Antiaggregatory Properties Research Articles

An Assembly Route to Inorganic Catalytic Nanoreactors Containing Sub‐10‐nm Gold Nanoparticles with Anti‐Aggregation Properties

Yolk–shell catalytic nanoreactors with sub-10-nm Au nanoparticles and a mesoporous ZrO2 or TiO2 shell are prepared from monodisperse hydrophobic Au nanoparticles through an assembly approach (see image). The nanoreactors demonstrate both a remarkable catalytic activity and anti-aggregation properties upon thermal treatment and recycling.

Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti-integrin antibody.

beta(3)-Integrins are involved in platelet aggregation via alpha(IIb)beta(3) [glycoprotein (GP)IIb-GPIIIa], and in angiogenesis via endothelial alpha(V)beta(3). Cross-reactive ligands with antiaggregatory and proangiogenic effects, both desirable in peripheral vasculopathies, have not yet been described. In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments, with emphasis on beta(3)-integrins. Recombinant Fab fragments were obtained by phage display technology. Specificity, affinity and IC(50) were determined by immunodot assays, enzyme-linked immunosorbent assay (ELISA), and Scatchard plot analysis, and by means of human umbilical vein endothelial cells (HUVECs). Functional analyses included ELISA for interaction with fibrinogen binding to GPIIb-GPIIIa, flow cytometry for measurement of activation parameters and competitive inhibition experiments, human platelet aggregometry, and proliferation, tube formation and the chorioallantoic membrane (CAM) assay for measurement of angiogenic effects. We observed specific and high-affinity binding to an intact GPIIb-GPIIIa receptor complex of two human Fab autoantibody fragments, with no platelet activation. Dose-dependent fibrinogen binding to GPIIb-GPIIIa and platelet aggregation were completely inhibited. One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody (mAb) 7E3, whereas the other Fab fragment bound to cultured HUVECs, suggesting cross-reactivity with alpha(V)beta(3), and also demonstrated proangiogenic effects in tube formation and CAM assays. These Fab fragments are the first entirely human anti-GPIIb-GPIIIa Fab fragments with full antiaggregatory properties; furthermore, they do not activate platelets. The unique dual-specificity anti-beta(3)-integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies.

The effect of beam-plasma modification of fibrin monomer on its biological properties

A change in the antiaggregation activity of fibrin monomer by its modification in helium and water vapor electron-beam plasmas was experimentally studied. It was found that fibrin-monomer modification products reduced the aggregation of platelets by a factor of 1.5 on average and that the acquiring of the antiaggregation properties was accompanied by the degradation of the native protein. It was demonstrated that the degradation process is controllable and products with a desired molecule mass can be obtained via the appropriate choice of the beam-plasma treatment parameters.

P2-130: Phenylthiazolyl-hydrazides: A lead for tau aggregation inhibitors and tau imaging?

The aggregation of tau protein into paired helical filaments is a key pathological feature of Alzheimer's disease. Several screening campaigns reported the “usual suspects” but few drug-like lead structures. Therefore, the HTS data on tau aggregation inhibition by small molecules created and reported by E. Mandelkow et al. were employed for an in silico approach. A ligand-based approach to tau protein aggregation into paired helical filaments resulted in successful scaffold hopping and the identification of phenythiazolyhydrazides (PTHs) as potent lead structure. Here we report the comparison of PTHs to selected curcumin derivatives and approved tetracyclines for their tau antiaggregatory properties and their affinity to fibrillar beta-amyloid in several assays and cellular models. The most potent fluorescent compounds were evaluated for beta-amyloid/tau PHF selectivity by fluorescence microscopy of human AD probes. Lead Optimization of PTHs. Several diverse PTH analogues were obtained by variation of the substituents R1-R4 to explore a preliminary SAR. Fluorescent substituents were incorporated without significantly compromising in vitro activity. Tau-biased ligands with enhanced affinity to tau paired helical filaments over beta-amyloid were identified by a panel of protein aggregation assays and fluorescence microscopy of human AD preparations. Phenylthiazolylhydrazides are potent inhibitors of tau-aggregation. The fluorescence microscopy of human AD preparation suggests a binding to growth-relevant sites of tau aggregates.

