suggesting that this unusual mechanism accounts for platelet activation by thrombin. Does this mechanism also mediate thrombin''s assorted actions on non-platelet cells? We now report that the novel thrombin receptor agonist peptide reproduces thrombin-induced events (specifically, phosphoinositide hydrolysis and mitogenesis) in CCL-39 hamster lung fibroblasts, a naturally thrombin- responsive cell line. Moreover, these thrombin-induced events could be recapitulated in CV-1 cells, normally poorly responsive to thrombin, after transfection with human platelet thrombin receptor cDNA. Our data show that important thrombin-induced cellular events are mediated by the same unusual mechanism of receptor activation in both platelets and fibroblasts, very likely via the same or very similar receptors.     

Orthovanadate decreases the leptin content in isolated mouse fat pads via proteasome activation

In a physiological milieu platelets continue to be exposed to agonists long after clot formation. We studied the regulation of postaggregation events consequent on protease-activated receptor (PAR) 1 ligation with either thrombin or the thrombin receptor-activating peptide (TRAP). Stimulation with TRAP (20 microM) but not with thrombin (1 U/ml) for 15 min evoked platelet disaggregation by about 30% and downregulation of high-affinity fibrinogen binding sites on integrin alpha(IIb)beta(3) to nearly prestimulation levels. Concurrently, only TRAP disorganized the actin-based cytoskeleton, with decrease in the cytoskeletal content of focal contact-associated proteins like integrin alpha(IIb)beta(3), Src, and focal adhesion kinase (FAK). While protein tyrosine kinases were activated during the initial period of platelet aggregation with either agonist, stimulation of protein tyrosine phosphatases determined the successive phase of reduced phosphotyrosine content. SHP-1, an abundant protein tyrosine phosphatase in the platelets, was tyrosine phosphorylated on challenge of PAR-1 and coprecipitated with two unidentified tyrosine phosphorylated proteins of 140 and 60 kDa; in addition, SHP-1 tyrosine phosphorylation (which is associated with enhanced phosphatase activity) was sustained until 15 min. Activity of calpain was upregulated following incubation with thrombin and not with TRAP. Collectively, these data suggest that signaling pathways elicited by PAR-1 agonists thrombin and TRAP are markedly different, which could have important implications on late platelet responses.     

Binding of Biotinylated Thrombin Receptor Peptide to Cloned Human Thrombin Receptor Overexpressed in Baby Hamster Kidney Cells

Abstract

Baby hamster kidney (BHK) cells transfected with an expression vector for the human thrombin receptor, and then treated with basic fibroblast growth factor, were found to express specific and saturable binding sites for biotinylated thrombin receptor peptide (SFLLRNPNDKYEPF). Analysis of the binding to live BHK cells yielded an equilibrium dissociation constant (Kd) of 3.0±0.3 μmol/l and a maximal binding capacity (Bmax) of 31.0±0.5 nmol/mg of protein. In competitive binding experiments, the thrombin receptor agonist peptide (SFLLRN), which is a strong inducer of human platelet aggregation, was the most potent competitor. In contrast, position 1 to 2 inverted peptides such as FSLLRNPNDKYEPF and FSLLRNP, which fail to induce for the platelet aggregation, were less potent. This simple and convenient binding assay system using the biotinylated thrombin receptor peptide as a labeled ligand and the cloned thrombin receptor overexpressed in BHK cells may be useful for exploring specific antagonists of the receptor.     

Protease-activated receptor-2 (PAR-2): structure-function study of receptor activation by diverse peptides related to tethered-ligand epitopes

