Platelet Aggregation Inhibitory Activity Research Articles

Three-Dimensional quantitative structure–Activity relationship analyses of RGD mimetics as fibrinogen receptor antagonists

In order to better understand the structural requirements of fibrinogen receptor antagonists, variations in the platelet aggregation inhibitory activity of a series of RGD mimetics were examined using techniques for the analysis of three-dimensional quantitative structure–activity relationship, such as CoMFA.

A comparative study of the function of phospholipases a2 fromagkistrodon acutus

Abstract: cDNAs encoding acidic phospholipases A2I and acid A2II, basic phospholipase A2 and Lys49- phospholipase A2 from Agkistrodon acutus were inserted into bacterial expression vectors and effectively expressed in E.coli RRl. Except for Lys49-phospholipase A2, the other phospholipase A2s have high enzymatic activities. Acidic phospholipas_eA2I has an inhibiting effect on platelet aggregation. All phospholipase A2s except acidic phospholipase A2II have hemolytic activities. The roles of various amino acid residues in the enzymatic, hemolytic and platelet aggregation inhibiting activities of phospholipase A2 are discussed.

Phospholipase A2 with Platelet Aggregation Inhibitor Activity from Austrelaps superbus Venom: Protein Purification and cDNA Cloning

Four phospholipase A2 (PLA2) enzymes (Superbins a, b, c, and d) with varying platelet aggregation inhibitor activities have been purified from Austrelaps superbus by a combination of gel filtration, ion-exchange, and reversed-phase high-pressure liquid chromatography. Purity and homogeneity of the superbins have been confirmed by high-performance capillary zone electrophoresis and mass spectrometry. The electron spray ionization mass spectrometry data showed that their molecular masses range from 13,140 to 13,236 Da. Each of the proteins has been found to be basic and exhibit varying degrees of PLA2 activity. They also displayed different platelet aggregation inhibitory activities. Superbin a was found to possess the most potent inhibitory activity with an IC50 of 9.0 nM, whereas Superbin d was found to be least effective with an IC50 of 3.0 μM. Superbins b and c were moderately effective with IC50 values of 0.05 and 0.5 μM, respectively. The amino-terminal sequencing confirmed the identity of these superbins. cDNA cloning resulted in the identification of 17 more PLA2 isoforms in A. superbus venom. It has also provided complete information on the precursor PLA2. The precursor PLA2 contained a 27-amino-acid signal peptide and 117- to 125-amino-acid PLA2 (molecular mass ranging from 13,000 to 14,000 Da). Two of these PLA2 enzymes resembled more closely (87%) Superbin a in structure. Two unique PLA2 enzymes containing an extra pancreatic loop also have been identified among the isoforms.

Platelet aggregation inhibitory activity of mutant proinsulin with C-peptide replaced by CRGDSC sequence

A CRGDSC peptide was introduced into an inactive human proinsulin molecule between the B30 site and A1 site to replace the C-peptide. The constructed RGD-proinsulin gene was overexpressed in E. coli and the protein purified. It showed an inhibitory activity of platelet aggregation with an IC50 value of 0.35 μM, while native insulin and proinsulin as controls did not exhibit any inhibitory activity. Meanwhile, the RGD-proinsulin demonstrated only 0.05% of insulin receptor binding activity and almost no insulin activity in in vivo assay.

Novel arylpyrazino[2,3-c][1,2,6]thiadiazine 2,2-dioxides as platelet aggregation inhibitors. 2. Optimization by quantitative structure-activity relationships.

In the previous paper (Part 1), we described the synthesis and antiplatelet activity of a series of phenyl- and heteroarylpyrazino[2,3-c][1,2,6]thiadiazine 2,2-dioxides. In this paper, we report the optimization of the platelet aggregation inhibitory activity by an iterative sequence of quantitative structure-activity relationship studies which encompassed synthesis and evaluation of the effects of structure variations at the 1-, 6-, and 7-positions of the heterocyclic system. A model has been established that correctly correlates antiplatelet activity in this series with the partial atomic charges calculated by a local density functional ab initio method. As a result of this study, the experimental platelet aggregation inhibitory activity of the lead compound was improved 300-fold.