Biochemical isolation of an insect haemocyte anti-aggregation protein from the venom of the endoparasitic wasp, Pimpla hypochondriaca, and identification of its gene

Pimpla hypochondriaca venom is complex and contains a number of different proteins and polypeptides that exert a variety of effects on insect physiology. In particular, it possesses factors with potent anti-haemocyte and immunosuppressive properties. In the current work, we describe the biochemical isolation of a single venom factor with insect haemocyte anti-aggregation properties. The protein was isolated using gel filtration and ion exchange chromatography, in conjunction with a qualitative in vitro haemocyte anti-aggregation assay to monitor activity and confirm identity. The protein has a molecular weight estimate of 33 kDa (determined by SDS PAGE under reducing conditions), and an N-terminal sequence of Asp-Ser-Asp-Ile-Tyr-Leu-Leu. The biochemically isolated protein has been demonstrated to inhibit haemocyte aggregation and to suppress encapsulation responses, using in vitro and in vivo assays, respectively. Furthermore, its gene has been identified as vpr3. The work is presented within the context of the role of P. hypochondriaca venom and the isolated protein in host immune suppression.

Antiaggregant effects of Arbutus unedo extracts in human platelets

Platelet hyperaggregability plays a pivotal role in the pathogenesis of cardiovascular diseases. Thrombin evokes aggregation through Ca 2+ mobilization, tyrosine phosphorylation and generation of reactive oxygen species (ROS). We have investigated the antiaggregant properties of Arbutus unedo extracts in human platelets. Changes in cytosolic Ca 2+ concentration and intracellular oxidants production were registered by espectrofluorimetry using fura-2 and dichlorodihydrofluorescein, respectively, platelet aggregation was assessed by aggregometry and protein tyrosine phosphorylation was detected by Western blotting. Platelet treatment with increasing concentrations (0.015–1.5 mg/mL) of crude aqueous, ethyl acetate or diethyl ether extracts reduced platelet aggregation evoked by thrombin (0.5 U/mL) and show a potent ROS scavenger activity, preventing thrombin-evoked endogenous generation of ROS. Treatment with Arbutus unedo extracts did not alter thrombin-evoked Ca 2+ release from the intracellular stores but reduced store-operated Ca 2+ entry induced by thrombin or by selective depletion of the two Ca 2+ stores in platelets, the dense tubular system and the acidic stores. In addition, platelet treatment with extracts reduced both basal and thrombin-stimulated protein tyrosine phosphorylation. We conclude that Arbutus unedo extracts show antiaggregant actions due to attenuation of Ca 2+ mobilization, ROS production and protein tyrosine phosphorylation and might be used for the treatment and/or prevention of cardiovascular diseases.

Characterization of the Intracellular Mechanisms Involved in the Antiaggregant Properties of Cinnamtannin B-1 from Bay Wood in Human Platelets

Cinnamtannin B-1, a natural A-type proanthocyanidin recently identified as a radical scavenger component of Laurus nobilis L., exerts antiaggregant and antiapoptotic effects in human platelets. Here, we have investigated the intracellular mechanisms involved in the antiaggregant effects of cinnamtannin B-1. Cinnamtannin B-1 showed a greater free radical scavenging activity than vitamin C, vitamin E, or Trolox, among other antioxidants and reduced thrombin-evoked tubulin reorganization and platelet aggregation. Thrombin-evoked activation of Btk and pp60(src) was also inhibited by cinnamtannin B-1. In conclusion, we show that cinnamtannin B-1 is a powerful oxygen radical scavenger that reduces thrombin-evoked microtubular remodeling and activation of the tyrosine kinases Btk and pp60(src), which leads to inhibition of platelet aggregation. These observations suggest that cinnamtannin B-1 may prevent thrombotic complications associated to platelet hyperaggregability and hyperactivity, although further studies are necessary to establish appropriate therapeutic strategies.

