New indole and pyridazinoindole analogs--synthesis and study as inhibitors of phosphodiesterases and as inhibitors of blood platelet aggregation

This paper presents the synthesis of new indole, pyridazino[4,5-b]-indole, and pyridazino[4,5-a]indole analogs as well as a study of their "in vitro" activity as inhibitors of different phosphodiesterases isolated from dog cardiac tissue, dog aorta, and bovine platelets; the study of their activity as inhibitors of platelet aggregation in guinea pig whole blood, with ADP and arachidonic acid (AA) as pro-aggregants, is also included. The selected compounds 8-benzyloxy-3,4-dihydro-1-(3,4,5-trimethoxy)benzylideneaminopyridazin o[4,5- b]indole 14g, and 8-benzyloxy-4-[(3,4-dimethyl)pyrazolyl]pyridazino[4,5-b]indo le 20 present an interesting profile as potential inodilators, with a complementary, beneficial activity as inhibitors of the aggregation, activities which could possibly be related to the inhibition of the PDE's. Among the other compounds studied, 8-benzyloxy-3,4-dihydro-1-[4-(methyl)piperazino]acetamidopyrida zino[4,5- b]indol-4-one 16c and 8-benzyloxy-3,4-dihydro-1-[4-(2- methoxyphenyl)piperazino]acetamidopyridazino[4,5-b]indol-4-o ne 16f stood out as inhibitors of platelet aggregation, with a mechanism that could possibly be related to the AA cascade.     

Thromboxane A2 synthase inhibition and thromboxane A2 receptor blockade by 2-[(4-cyanophenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15) in rat platelets

The effects of 2-[(4-acetylphenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15), a synthetic 1,4-naphthoquinone derivative, on platelet activity and its mechanism of action were investigated. NQ-Y15 caused a concentration-dependent inhibition of the aggregation induced by thrombin, collagen, arachidonic acid (AA), and A23187. The IC50 values of NQ-Y15 on thrombin (0.1 U/mL)-, collagen (10 microg/mL)-, AA (50 microM)-, and A23187 (2 microM)-induced aggregation were 36.2 +/- 1.5, 6.7 +/- 0.7, 35.4 +/- 1.7, and 93.1 +/- 1.4 microM, respectively. NQ-Y15 also inhibited thrombin-, collagen-, AA-, and A23187-stimulated serotonin secretion in a concentration-dependent manner. However, a high concentration (100 microM) of NQ-Y15 showed no significant inhibitory effect on ADP-induced primary aggregation, which is independent of thromboxane A2 (TXA2) production in rat platelets. In fura-2-loaded platelets, the elevation of intracellular free calcium concentration stimulated by AA, thrombin, and 4-bromo-A23187 was inhibited by NQ-Y15 in a concentration-dependent manner. The formation of TXA2 caused by AA, thrombin, and collagen was inhibited significantly by NQ-Y15. NQ-Y15 inhibited TXA2 synthase in intact rat platelets, since this agent reduced the conversion of prostaglandin (PG) H2 to TXA2. Similarly, NQ-Y15 selectively inhibited the TXA2 synthase activity in human platelet microsomes, whereas it had no effect on activity of phospholipase A2, cyclooxygenase, and PGI2 synthase in vitro. NQ-Y15 inhibited platelet aggregation induced by the endoperoxide analogue U46619 in human platelets, indicating TXA2 receptor antagonism, possibly of a competitive nature. These results suggest that the antiplatelet effect of NQ-Y15 is due to a combination of TXA2 synthase inhibition with TXA2 receptor blockade, and that it may be useful as an antithrombotic agent.     

Novel non-peptide fibrinogen receptor antagonists. 1. Synthesis and glycoprotein IIb-IIIa antagonistic activities of 1,3,4-trisubstituted 2-oxopiperazine derivatives incorporating side-chain functions of the RGDF peptide