Synthesis and In-vitro Evaluation of Platelet Aggregation Inhibitory Activity of Paeonol and its Analogues

Paeonol (1-(2-hydroxy-4-methoxyphenyl)ethanone) and a series of substituted 1-(2-hydroxy-phenyl)ethanone derivatives were synthesized and screened as inhibitors of platelet aggregation. The compounds with the greatest anti-platelet potential among the series tested were 1-(2,5-dihydroxyphenyl)ethanone (65-36% inhibition at 300 μM against 5 μM ADP), paeonol (36.31%), 1-(2-hydroxy-5-methoxyphenyl)ethanone (24.47%), 1-(2-hydroxy-5-nitro-phenyl) ethanone (30.40%) and 1-(5-chloro-2-hydroxy-4-methylphenyl)ethanone (24.43%).

RGD-containing trypsin with both platelet aggregation inhibitory activity and proteolytic activity.

Arg-Gly-Asp (RGD) motif mediates cell adhesion as a major determinant in interactions of disintegrins with cell surface receptors. In order to obtain a mutant trypsin with both high affinity to integrins and retained proteolytic activity, RGDS and RGD were inserted into a rigid turn region and a flexible loop of trypsin, respectively. Wild type trypsin and substituted mutant trypsins, 37RGDS and 77RGD, were expressed in E. coli and purified. Kinetic properties, autolytic stability as well as platelet aggregation inhibitory activity of both 37RGDS and 77RGD were determined. 37RGDS and 77RGD give retained proteolytic activities of 34% and 87%, respectively, and both become less stable to autolysis. 37RGDS shows an obvious inhibition rate of 29% for platelet aggregation and 77RGD gives a rather weak rate of 14% at the same protein concentration of 3.5 microM, while the wild type trypsin shows no inhibitory activity.

Isolation, Sequence Analysis, and Biological Activity of Atrolysin E/D, the Non-RGD Disintegrin Domain fromCrotalus atroxVenom

Crotalid snake venom metalloproteinases often have associated with them nonproteinase domains that may be processed from the mature proteinases. Nascent atrolysin E, from the western diamondback rattlesnake,Crotalus atrox,has a metalloproteinasedomain and a non-RGD disintegrin domain that is lacking in the mature metalloproteinase. In this studywe report on the isolation, sequence analysis, andbiological activity of the 7.4-kDa atrolysin E disintegrin domain (atrolysin E/D). Atrolysin E/D represents approximately 0.2% of the total protein fromthe crude venom. The protein begins with a glycinyl residue found in the latter part of the spacer region.The sequence of atrolysin E/D is identical to thatof the non-RGD disintegrin domain of atrolysin E.The structure is termed a non-RGD disintegrin sincein lieu of the characteristic RGD sequence, a Met-Val-Asp (MVD) is found instead. Nevertheless, the protein is a potent inhibitor of both collagen- and ADP-stimulated platelet aggregation with IC50values of 4 and 8 nM, respectively. A cyclized synthetic peptide, Ac-CRVSMVDRNDDTC-NH2, which represents the sequence of the atrolysin E/D non-RGD loop, was demonstrated to be an effective inhibitor of platelet aggregation. Therefore, this region of atrolysin E/D's structure, as in the disintegrins proper, is important for the biological activity of the protein. Thus, like the non-RGD disintegrin barbourin fromSistrurus miliarius barbouri,a RGD sequence in the context of the disintegrin protein backbone is not an absolute requirement for platelet aggregation inhibitory activity. These data underscore the biochemical and functional complexity of crotalid snake venoms due to differential proteolytic processing of the precursor metalloproteinases and exemplify how the processed fragments may contribute to the observed pathological effects of the venom.

GPIIb/IIIa integrin antagonists with the new conformational restriction unit, trisubstituted beta-amino acid derivatives, and a substituted benzamidine structure.