Protease-activated receptor-2 (PAR-2) is a tethered-ligand, G-protein-coupled receptor that is activated by proteolytic cleavage or by small peptides derived from its cleaved N-terminal sequence, such as SLIGRL-NH2. To assess specific PAR activity, we developed an immortalized murine PAR-1 (-/-) cell line transfected with either human PAR-2 or PAR-1. A "directed" library of more than 100 PAR agonist peptide analogues was synthesized and evaluated for PAR-2 and PAR-1 activity to establish an in-depth structure-function profile for specific action on PAR-2. The most potent agonist peptides (EC50 = 2-4 microM) had Lys at position 6, Ala at position 4, and pFPhe at position 2; however, these also exhibited potent PAR-1 activity (EC50 = 0.05-0.35 microM). We identified SLIARK-NH2 and SL-Cha-ARL-NH2 as relatively potent, highly selective PAR-2 agonists with EC50 values of 4 microM. Position 1 did not tolerate basic, acidic, or large hydrophobic amino acids. N-Terminal capping by acetyl eliminated PAR-2 activity, although removal of the amino group reduced potency by just 4-fold. At position 2, substitution of Leu by Cha or Phe gave equivalent PAR-2 potency, but this modification also activated PAR-1, whereas Ala, Asp, Lys, or Gln abolished PAR-2 activity; at position 3, Ile and Cha were optimal, although various amino acids were tolerated; at position 4, Ala or Cha increased PAR-2 potency 2-fold, although Cha introduced PAR-1 activity; at position 5, Arg or Lys could be replaced successfully by large hydrophobic amino acids. These results with hexapeptide C-terminal amides that mimic the native PAR-2 ligand indicate structural modes for obtaining optimal PAR-2 activity, which could be useful for the design of PAR-2 antagonists.     

A new role for FcgammaRIIA in the potentiation of human platelet activation induced by weak stimulation

The low affinity receptor for immunoglobulin G, FcgammaRIIA, is expressed in human platelets, mediates heparin-induced thrombocytopenia and participates to platelet activation induced by von Willebrand factor. In this work, we found that stimulation of platelets with agonists acting on G-protein-coupled receptors resulted in the tyrosine phosphorylation of FcgammaRIIA, through a mechanism involving a Src kinase. Treatment of platelets with the blocking monoclonal antibody IV.3 against FcgammaRIIA, but not with control IgG, inhibited platelet aggregation induced by TRAP1, TRAP4, the thromboxane analogue U46619, and low concentrations of thrombin. By contrast, platelet aggregation induced by high doses of thrombin was unaffected by blockade of FcgammaRIIA. We also found that the anti-FcgammaRIIA monoclonal antibody IV.3 inhibited pleckstrin phosphorylation and calcium mobilization induced by low, but not high, concentrations of thrombin. In addition, thrombin- or U46619-induced tyrosine phosphorylation of several substrates typically involved in FcgammaRIIA-mediated signalling, such as Syk and PLCgamma2, was clearly reduced by incubation with anti-FcgammaRIIA antibody IV.3. Upon stimulation with thrombin, FcgammaRIIA relocated in lipid rafts, and thrombin-induced tyrosine phosphorylation of FcgammaRIIA occurred within these membrane domains. Controlled disruption of lipid rafts by depleting membrane cholesterol prevented tyrosine phosphorylation of FcgammaRIIA and impaired platelet aggregation induced by U46619 or by low, but not high, concentrations of thrombin. These results indicate that FcgammaRIIA can be activated in human platelets downstream G-protein-coupled receptors and suggest a novel general mechanism for the reinforcement of platelet activation induced by low concentrations of agonists.     

Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation.

We isolated a cDNA encoding a functional human thrombin receptor by direct expression cloning in Xenopus oocytes. mRNA encoding this receptor was detected in human platelets and vascular endothelial cells. The deduced amino acid sequence revealed a new member of the seven transmembrane domain receptor family with a large amino-terminal extracellular extension containing a remarkable feature. A putative thrombin cleavage site (LDPR/S) resembling the activation cleavage site in the zymogen protein C (LDPR/I) was noted 41 amino acids carboxyl to the receptor's start methionine. A peptide mimicking the new amino terminus created by cleavage at R41 was a potent agonist for both thrombin receptor activation and platelet activation. "Uncleavable" mutant thrombin receptors failed to respond to thrombin but were responsive to the new amino-terminal peptide. These data reveal a novel signaling mechanism in which thrombin cleaves its receptor's amino-terminal extension to create a new receptor amino terminus that functions as a tethered ligand and activates the receptor.     

Design and synthesis of para-fluorophenylalanine amide derivatives as thrombin receptor antagonists.