Based on the lead tetrapeptide RGDF, two possible non-peptide glycoprotein (GP) IIb-IIIa antagonists possessing an (S)-2-oxopiperazine-3-acetic acid moiety as a scaffold incorporating the indispensable Asp fragment were prepared, and (S)-4-[[trans-[4-(guanidinomethyl)-cyclohexyl]carbonyl]glycyl]-2- oxopiperazine-1,3-diacetic acid, 1a, was identified as a potential lead. A series of 3-substituted 2-oxopiperazine-1-acetic acids bearing the Arg-Gly equivalent at the 4-position were prepared and evaluated for their ability to prevent platelet aggregation and for their binding affinity for the GP IIb-IIIa receptor purified from human HEL cells. (S)-4-[(4-Amidinobenzoyl)glycyl]-3-[(methoxycarbonyl)methyl]- 2-oxopiperazine-1-acetic acid, 9 (TAK-029), inhibited in vitro human platelet aggregation with an IC50 value of 0.03 microM and GP IIb-IIIa-fibrinogen binding with an IC50 value of 0.49 nM. The [4-(2-aminoethyl)benzoyl]glycyl derivative 26 showed activity comparable to that of 9 (IC50 = 0.093 microM, guinea pig platelet aggregation assay). Compound 9 dose-dependently inhibited ex vivo platelet aggregation in guinea pigs (0.03 and 0.1 mg/kg, i.v.), and long-lasting inhibition of platelet aggregation was observed upon oral administration of 9 (3 mg/kg) to guinea pigs. On the other hand, the activity of 26 disappeared within 1 h after a dose of 1 mg/kg (i.v.). Compound 9 may therefore be useful in the clinical treatment of arterial thrombotic diseases.     

Modulatory effect of 8-iso-PGE2 on platelets

1. 8-Iso-PGE2 induced either reversible or irreversible aggregation of platelets in human platelet-rich plasma (PRP) or in the suspension of washed platelets (WP). The values of EC50 for irreversible aggregation in PRP and WP were 4 and 2 microM, respectively. 2. In rabbit PRP, 8-iso-PGE2 (0.1-100 microM) itself did not induce or induced only reversible aggregation. 3. 8-Iso-PGE2 (0.1-20 microM) potentiated adenosine diphosphate-(ADP) induced platelet aggregation in both human and rabbit. The same effect also was found for adrenaline-induced platelet aggregation in rabbit. 4. The lower concentrations (0.2-0.5 microM) of 8-iso-PGE2 decreased, and higher concentrations (1-2 microM) increased platelet aggregating factor- (PAF) induced aggregation in human PRP. In rabbit PRP, 8-iso-PGE2 (0.02-200 microM) had only a decreasing effect on PAF-induced aggregation. 5. The results suggest that low concentrations of 8-iso-PGE2 can amplify or weaken platelet aggregation induced by various aggregatory agents.     

Analogues of 8-[[6-(diethylamino) hexyl]amino]-6-methoxy-4-methylquinoline as candidate antileishmanial agents

8-[[6-(Diethylamino)hexyl]amino]-6-methoxy-4-methylquinoline (I) has been shown to be highly effective against Leishmania donovani in hamsters, being approximately 475 times as active as the standard meglumine antimoniate. Several nuclear and side-chain analogues of I have been prepared in an attempt to further enhance the antileishmanial activity of this class of compounds. The compounds were tested against L. donovani in the golden hamster. Although several analogues had meglumine antimoniate indexes in excess of 300, none was superior to the model compound.     

Cyclopiazonic acid and thapsigargin induce platelet aggregation resulting from Ca2+ influx through Ca2+ store-activated Ca2+-channels

The effects of cyclopiazonic acid and thapsigargin, selective inhibitors of the endoplasmic reticulum Ca2+-ATPase pump, on the platelet aggregation were investigated using washed rat platelets prepared by chromatography on Sepharose 2B columns. In Ca2+-free medium, cyclopiazonic acid and thapsigargin did not induce aggregation, but in the presence of 1 mM Ca2+, platelet aggregation was induced in a concentration-dependent manner. Cyclopiazonic acid- and thapsigargin-induced platelet aggregation was blocked by 1 mM Ni2+ but not by 100 microM indomethacin or 1 microM nifedipine. In aequorin-loaded platelets, cyclopiazonic acid and thapsigargin caused sustained elevation of the cytosolic Ca2+ concentration, an effect which was blocked by Ni2+, a non-selective Ca2+ channel blocker and SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenyl]-1H-imidazole hydrochloride), a putative receptor-operated Ca2+ channel antagonist. The above results indicated that both cyclopiazonic acid and thapsigargin induced platelet aggregation and elevation of cytosolic Ca2+ concentration, that extracellular Ca2+ was essential for cyclopiazonic acid- and thapsigargin-induced platelet aggregation, and that platelet aggregation may be associated with Ca2+ influx through Ca2+ store-activated Ca2+ channels.     