4‐(Isoindolin‐5‐yl) amino‐4‐oxobutyryl‐β‐alkoxyphenyl‐β‐alanines, New RGDF Mimetics. Synthesis, Affinity to Fibrinogen Receptors, Antiaggregatory Properties, and Their Correlation with the Hydrophobicity.

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Thiosulfinates modulate platelet activation by reaction with surface free sulfhydryls and internal thiol-containing proteins

Thiosulfinates are characteristic flavors of Allium vegetables, with a highly reactive S–S=O group, that we previously showed to inhibit platelet aggregation through calpain-dependent mechanisms. With the aim to clarify the mode of action of these redox phytochemicals, we studied their effect on extracellular free sulfhydryls in relation to their effect on platelet responses (Ca2+ signals, release reaction, and aIIbβ3 integrin activation state). At the platelet surface, thiosulfinate dose-dependently increased the basal level of free sulfhydryls, independently of protein disulfide isomerase activity. This generation of new free sulfhydryls was associated with: (i) a three fold increase in labeling of resting platelets with an anti ligand-induced binding site antibody and (ii) marked inhibition of subsequent aIIbβ3 activation by agonists. Thiosulfinates increased the basal intracellular Ca2+ level of platelets. In activated platelets, they markedly inhibited the Ca2+ mobilization independently of the external Ca2+, the calpain-induced SNAP-23 cleavage and the granule release. In platelet free systems, thiosulfinates inhibited the activity of purified calpain and the free sulfhydryl of glutathione without any reducing properties on disulfides. The results demonstrate for the first time that thiosulfinates rapidly interact with sulfhydryls both at the platelet surface and inside the cell on intracellular cysteine-proteins, especially calpain. Inhibition of free cysteine and glutathione in whole blood may also contribute to their anti-aggregant properties. Such sulfur compounds are of interest for the development of a new class of antithrombotic agents.

Platelet Aggregability in Patients with Hypertension Treated with Angiotensin II Type 1 Receptor Blockers

Cardiovascular events associated with hypertension often involve thrombosis. Increased platelet activity is one of the risk factors of cardiovascular diseases. Antithrombotic properties of antihypertensive agents are not fully characterized. Angiotensin II type 1 receptor blockers (ARBs) are widely used for the treatment of hypertension. Some ARBs can provoke antiaggregatory effects on platelets in vitro. Whether ARBs can inhibit platelet aggregation was tested in hypertensive patients in vivo. Platelet aggregation was assessed by the highly sensitive particle counting method using laser-light scattering. Large platelet aggregation induced by adenosine diphosphate (ADP, 3 microM) was 2.6+/-0.4 (x10(7)) (SE) in hypertensive patients treated with losartan (72+/-3 years old, n=10) while it was 3.9+/-0.6 in hypertensive patients treated with candesartan (70+/-5 years old, n=6; p=0.056). Large platelet aggregation induced by thromboxane A2 receptor agonist, U46619 (10 microM), was 2.8+/-0.5 (x10(7)) in hypertensive patients treated with losartan while it was 5.1+/-0.9 in hypertensive patients treated with candesartan (p=0.033). Clinical characteristics including the control of blood pressure did not differ between the two groups (losartan 136+/-5/73+/-3 mmHg vs. candesartan 135+/-4/76+/-5). Thus, losartan may have the possibility to inhibit platelet activation in patients with hypertension independent of blood pressure reduction. Antiaggregatory properties may be independent of angiotensin II type 1 receptor or of antihypertensive actions. The favorable effects of losartan on reduction of adverse cardiovascular events among hypertensive patients may be at least partly mediated by inhibition of platelet activation.