Ethyl N-[3-(2-fluoro-4-(thiazolidin-3-yl(imino)methyl)benzoyl)amino-2, 2-dimethylpentanoyl]piperidine-4-acetate 40 (NSL-96184) is a highly potent and orally active fibrinogen receptor antagonist, which is characterized by the presence of the trisubstituted beta-amino acid residue, 3-ethyl-2,2-dimethyl-beta-alanine. This compound was developed on the basis of the SAR study of N-[3-(N-4-amidinobenzoyl)amino-2, 2-dimethyl-3-phenylpropionyl]piperidine-4-acetic acid 1(NSL-95301) with the derivatization focused on the central trisubstituted beta-amino acid unit as well as the basic amidinobenzoyl unit, and the esterification of the carboxyl group for prodrug composition. Compound 1, which was reported in our previous study, was discovered by the application of combinatorial chemistry. The molecular modeling study suggests that the trisubstituted beta-amino acid unit is responsible for fixing the molecule to its active conformation. Compound 40 showed an excellent profile in the in vitro and in vivo studies for its human platelet aggregation inhibitory activity and oral availability in guinea pigs. This oral availability largely depends on the modification of the amidino group with a cyclic secondary amine, i.e., thiazolidine in 40. In in vivo studies, the onset of the antiplatelet action of 40 is very fast after oral administration, whereas its duration of action is relatively short. These results suggest that 40 has an excellent therapeutic potential, especially for antithrombotic treatment in the acute phase. 3-Substituted-2,2-dimethyl-beta-amino acid residues would serve as new and useful linear templates to restrict the conformational flexibility of peptidomimetics.

A Constrained Diketopiperazine as a New Scaffold for the Synthesis of Peptidomimetics

As a new scaffold for peptidomimetic synthesis, a highly constrained bifunctional diketopiperazine, 4, has been prepared by smooth N-alkylation with tert-butyl bromoacetate. As a first application, we describe herein the synthesis of new peptidomimetics of the Arg-Gly-Asp (RGD) sequence. The product 30, which shows a selective platelet-aggregation inhibiting activity, can be used as a lead for the preparation of more potent products.

Asymmetric synthesis of the sulfoxide metabolite of ON-579 by the kagan protocol

Abstract A synthesis of both enantiomers of bio-active metabolite of ON-579; 4-[2-(4-chlorophenyl-sulfonylamino)-ethylsulfinyl]-2,6-difluorophnoxyacetic acid was accomplished by the asymmetric oxidation of ON-579 methyl ester followed by hydrolysis. Difference in TXA 2 receptor antagonizing activity was noted for the enantiomers in an U46619-induced rabbit platelet aggregation inhibitory activity.

Sequence and Biological Activity of Catrocollastatin-C: A Disintegrin-Like/Cysteine-Rich Two-Domain Protein fromCrotalus atroxVenom

In this study we report on the isolation and biological characterization of a 23.6-kDa protein from the venom of the western diamondback rattlesnake,Crotalus atrox.Primary structural analysis shows the protein to be composed of a spacer/disintegrin-like domain and a cysteine-rich domain. The sequence is identical to the same carboxy-terminal domains found in theC. atroxmetalloproteinase, catrocollastatin, and hence we termed the protein catrocollastatin-C. We estimate that catrocollastatin-C represents at least 0.5% of the total protein inC. atroxvenom. The protein is an inhibitor of collagen-stimulated but not ADP-stimulated platelet aggregation. Reduction and alkylation of catrocollastatin-C causes a loss of platelet aggregation inhibitor activity. A synthetic, cyclic peptide designed from the catrocollastatin-C disintegrin-like domain has potent platelet aggregation inhibitory activity. This suggests that the corresponding region in the disintegrin-like domain of the protein is at least partially responsible for the inhibition of platelet aggregation previously reported for the protein. These studies underscore the biochemical and functional complexity of crotalid snake venoms due to differential proteolytic processing of precursor proteins and how the processed precursor fragments may contribute to the observed pathological effects of the venom.