An antagonist specific for the thrombin receptor is expected to be a remedy for thrombosis. Structure-activity studies of thrombin receptor-tethered ligand SFLLRNP have revealed the importance of the Phe-2-phenyl group in receptor recognition and the replacement of the Phe-2 by para-fluorophenylalanine [(p-F)Phe] was found to enhance its activity [Nose, T. et al. (1993) Biochem. Biophys. Res. Commun. 193, 694-699]. In order to obtain a small sized antagonist, a series of (p-F)Phe derivatives was designed and synthesized novel structural elements essential for receptor interactions being introduced at both the N and C-termini. beta-Mercaptopropionyl (betaMp) or its derivative activated by S-3-nitro-2-pyridinesulphenyl (Npys) was introduced at the N-terminus, and phenylmethyl amines were coupled to the C-terminus. All compounds were inactive when assayed for human platelet aggregation, indicating that they are not agonists. beta-Mercaptopropionyl derivatives were also inactive as antagonists. However, Npys-containing analogs were found to inhibit the agonist activity of SFLLRNP. In particular, SNpys-betaMp-(p-F)Phe-NH-R [R = -CH(C6H5)2 and -CH2-CH-(C6H5)2] potently suppressed platelet aggregation. The results suggested that (p-F)Phe can be used as a structural core to construct an effective antagonist conformation.     

Inhibition of calpain blocks platelet secretion, aggregation, and spreading

Previous studies have indicated that the Ca(2+)-dependent protease, calpain, is activated in platelets within 30-60 s of thrombin stimulation, but specific roles of calpain in platelets remain to be identified. To directly test the functions of calpain during platelet activation, a novel strategy was developed for introducing calpain's specific biological inhibitor, calpastatin, into platelets prior to activation. This method involves treatment of platelets with a fusion peptide, calpastat, consisting of the cell-penetrating signal sequence from Kaposi's fibroblast growth factor connected to a calpain-inhibiting consensus sequence derived from calpastatin. Calpastat specifically inhibits thrombin peptide (SFLLR)-induced alpha-granule secretion (IC(50) = 20 microM) during the first 30 s of activation, thrombin-induced platelet aggregation (IC(50) = 50 microM), and platelet spreading on glass surfaces (IC(50) = 34 microM). Calpastat-Ala, a mutant peptide in which alanine is substituted at conserved calpastatin residues, lacks calpain inhibitory activity and fails to inhibit secretion, aggregation, or spreading. The peptidyl calpain inhibitors calpeptin, MDL 28,170 (MDL) and E64d also inhibit secretion, aggregation and spreading, but require 3-10-fold higher concentrations than calpastat for biological activity. Together, these findings demonstrate that calpain regulates platelet secretion, aggregation, and spreading and indicate that calpain plays an earlier role in platelet activation following thrombin receptor stimulation than had been previously detected.     

蛋白激酶C在血小板聚集中的作用

利用￣（３２）Ｐ－ＮａＨ２ＰＯ４标记猪血小板,以蛋白激酶Ｃ的４０ｋＤ底物为蛋白激活的标志．用血小板激动剂在聚集浓度范围内处理血小板,结果表明,除了不能使猪血小板聚集的肾上腺素外,凝血酶等激动剂都使血小板４０ｋＤ底物蛋白磷酸化明显增加,同时３８ｋＤ,２６ｋＤ蛋白质磷酸化也明显增加,且４０ｋＤ底物磷酸化与血小板聚集有平行增加关系．蛋白激酶Ｃ在血小板聚集中可能起着重要的调节作用。     

Plasmin-mediated activation of platelets occurs by cleavage of protease-activated receptor 4