Identification of domains responsible for von Willebrand factor type VI collagen interaction mediating platelet adhesion under high flow

We have identified type VI collagen (Col VI) as a primary subendothelial extracellular matrix component responsible for von Willebrand factor (vWF)-dependent platelet adhesion and aggregation under high tensile strength. Intact tetrameric Col VI was the form of the collagen found to be capable of promoting vWF-mediated platelet adhesion/aggregation under this shear condition, whereas removal of the predominant portion of the terminal globules by pepsin treatment abrogated its activity. The inability of the pepsin-digested Col VI to support any platelet interaction at high flow was because of the failure of the A3(vWF) domain to bind to this form of collagen, suggesting a stringent requirement of a tridimensional conformation or of intactness of its macromolecular structure. In contrast, the A1(vWF) domain bound to both intact and pepsin-digested Col VI tetramers but, in accordance with the cooperating function of the two vWF domains, failed to support platelet adhesion/aggregation under high shear onto Col VI by itself. The putative A1(vWF) binding site resided within the A7(VI) module (residues 413-613) of the globular amino-terminal portion of the alpha3(VI) chain. Soluble recombinant A7(VI) polypeptide strongly perturbed the vWF-mediated platelet adhesion to Col VI under high shear rates, without affecting the binding of the vWF platelet receptor glycoprotein Ibalpha to its cognate ligand A1(vWF). The findings provide evidence for a concerted action of the A1(vWF) and A3(vWF) domains in inducing platelet arrest on Col VI. This is accomplished via an interaction of the A1(vWF) domain with a site contained in the alpha3 chain A7(VI) domain and via a conformation-dependent interaction of the A3(vWF) domain with the intact tetrameric collagen. The data further emphasize that Col VI microfilaments linking the subendothelial basement membrane to the interstitial collagenous network may play a pivotal role in the hemostatic process triggered upon damage of the blood vessel wall.     

Regional brain-derived neurotrophic factor mRNA and protein levels following transient forebrain ischemia in the rat

A new series of 2-arylmethyl-1,4-benzoquinones (2) was synthesized for evaluation of their pharmacological activities. These compounds showed significant inhibition of platelet aggregation induced by arachidonic acid (AA) and some of them possessed a protective effect against endothelial cell injury caused by hydrogen peroxide.     

Inhibition of platelet activation by the Alzheimer's disease amyloid precursor protein

The amyloid precursor protein (APP) of Alzheimer's disease is abundantly expressed in the platelet alpha-granule where its role remains unclear. This study describes a novel function for APP in regulating human platelet activation. Preincubation of platelet-rich plasma with recombinant secreted APP (sAPP) isoforms dose-dependently inhibited platelet aggregation and secretion induced by ADP or adrenaline. Similarly, sAPP potently inhibited low-dose thrombin-induced activation in washed platelet suspensions, indicating that the activity does not require plasma cofactors. There were no functional differences between sAPP forms with or without the Kunitz protease inhibitor domain or derived from either alpha- or beta-secretase cleavage. In fact, the N-terminal cysteine-rich region of APP (residues 18-194) was as effective as the entire sAPP region in the inhibition of platelet activation. The inhibitory activity of sAPP correlated with a significant reduction in the agonist-induced production of the arachidonic acid (AA) metabolites thromboxane B2 and prostaglandin E2. However, sAPP did not affect AA-induced platelet aggregation or secretion, indicating the enzymatic conversion of AA was not inhibited. The addition of a threshold dose of AA reversed the sAPP-inhibition of agonist-induced platelet activation. This suggests that sAPP decreases the availability of free AA, although the mechanism is not yet known. These data provide evidence that the release of sAPP upon platelet degranulation may result in negative feedback regulation during platelet activation.     