Lipid Peroxidation and Preeclampsia (Part 1)

The influences of lipid peroxidation on the impairment of placental microcirculation in preeclampsia were studied.Lipid peroxides and vitamin E concentrations in the blood and the placental brush border membrane (PBBM) of the women with preeclampsia were measured by the TBA and HPLC methods, and the platelet aggregation inhibitory activity and ADP degrading activity of PBBM were analyzed using Born's Method and 14C-ADP degrading assay. Lipid peroxides concentrations both in the blood and in the PBBM were significantly increased (p<0.001, p<0.01 respectively), but plasma vitamin E concentrations were significantly reduced (p<0.01) in the women with preeclampsia. Significant reduction of platelet aggregation inhibitory activity (p<0.005) and ADP degrading activity (p<0.001) of PBBM were observed in preeclampsia. The influences of artificial peroxidation of PBBM by tertiary-butylhydroperoxide (t-BHP) on the platelet aggregation inhibitory activity and ADP degrading activity of PBBM were investigated in vitro assay using normal PBBM. Significant reduction of platelet aggregation inhibitory activity (p<0.005) and ADP degrading activity (p<0.001) of PBBM were observed in this artificially peroxidated PBBM. The effects of vitamin E on the artificial peroxidation of t-BHP were also investigated in the same in vitro assay. Vitamin E prevented the artificial peroxidation of PBBM, and at the same time the platelet aggregation inhibitory activity and ADP degrading activity of PBBM were reduced depending on the concentration (p<0.005, p<0.001). Furthermore, high concentrated vitamin E (500 μg/ml) demonstrates platelet aggregation inhibitory activity by itself, and low concentrated vitamin E (20μg/ml) promotes the potential platelet aggregation inhibitory activity of PBBM.These results suggests that lipid metabolism in the women with preeclampsia are under severe oxidative stress, and that in preeclampsia, the lipid peroxidation of PBBM plays an important role on the impaired placental microcirculation by reducing the platelet aggregation inhibitory activity, and vitamin E preserves the platelet aggregation inhibitory activity by preventing the lipid peroxidation of PBBM or by another mechanism.

Chiral resolution and absolute configuration of the enantiomers of 5-acetyl-2-methyl-4-methylsulfinyl-6-phenyl-3(2H)-pyridazinone and evaluation of their platelet aggregation inhibitory activity.

In a series of 5-acyl-6-phenyl-2,4-substituted-3(2H)-pyridaziones the derivative 1a, with a sulfur stereogenic center, had the most potent activity as human platelet aggregation inhibitor. The resolution of rac-1a was successfully performed by chiral chromatography on Chiralcel OD-R, OD-H, and Chiralpak AD columns and scaled up to a preparative level. The absolute configuration of (-)-(S)-1a was determined by X-ray crystallographic analysis. In vitro human platelet aggregation inhibitory activity was evaluated. Both the enantiomers showed IC50 values in the same micromolar range, but the (-)-(S) isomer was slightly more potent [(S)/(R) potency ratio was 4/1].

Synthesis biological evaluation of alkyl, alkoxy, alkylthio, or amino-substituted 2,3-dihydro-1,5-benzothiazepin-4(5H)-ones.

2,3-Dihydro-1,5-benzothiazepin-4(5H)-ones substituted with an alkyl, alkoxy, alkylthio, hydroxy, or amino group on the fused benzene ring of the 1,5-benzothiazepine skeleton were synthesized and their vasodilating, antihypertensive, and platelet aggregation-inhibitory activities were investigated. (-)-cis-3-Acetoxy-5-[2-(di-methylamino) ethyl]-2,3-dihydro-8-methyl-2-(4-methylphenyl)-1,5-benzothiazepin- 4(5H)-one ((-)-13e) was selected for further studies as a potent inhibitor of platelet aggregation.