The activation of plasmin from its circulating precursor plasminogen is the mechanism of several clot-busting drugs used to clinically treat patients who have suffered a stroke; however, plasmin thus generated has been shown to activate platelets directly. There has been speculation as to whether plasmin interacts with the protease-activated receptors (PARs) because of its similarity in amino acid specificity with the classic platelet activator thrombin. We have investigated whether plasmin activates platelets via PAR activation through multiple complementary approaches. At concentrations sufficient to induce human platelet aggregation, plasmin released very little calcium compared with that induced by thrombin, the PAR-1 agonist peptide SFLLRN, or the PAR-4 agonist peptide AYPGKF. Stimulation of platelets with plasmin initially failed to desensitize additional stimulation with SFLLRN or AYPGKF, but a prolonged incubation with plasmin desensitized platelets to further stimulation by thrombin. The desensitization of PAR-1 had no effect on plasmin-induced platelet aggregation and yielded an aggregation profile that was similar to plasmin in response to a low dose of thrombin. However, PAR-4 desensitization completely eliminated aggregation in response to plasmin. Inclusion of the PAR-1-specific antagonist BMS-200261 inhibited platelet aggregation induced by a low dose of thrombin but not by plasmin. Additionally, mouse platelets naturally devoid of PAR-1 showed a full aggregation response to plasmin in comparison to thrombin. Furthermore, human and mouse platelets treated with a PAR-4 antagonist, as well as platelets isolated from PAR-4 homozygous null mice, failed to aggregate in response to plasmin. Finally, a protease-resistant recombinant PAR-4 was refractory to activation by plasmin. We conclude that plasmin induces platelet aggregation primarily through slow cleavage of PAR-4.     

Mechanisms of Arg-Pro-Pro-Gly-Phe inhibition of thrombin

Investigations determined the mechanism(s) by which Arg-Pro-Pro-Gly-Phe (RPPGF) inhibits thrombin-induced platelet activation. High concentrations of RPPGF inhibit thrombin-induced coagulant activity. RPPGF binds to the active site of thrombin by forming a parallel beta-strand with Ser214-Gly216 and interacts with His57, Asp189, and Ser195 of the catalytic triad. RPPGF competitively inhibits alpha-thrombin from hydrolyzing Sar-Pro-Arg-paranitroanilide with a Ki = 1.75 +/- 0.03 mM. Other mechanisms were sought to explain why RPPGF inhibits thrombin activation of platelets at concentrations below that which inhibits its active site. Soluble RPPGF blocks biotinylated NATLDPRSFLLR of the thrombin cleavage site on protease-activated receptor (PAR)1 from binding to the peptide RPPGC (IC50 = 20 microM). The soluble recombinant extracellular domain of PAR1 (rPAR1EC) blocks biotinylated RPPGF binding to rPAR1EC (IC50 = 50 microM) bound to microtiter plates, but rPAR1EC deletion mutants missing the sequence LDPR or PRSF do not. RPPGF and related forms prevent the thrombin-like enzyme thrombocytin from proteolyzing rPAR1EC at concentrations that do not block thrombocytin's active site. These studies indicate that RPPGF is a bifunctional inhibitor of thrombin: it binds to PAR1 to prevent thrombin cleavage at Arg41 and interacts with the active site of alpha-thrombin.     

Thrombin receptor-activating peptides (TRAPs): investigation of bioactive conformations via structure-activity, spectroscopic, and computational studies

The thrombin receptor (PAR-1) is an unusual transmembrane G-protein coupled receptor in that it is activated by serine protease cleavage of its extracellular N-terminus to expose an agonist peptide ligand, which is tethered to the receptor itself. Synthetic peptides containing the agonist motif, such as SFLLRN for human PAR-1, are capable of causing full receptor activation. We have probed the possible bioactive conformations of thrombin receptor-activating peptides (TRAPs) by systematic introduction of certain conformational perturbations, involving alpha-methyl, ester psi(COO), and reduced-amide psi(CH2N) scans, into the minimum-essential agonist sequence (SFLLR) to probe the importance of the backbone conformation and amide NH hydrogen bonding. We performed extensive conformational searches of representative pentapeptides to derive families of putative bioactive structures. In addition, we employed 1H NMR and circular dichroism (CD) to characterize the conformational disposition of certain pentapeptide analogues experimentally. Activation of platelet aggregation by our pentapeptide analogues afforded a structure-function correlation for PAR-1 agonist activity. This correlation was assisted by PAR-1 receptor binding data, which gauged the affinity of peptide ligands for the thrombin receptor independent of a functional cellular response derived from receptor activation (i.e. a pure molecular recognition event). Series of alanine-, proline-, and N-methyl-scan peptides were also evaluated for comparison. Along with the known structural features for PAR-1 agonist peptides, our work adds to the understanding of peptide topography relative to platelet functional activity and PAR-1 binding. The absolute requirement of a positively charged N-terminus for strong agonist activity was contradicted by the N-terminal hydroxyl peptide psi(HO)S-FLLR-NH2. The amide nitrogen between residues 1 and 2 was found to be a determinant of receptor recognition and the carbonyl groups along the backbone may be involved in hydrogen bonding with the receptor. Position 3 (P3) of TRAP-5 is known to tolerate a wide variety of side chains, but we also found that the amide nitrogen at this position can be substituted by an oxygen, as in SF-psi(COO)-LLR-NH2, without diminishing activity. However, this peptide bond is sensitive to conformational changes in that SFPLR-NH2 was active, whereas SF-NMeL-LR-NH2 was not. Additionally, we found that position 3 does not tolerate rigid spacers, such as 3-aminocyclohexane-1-carboxylic acid and 2-aminocycloalkane-1-carboxylic acid, as analogues 1A, 1B, 2A, 2B, 3, 4, 5A and 5B lack agonist activity. On the basis of our results, we suggest that an extended structure of the agonist peptide is principally responsible for receptor recognition (i.e. binding) and that hydrophobic contact may occur between the side chains of the second (Phe) and fourth (Leu) residues (i.e. P2-P4 interaction).     