4-(Isoindolin-5-yl)amino-4-oxobutyryl-β-alkoxyphenyl-β-alanines, new RGDF mimetics. Synthesis, affinity to fibrinogen receptors, antiaggregatory properties, and their correlation with the hydrophobicity

A new series of RGDF mimetics, derivatives of 4-(isoindolin-5-yl)amino-4-oxobutanoic acid, was synthesized. The compounds obtained inhibit efficiently the thrombocytes aggregation in experiments in vitro; their biological targets are fibrinogen receptors. The antiaggregatory activity and the affinity correlate with the hydrophobicity of the compounds.

Aged Garlic Extract and Its Constituents Inhibit Platelet Aggregation through Multiple Mechanisms1–3

Aged garlic extract (AGE) is a complex mixture. Its constituents include allin, cycloalliin, S-allyl-L-cysteine, S-methyl-L-cysteine, S-ethylcysteine, S-1-proponyl-L-cysteine, S-allylmercapto-L-cysteine, fructosyl-arginine, and beta-chlorogenin. It also contains L-arginine, L-cysteine, and L-methionine. AGE reduces the parameters associated with cardiovascular disease, including the inhibition of platelet aggregation. However, the underlying mechanisms have yet to be established and are the subject of this study. AGE inhibited platelet aggregation in a concentration-dependent manner, and this achieved significance between concentrations of 1.56-25% (v:v). The constituents of AGE, when tested as a mixture, displayed a biphasic pattern of inhibition, although this was not significant. L-Methionine displayed anti-aggregatory properties at concentrations of 0.00078, 0.00625, and 0.1 mmol/L, whereas L-arginine and L-cysteine were effective at 9 mmol/L. However, when the constituents and the amino acids were tested together, significant inhibition of platelet aggregation was observed only at a concentration of 1 mmol/L. AGE also displayed disaggregatory properties at concentrations of 12.5 and 25% (v:v). The constituents and the amino acids, when tested as a mixture, displayed disaggregatory properties at concentrations of 0.25 and 1 mmol/L. In contrast, a diethyl-ether extract of AGE had no effect on platelet aggregation. When platelets were stimulated with either A23187 or ADP, an increase in intraplatelet Ca2+ accompanied platelet aggregation. This increase in Ca2+ was abolished in the presence of AGE. It is likely that AGE works in a synergistic manner and exerts multiple effects on the biochemical pathways involved in platelet aggregation.

Mini-αB-Crystallin: A Functional Element of αB-Crystallin with Chaperone-like Activity

Alpha-crystallin is a member of the family of small heat-shock proteins (sHSP) and is composed of two subunits, alphaA-crystallin and alphaB-crystallin, which exhibit molecular chaperone-like properties. In a previous study, we found that residues 70-88 in alphaA-crystallin can function like a molecular chaperone by preventing the aggregation and precipitation of denaturing substrate proteins [Sharma, K. K., et al. (2000) J. Biol. Chem. 275, 3767-3771]. In this study, we show that the complementary sequence in alphaB-crystallin, residues 73-92 (DRFSVNLDVKHFSPEELKVK), is the functional chaperone site of alphaB-crystallin. Like the mini-alphaA-crystallin chaperone, the mini-alphaB-crystallin chaperone interacts with 1,1'-bi(4-anilino) naphthalene-5,5'-disulphonic acid (bis-ANS) and also possesses significant beta-sheet and random coil structure. Deletion of four residues (DRFS) from the N-terminus or deletion of C-terminus LKVK residues from the 73-92 peptide abolishes the chaperone-like activity against denaturing alcohol dehydrogenase. However, removal of DRFS or HFSPEELKVK is necessary to completely abolish the antiaggregation property of the peptide in insulin reduction assay. Substitution of Asp at a site corresponding to D80 in alphaB-crystallin with d-Asp or beta-Asp results in a significant loss of chaperone-like activity. Kynurenine modification of His in the peptide abolishes the antiaggregation property of the mini-chaperone. These data suggest that the 73-92 region in alphaB-crystallin is one of the substrate binding sites during chaperone activity.