In vitro platelet function in controlled ovarian hyperstimulation cycles

OBJECTIVE: To determine the effects of elevated endogenous E2 levels on in vitro platelet function in patients undergoing controlled ovarian hyperstimulation (COH). DESIGN: Women with normal ovulatory cycles and patients undergoing COH on cycle day 3 and near ovulation (preovulatory follicles were at least 16 mm in diameter) were studied. Serum E2, Thrombostat 4000, (V. d. Goltz, Seeon, Germany), von Willebrand factor antigen (vWF-Ag), and platelet aggregation and adenosine triphosphate (ATP) release to adenosine diphosphate (ADP), collagen (COL), and arachidonic acid (AA) were measured. SETTING: University-based outpatient infertility clinic. PATIENT(S): Twenty-two consenting infertile women undergoing COH cycles and 14 women with documented ovulatory cycles. MAIN OUTCOME MEASURE(S): Whole blood platelet aggregation with ADP, COL, AA, and Thrombostat 4000. RESULTS(S): Estradiol levels rose significantly at peak times (P = 0.011). No changes were noted in in vitro platelet function measured by the Thrombostat 4000 and by whole blood platelet aggregation with ADP and AA and in ATP release with ADP, COL, or AA. Aggregation with collagen was increased because of likely elevations in vWF-Ag levels. CONCLUSION(S): No significant changes in in vitro platelet function were noted in 19 women undergoing COH with E2 levels two to three times that observed in oral contraceptive or hormone replacement therapy users, suggesting no increased risk for arterial thromboembolism.     

Nuclear localization signal-independent and importin/karyopherin-independent nuclear import of beta-catenin

Increased platelet aggregation has been suggested to play a role in the accelerated atherosclerosis of diabetics. However the physiological relevance of the aggregation tests has been questioned. The purpose of this study was to determine platelet activation in diabetic patients, using a novel device--the cone and plate(let) analyzer--to measure shear-induced platelet adhesion and aggregation on extracellular matrix (ECM). Whole blood platelet adhesion and aggregation in patients with noninsulin-dependent diabetes mellitus (n=82) and in nondiabetic controls (n=71) were compared. Clinical and laboratory characteristics of the diabetic patients were analyzed for possible correlation with parameters of platelet activity. Patients with diabetes had a significantly increased platelet activation compared to nondiabetic subjects, demonstrated by an increased adhesion to the ECM (surface coverage, 23% [95% confidence interval, 22-25%] vs. 19% [95% confidence interval, 18-20%], respectively) and an increased average size of the ECM-bound aggregates (54 microm2 [95% confidence interval, 51-57 microm2] vs. 47 microm2 [95% confidence interval, 43-51 microm2], respectively). Platelet adhesion in the diabetic group was found to correlate with triglyceride levels (r=0.36) and hematocrit values (r=0.31) and inversely with high-density lipoprotein cholesterol levels (r=0.30). There were no correlation, however, between parameters of platelet reactivity and duration of diabetes, vascular complications and low-density lipoprotein levels. Our data demonstrate an increased platelet adhesion and aggregation in diabetic patients and suggest a modulatory role of diabetic dyslipidemia.     

Platelet activation by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate is mediated by its binding to a putative ADP receptor, aggregin

Platelet responses induced by ADP are mediated by a unique P21-purinergic receptor. Although a variety of ADP analogs, substituted at C2, have been used to delineate pharmacological properties of the ADP-binding site(s), the identity of the receptor protein has not been firmly established. 2-(4-Bromo-2,3-dioxobutylthio)- ADP [2-BrCH2(CO)2CH2-S-ADP], a well-characterized ADP analog, has been previously used as an affinity label to examine the structure/function relationship of ADP-requiring enzymes [Kapetanovic, E., Bailey, J.B. & Colman, R.F. (1985) Biochemistry 24, 7586-7593]. We found that it induced platelet shape change, aggregation, exposure of fibrinogen binding sites, secretion and mobilization of intracellular calcium, but was less potent than ADP. Under non-stirring conditions, incubation of platelets with this analog for longer time periods blocked ADP-induced shape change, aggregation, and the ability to ADP to antagonize the rise in intracellular levels of cAMP induced by iloprost (a prostaglandin I2 analog). Of a variety of agonists examined, only ADP-induced aggregation was almost completely inhibited in platelets irreversibly modified by the analog. An autoradiogram of the gel obtained by SDS/PAGE of solubilized platelets modified by the ADP analog followed by reduction of the dioxo group by NaB[3H], showed the presence of a single radiolabeled protein band at 100 kDa. Platelets incubated first with either ADP, ATP, or 2-methylthio-ADP were not labeled by 2-BrCH2(CO)2CH2S-ADP and NaB[3H]4-8-BrCH2(CO)2CH2-S-ADP was previously shown by us to irreversibly antagonize ADP-induced platelet responses by selectively modifying aggregin. Incubation of platelets with 2-BrCH2(CO)2CH2S-ADP completely blocked labeling of aggregin in platelets by 8-BrCH2(CO)2CH2S-[32P]ADP. These results show that 2-BrCH2(CO)2CH2S-ADP initially interacts reversibly with aggregin (100kDa), a putative ADP receptor, and induces platelet shape change and aggregation, and at longer periods of incubation reacts irreversibly to block the ability of ADP to antagonize stimulated adenylate cyclase activity. In contrast, 6-BrCH2(CO)2CH2S-ADP was found to be a weak and reversible inhibitor of ADP-induced platelet aggregation. Prior incubation of platelets with the latter analog reduced labeling of aggregin by 8-BrCH2(CO)2CH2S-[32P]ADP. Taken together, the results further show that substitution by the BrCH2(CO)2CH2 group at the C2 and C8 positions is tolerated, while the presence of a free amino function at the C6 position is essential for its interaction with aggregin.     