Design and modeling of new platelet-activating factor antagonists. 2. Synthesis and biological activity of 1,4-bis-(3′,4′,5′-trimethoxybenzoyl)-2-alkyl and 2-alkyloxymethylpiperazines

In the continuation of our investigations on the structure of platelet-activating factor (PAF)-receptor, 25 additional 2-substituted 1,4-bis-(poly- and mono methoxybenzoyl)-piperazines were synthesized and their in vitro biological activities measured. Substituent at position 2 is representative of the classical balance lipophilicity/hydrophilicity, i.e. alkyl, phenylalkyl, alkoxy and polyalkoxy groups. A potent PAF-induced platelet aggregation inhibitory activity measured in PRP medium is obtained with 5c, IC 50 = 6 × 10 −8 M, which displaces the [ 3H]PAF from platelet membrane with an EC 50 = 6 × 10 −8 M, and compound 4 presents an EC 50 of 3 × 10 −8 M. Examination of structure-activity relationships shows that molecules bearing a hydrophilic or slightly hydrophobic appendix in position-2 are still potent; their IC 50 being included between 10 −6 and 10 −7 M. After quantitative analysis, it seems that in PRP medium, the role of serum albumin must be taken into account instead of a pure hydrophobic interaction of the appendix Z into the receptor. The role of the methoxy groups in producing a potent antagonistic activity is demonstrated by syntheses of several 2-octylpiperazine analogs. These specific features will be quatitatively analysed in the following related publication (part 3).

Development of the new potent non-peptide gpIIb/IIIa antagonist NSL-95301 by utilizing combinatorial technique

The synthetic study of new non-peptide gpIIb/IIIa antagonist, NSL-95301, is presented. The combinatorial technique was engaged in the lead compound discovery process and optimization process to find (+)-NSL-95301. The IC 50 value of collagen induced platelet aggregation inhibitory activity is 92 nM.

Pyridazines. XIII. Synthesis of 6-aryl-5-oxygenated substituted-3(2H)-pyridazinones and evaluation as platelet aggregation inhibitors.

Several 6-aryl-5-oxygenated substituted pyridazinones have been synthesized and evaluated in vitro for inhibition of platelet aggregation induced by adenosine 5'-diphosphate (ADP), thrombin and collagen. All the tested compounds (except 8 and 9) inhibited platelet aggregation in a dose-dependent manner. The IC50 of the most active substance, compound 2b, was around 60 microM against ADP and collagen as inducers. The inhibition of platelet aggregation caused by test compounds was dependent on the level of oxidation of the function at the 5-position, with the order of IC50 values being R-OH (2a, b, 5) < R-CHO (6, 7) < < R-COOH (8, 9). None of the tested compounds increased the intracellular levels of cAMP, indicating a lack of inhibitory activity on cAMP phosphodiesterase (PDE III) in intact cells. These results suggest that the group present at the 5 position of 6-aryl-5-substituted pyridazinones determines the platelet aggregation-inhibitory activity, and that a mechanism other than PDE inhibition is responsible for this effect.

Importance of the structure of the RGD-containing loop in the disintegrins echistatin and eristostatin for recognition of αIIbβ3 and αvβ3 integrins

Echistatin and eristostatin are structurally homologous disintegrins which exhibit significant functional differences in interaction with various integrins. We hypothesized that this may reflect differences in the sequences of their RGD loops: 20CKRARGDDMDDYC 32 and 23CRVARGDWNDDYC 35, respectively. Mapping of eristostatin peptides obtained by proteolytic digestion suggested that it has the same alignment of SS bridges as echistatin. Synthetic echistatin D27W resembled eristostatin since it had increased platelet aggregation inhibitory activity, increased potency to block fibrinogen binding to αIIbβ3, and decreased potency to block vitronectin binding to αvβ3 as compared to wild-type echistatin. Since eristostatin and echistatin have a similar pattern of disulfide bridges, we constructed molecular models of eristostatin based on echistatin NMR coordinates. The RGD loops of eristostatin and echistatin D27W were wider than echistatin's due to the placement of tryptophan (rather than aspartic acid) immediately after the RGD sequence. We propose a hypothesis that the width and shape of the RGD loop are important ligand structural features that affect fitting of ligand to the binding pocket of αIIbβ3 and αvβ3.

Design and synthesis of new antagonist peptides for platelet GPIIb/IIIa receptor as anti-thrombotic agents

The structure-activity relationships of N-terminal modified RGD peptides against platelet aggregation inhibitory activity were studied, and Orotyl-Ser-Arg-Gly-Asp-Trp (NSL-9403) was found to be a new and effective anti-thrombotic agent during extracorporeal circulation.