The Oxidation State of DJ-1 Regulates its Chaperone Activity Toward α-Synuclein

DJ-1 has been reported to have chaperone activity by preventing the aggregation of some proteins, and by structural analogy to Hsp31. The L166P mutation has been linked to a familial early onset form of Parkinson's disease (PD). Since the aggregation of alpha-synuclein is believed to be a critical step in the etiology of PD, we have investigated the interaction of wild-type DJ-1 and its oxidized forms with alpha-synuclein. Native (unoxidized) DJ-1 did not inhibit alpha-synuclein fibrillation, and no evidence for stable interactions between alpha-synuclein and native DJ-1 was observed. However, DJ-1 is very susceptible to oxidation by the addition of two oxygen atoms to form the sulfinic acid of Cys106 (2O DJ-1) (no 1O oxidized state is detectable). 2O DJ-1 was readily prepared by the addition of H(2)O(2) at concentrations up to a 20-fold molar excess. The oxidation of Cys106 to the sulfinic acid had minimal effect on the structural properties of DJ-1. However, 2O DJ-1 was very effective in preventing the fibrillation of alpha-synuclein, and only this form of DJ-1 appears to have significant anti-aggregation properties against alpha-synuclein. Further oxidation of DJ-1 leads to loss of some secondary structure, and to loss of the ability to inhibit alpha-synuclein fibrillation. Our observations confirm the suggestion that DJ-1 may act as an oxidative-stress-induced chaperone to prevent alpha-synuclein fibrillation. Since oxidative stress has been associated with PD, this observation may explain why mutations of DJ-1 could be a contributing factor in PD, and also indicates that excess oxidative stress could also lead to enhanced alpha-synuclein aggregation and hence PD.

Synthesis and pharmacological activity of 2-methoxyphenyl-and 2-oxyphenyl-substituted 1-dialkylaminoalkylimidazo[1,2-a]-benzimidazoles

A series of 2-methoxy-and 2-oxyphenyl-1-dialkylaminoalkylimidazo[1,2-a]benzimidazoles have been synthesized and characterized with respect to pharmacological properties. Some of the synthesized compounds exhibit antioxidant, membranotropic, antiaggregant, hemorheological, anticalmodulin, and hypotensive properties, and behave as antagonists of serotonin (5-HT2,3) and purine (P2Y1) receptors. The obtained data show good prospects for the synthesis and screening of biologically active substances in this class of compounds.

Synthesis and Pharmacological Activity of 2-Methoxyphenyl-Substituted 9-Dialkylaminoethylimidazo[1,2-a]benzimidazoles

A series of 2-methoxyphenyl-9-dialkylaminoethyl derivatives of imidazo[1,2-a]benzimidazole have been synthesized and their pharmacological properties have been studied. Some of the synthesized compounds exhibit antioxidant, radioprotector, antiarrythmic, spasmolytic, antiaggregant, anticalmodulin, and antisecretory properties. Some substances exhibit the properties of phosphodiesterase inhibitors, decrease calcium ion transport through membranes, increase myocardium resistance to hypoxia, and reduce the arterial pressure. The obtained data show good prospects for the synthesis and screening of biologically active substances in this chemical group.