New chiral inhibitors of induced platelet aggregation: the enantiomeric specificity of (R)- and (S)-methyl and ethyl esters of 1-(4-[(1-hydroxycarbonyl)-ethoxy]-benzyl)-1H-1,2,3-triazole as a tool for determining their biological target

The synthesis of title enantiomers was accomplished and their biological behaviour as inhibitors of rabbit platelet aggregation process induced by ADP and arachidonic acid was determined. Structure-activity comparison with that of SM-12502 [(2R,5S)-(+) 3,5-dimethyl-2-(3-pyridyl)-thiazolidin-4-one hydrochloride] and Dazoxiben [4-[2-(1H-imidazol-1-yl)-ethoxy]-benzoic acid] allowed us to formulate the possible capability for the synthesized compounds to interact with the biological targets of the model molecules.     

Role of chondroitin 4-sulphate as a receptor for polycation induced human platelet aggregation

1. Proteoglycans provide negatively charged sites on the surface of platelets, leukocytes and endothelial cells. Since chondroitin 4-sulphate is the main proteoglycan present on the platelet surface, the role of this molecule in mediating the activation of human platelets by polylysine was studied. 2. Platelets were desensitized with phorbol 12-myristate 13-acetate (PMA, 10 nM) 5 min before the addition of polylysine to platelet-rich plasma (PRP). Changes in the intracellular Ca2+ concentration were measured in fura2-am (2 microM) loaded platelets and protein phosphorylation was assessed by autoradiography of the electrophoretic profile obtained from [32P]-phosphate labelled platelets. The release of dense granule contents was measured in [14C]-5-hydroxytryptamine loaded platelets and the synthesis of thromboxane (TXA2) was assessed by radioimmunoassay. Surface chondroitin 4-sulphate proteoglycan was degraded by incubating platelets with different concentrations of chondroitinase AC (3 min, 37 degrees C). The amount of chondroitin 4-sulphate remaining in the platelets was then quantified after proteolysis and agarose gel electrophoresis. 3. The addition of PMA to PRP before polylysine inhibited the aggregation by 88 +/- 18% (n = 3). Staurosporine (1 microM, 5 min) prevented the PMA-induced inhibition. Chondroitinase AC (4 pu ml-1 to 400 muu ml-1, 3 min) abolished the polylysine-induced aggregation in PRP but caused only a discrete inhibition of ADP-induced aggregation. The concentration of chrondroitin 4-sulphate in PRP (0.96 +/- 0.2 microgram/10(8) platelets, n = 3) and in washed platelets (WP; 0.35 +/- 0.1 microgram/10(8) platelets, n = 3) was significantly reduced following incubation with chondroitinase AC (PRP = 0.63 +/- 0.1 microgram/10(8) platelets and WP = 0.08 +/- 0.06 microgram/10(8) platelets). 4. Washed platelets had a significantly lower concentration of chondroitin 4-sulphate than platelets in PRP. The addition of polylysine to WP induced a rapid increase in light transmission which was not accompanied by TXA2 synthesis or the release of dense granule contents. This effect was not inhibited by sodium nitroprusside (SNP), iloprost, EDTA or the peptide RGDS. This event was accompanied by the discrete phosphorylation of plekstrin and myosin light chain, which were inhibited by staurosporine (10 microM, 10 min). The hydrolysis of platelet surface chondroitin 4-sulphate strongly reduced the polylysine-induced phosphorylation. 5. Our results indicate that polylysine activates platelets through a specific receptor which could be the proteoglycan chondroitin 4-sulphate present on the platelet membrane.     