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation,platelet aggregation,and P-selectin expression

In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.     

Macrocyclic hexapeptide analogues of the thrombin receptor (PAR-1) activation motif SFLLRN

The thrombin receptor (PAR-1) is activated by alpha-thrombin to stimulate various cell types, including platelets, through the tethered-ligand sequence SFLLRN. Macrocyclic peptide analogues of SFLLRN were synthesized and evaluated in vitro. In general, the compounds were much less potent in inducing platelet aggregation relative to SFLLRN-NH2 and did not act as antagonists of alpha-thrombin. Derivative 3c was the most potent macrocycle in activating PAR-1, with an EC50 of 24 microM.     

Differential effect of pH on thromboxane A2/prostaglandin H2 receptor agonist and antagonist binding in human platelets.

The effects of changes in pH on the binding of agonists and antagonists to the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were determined. Competition binding studies were performed with the TXA2/PGH2 mimetic [1S-1 alpha,2 beta (5Z), 3 alpha(1E,3R*),4 alpha)]-7-[3-(3-hydroxy-4'-iodophenoxy)-1-buteny) 7-oxabicyclo-[2.2.1]-heptan-2-yl]-5-heptenoic acid ([125I]BOP). The pH optimum for binding of [125I] BOP to washed human platelets was broad with a range of pH 4-6 in contrast to that of the TXA2/PGH2 receptor antagonist 9,11-dimethyl-methano-11,12-methano-16-(3-iodo-4-hydroxyl)-13-aza-15 alpha,beta-omega-tetranorthromboxane A2 ([125I]PTA-OH) which was 7.4. Scatchard analysis of [125I]BOP binding in washed platelets at pH 7.4, 6.0, and 5.0 revealed an increase in affinity (Kd = 1.16 +/- 0.06, 0.64 +/- 0.09, and 0.48 +/- 0.05 nM, respectively) and an increase in the number of receptors (Bmax = 2807 +/- 415, 5397 +/- 636, and 7265 +/- 753 sites/platelet, respectively). The potency of I-BOP to induce shape change in washed platelets at pH 6.0 was also significantly increased from an EC50 value of 0.34 +/- 0.016 nM at pH 7.4 to 0.174 +/- 0.014 nM at pH 6.0 (n = 6, p less than 0.05). In contrast, the EC50 value for thrombin was unaffected by the change in pH. In competition binding studies with [125I]BOP, the affinity of the agonists U46619 and ONO11113 were increased at pH 6.0 compared to 7.4. In contrast, the affinity of the TXA2/PGH2 receptor antagonists I-PTA-OH, SQ29548, and L657925 were either decreased or unchanged at pH 6.0 compared to 7.4. Diethyl pyrocarbonate and N-bromosuccinimide, reagents used to modify histidine residues, reversed the increase in affinity of [125I]BOP at pH 6.0 to values equivalent to those at pH 7.4. In solubilized platelet membranes, the effects of NBS were blocked by coincubation with the TXA2/PGH2 mimetic U46619. The results suggest that agonist and antagonist binding characteristics are different for the TXA2/PGH2 receptor and that histidine residue(s) may play an important role in the binding of TXA2/PGH2 ligands to the receptor.