Not all statins interfere with clopidogrel during antiplatelet therapy

Clopidogrel and statins are frequently coadministered in patients with ischemic heart diseases. Recent reports suggested that clopidogrel's effectiveness in inhibiting adenosine diphosphate (ADP)-induced platelets aggregation is attenuated by co-administration of certain statins. The objective of the present study was to define which statin might interfere with the antiaggregation property of clopidogrel. We designed a pharmacokinetic study and tested ex vivo platelet function on 21 healthy volunteers who received clopidogrel and all currently commercially available statins: rosuvastatin [10 mg o.d.], simvastatin [20 mg o.d.], fluvastatin [80 mg o.d.], pravastatin [40 mg o.d.], and atorvastatin [20 mg o.d.]. Each statin was administered for 7 days followed by 1 week of wash-out period with clopidogrel treatment alone. Detection of the statins in the plasma was performed on all blood samples, using HPLC analytical method. All individuals, except one, were responders to clopidogrel with inhibition of ex vivo ADP induced platelet aggregation. All statins, except pravastatin, were detectable in the plasma at the end of each treatment period in all patients, and no statin was detectable after any of the wash-out periods. Clopidogrel was significantly less efficient to prevent platelet aggregation when coadministrated with simvastatin or fluvastatin. No difference was observed in clopidogrel efficacy when coadministered with rosuvastatin, pravastatin or atorvastatin. This is the first study investigating clopidogrel-statin interactions on ex vivo platelet function with all commercially available statins and which were administered to the same individuals. It demonstrates in healthy volunteers that at the doses used in this study, simvastatin and fluvastatin, but not atorvastatin, pravastatin or rosuvastatin interfere with the anti-aggregation effect of clopidogrel.

(252) Correlation of Pungency, Thiosulfinates, Antiplatelet Activity, and Total Soluble Solids in Two Garlic Families

Allium plants possess organosulfur compounds and carbohydrates that provide unique flavor and health-enhancing properties. In previous studies of onion F3 families, significant phenotypic and genetic correlations have been reported between pungency, in vitro antiplatelet activity (IVAA), and soluble solids content (SSC); although in other studies SSC and pungency have not always been correlated. In this study we analyzed SSC, pungency, garlic-induced in vitro antiplatelet activity and the content of three predominant thiosulfinates in bulbs from two garlic families obtained from unrelated self-pollinated plants. A strong positive correlation was observed between pungency and IVAA for both sample sets, indicating that it will be difficult to develop garlic populations with low pungency and high IVAA. Allicin was the most abundant thiosulfinate and its content was positively correlated with pungency and IVAA (r= 0.70 and 0.74, respectively). The thiosulfinates AllS(O)SPropenyl and AllS(O)SMe were also positively correlated with pungency and IVAA. When compared with IVAA, AllS(O)SMe had higher r values than AllS(O)SPropenyl (0.88 and 0.50, respectively). These differences could reflect differential platelet anti-aggregatory properties of different thiosulfinates. SSC was not correlated with IVAA, pungency, or thiosulfinates content, suggesting that soluble solids in garlic can be independently selected.

Synthesis and antiplatelet activity of thioaryloxyacids analogues of clofibric acid

The thiophene-, benzothiazole- and pyridine-thioaryloxyacids analogues of clofibric acid were synthesized and their antiplatelet activity was screened. Some compounds exhibited antiaggregating properties. The platelet-related haemostasis was measured on a PFA-100® analyzer using bull blood.

Molecular mechanism of the inhibition of cytochrome c aggregation by Phe-Gly

Experimental and computational studies suggest that few general principles govern protein/protein interactions and aggregation. The knowledge of these rules may be exploited to design peptides that are able to interfere with the self-assembly and aggregation of proteins. This work is aimed to verify the validity of this hypothesis by investigating the interaction of cytochrome c with Phe and Gly amino acids, Ala-His (carnosine), and two water-soluble dipeptides Phe-Gly and Gly-Phe. The combined use of 1H NMR, MD, and DSC has shown that: (i) at neutral pH, only Phe-Gly is able to prevent the thermally induced aggregation of cytochrome c; (ii) Phe-Gly interacts with Gly45 and Phe46 residues of the protein, either when the protein is in the folded or in the unfolded state; and (iii) the interaction of Phe-Gly with cytochrome c is sequence-dependent. These results support the hypothesis that the basic principles that describe protein aggregation can be used for the design of peptides with antiaggregating properties.