Characterization of P2x1 purinoreceptors on rat platelets: effect of clopidogrel

This study aimed to determine the binding characteristics of [3H]alpha,beta-Me-ATP, a specific ligand of the P2x1 receptors to rat platelets, and to investigate the effect of clopidogrel, a thienopyridine compound which has been found to selectively inhibit ADP-induced platelet aggregation and adenylyl cyclase ex vivo. Binding of [3H]alpha,beta-Me-ATP to rat platelets was time-dependent and saturable. Scatchard analysis of the saturation binding data indicated that [3H]alpha,beta-Me-ATP bound to one population of specific binding sites with high affinity (KD = 23.6 +/- 1.6 nM; Bmax = 690 +/- 24 fmole/10[8]cells) (n=3). Unlabelled alpha,beta-Me-ATP as well as 2-MeS-ADP and ADP competitively inhibited the specific binding of [3H]alpha,beta-Me-ATP with IC50 values of 19.0 +/- 6.6, 103 +/- 20 and 1120 +/- 80 nM respectively (n=3). Other nucleotide analogues such as ATP, ATP-gammaS, UTP and GTP also antagonized [3H]alpha,beta-Me-ATP binding. When administered orally (10mg/kg, p.o.), clopidogrel inhibited ADP- or 2-MeS-ADP-induced platelet aggregation but did not affect the binding of [3H]alpha,beta-Me-ATP to rat platelets ex vivo. In vitro, alpha,beta-Me-ATP did not induce the aggregation or shape change of rat platelets and did not interfere with ADP-induced platelet aggregation.     

Heterogenous inhibition of platelet aggregation by monoclonal antibodies binding to multiple sites on GPIIIa

Six monoclonal IgG1-k antibodies (LK2, LK3r, LK4-55, LK5, LK6-55, LK7r) were raised against platelet membrane GPIIIa in order to study the structure-function relationship of this molecule. Antibodies were selected on their ability to react with GPIIIa by ELISA on adherent platelets, by immunoblot on platelet lysates and by fluorescence flow cytometry on intact platelets. Fluorescence reactivity varied from 3- to 202-fold greater than isotype control fluorescence. Two MoAbs reacted on immunoblot under reduced conditions (LK7r and LK3r). Two reacted with a 55 kD chymotrypsin/subtilisin digest of GPIIIa which is likely to exclude amino acids 121-348 (LK4-55 and LK6-55). Four of the MoAbs (LK5, LK3r, LK2 and LK4-55) inhibited tyrosine phosphorylation of one to four distinct bands on immunoblot. LK4-55 reacted with an N-terminal 66 amino acid fusion protein of GPIIIa near the PLA epitope (Leu 33). LK7r reacted with a 212-222 peptide reported to be an RGD fibrinogen binding site. LK2 reacted near a disintegrin-RGD binding site. Except for LK5, all inhibited ADP, collagen and thrombin-induced platelet aggregation in a heterogeneous fashion. Percentage inhibition of 125I-fibrinogen binding to platelets varied from 18% to 98%. No correlation was noted between inhibition of fibrinogen binding, location of MoAb binding on GPIIIa, reactivity of MoAb binding with GPIIIa, inhibition of thrombin-induced tyrosine phosphorylation or inhibition of platelet aggregation induced by ADP, collagen or thrombin. Thus MoAbs, binding to platelet GPIIIa at different sites, inhibit platelet aggregation in a heterogeneous manner.     

Structural determinants for binding to CGRP receptors expressed by human SK-N-MC and Col 29 cells: studies with chimeric and other peptides

Structure-activity relationships for the binding of human alpha-calcitonin gene-related peptide 8-37 (halphaCGRP8-37) have been investigated at the CGRP receptors expressed by human SK-N-MC (neuroblastoma) and Col 29 (colonic epithelia) cells by radioligand binding assays and functional assays (halphaCGRP stimulation of adenylate cyclase). On SK-N-MC cells the potency order was halphaCGRP8-37 > halphaCGRP19-37 = AC187 > rat amylin8-37 > halpha[Tyr0]-CGRP28-37 (apparent pKBs of 7.49+/-0.25, 5.89+/-0.20, 6.18+/-0.19, 5.85+/-0.19 and 5.25+/-0.07). The SK-N-MC receptor appeared CGRP1-like. On Col 29 cells, only halphaCGRP8-37 of the above compounds was able to antagonize the actions of halphaCGRP (apparent pKB=6.48+/-0.28). Its receptor appeared CGRP2-like. halpha[Ala11,18]-CGRP8-37, where the amphipathic nature of the N-terminal alpha-helix has been reduced, bound to SK-N-MC cells a 100 fold less strongly than halphaCGRP8-37. On SK-N-MC cells, halphaCGRP8-18,28-37 (M433) and mastoparan-halphaCGRP28-37 (M432) had apparent pKBs of 6.64+/-0.16 and 6.42+/-0.26, suggesting that residues 19-27 play a minor role in binding. The physico-chemical properties of residues 8-18 may be more important than any specific side-chain interactions. M433 was almost as potent as halphaCGRP8-37 on Col 29 cells (apparent pKB=6.17+/-0.20). Other antagonists were inactive.     

A potent 5-hydroxytryptamine receptor (5-HT2A) antagonist, DV-7028, delays arterial thrombosis development in rats

In our study, we demonstrated that DV-7028: (3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-6, 7,8,9-tetrahydro-2H-pyrido [1,2,-a]-1,3,5-triazine-2, 4(3H)-dione maleate)--a selective 5-HT2A receptor antagonist, inhibited thrombus formation in the arterial thrombosis model and was completely ineffective in the prevention of venous thrombosis in the rat. In washed platelets prelabelled with 3H-serotonin, DV-7028 inhibited, in a dose-dependent manner, the collagen-induced secretion of serotonin. However, the uptake of serotonin into platelets was not affected by this substance. Administration of DV-7028 also inhibited platelet aggregation in the whole blood and platelet-rich plasma (PRP) induced by collagen, and diminished serotonin-induced aggregation of rat platelets in the presence of a sensitizing but nonaggregating amount of ADP, whereas it did not modify aggregation in PRP when induced by ADP. DV-7028 caused a concentration-dependent, almost parallel shift to the right of the concentration-response to serotonin for its pressor effect in the rat perfused tail artery. The present data demonstrate that DV-7028 exhibits 5-HT2A receptor antagonistic properties in the rat cardiovascular system, exhibits antithrombotic effect in the model of arterial but not venous thrombosis in rats. These results constitute further evidence of the possible importance of serotonin as a mediator of platelet thrombosis in arteries. Moreover, they can provide a useful tool for the prevention of various thrombotic diseases.     

Inhaled nitric oxide inhibits human platelet aggregation, P-selectin expression, and fibrinogen binding in vitro and in vivo

BACKGROUND: Recent data suggest that inhaled NO can inhibit platelet aggregation. This study investigates whether inhaled NO affects the expression level and avidity of platelet membrane receptors that mediate platelet adhesion and aggregation. METHODS AND RESULTS: In 30 healthy volunteers, platelet-rich plasma was incubated with an air/5% CO2 mixture containing 0, 100, 450, and 884 ppm inhaled NO. ADP- and collagen-induced platelet aggregation, the membrane expression of P-selectin, and the binding of fibrinogen to the platelet glycoprotein (GP) IIb/IIIa receptor were determined before (t0) and during the 240 minutes of incubation. In addition, eight patients suffering from severe adult respiratory distress syndrome (ARDS) were investigated before and 120 minutes after the beginning of administration of 10 ppm inhaled NO. In vitro, NO led to a dose-dependent inhibition of both ADP-induced (3+/-3% at 884 ppm versus 70+/-6% at 0 ppm after 240 minutes; P<.001) and collagen-induced (13+/-5% versus 62+/-5%; P<.01) platelet aggregation. Furthermore, P-selectin expression (36+/-7% of t0 value; P<.01) and fibrinogen binding (33+/-11%; P<.01) were inhibited. In patients with ARDS, after two who did not respond to NO inhalation with an improvement in oxygenation had been excluded, an increase in plasma cGMP, prolongation of in vitro bleeding time, and inhibition of platelet aggregation and P-selectin expression were observed, and fibrinogen binding was also inhibited (19+/-7% versus 30+/-8%; P<.05). CONCLUSIONS: NO-dependent inhibition of platelet aggregation may be caused by a decrease in fibrinogen binding to the platelet GP IIb/IIIa receptor.     

Red-wine polyphenols and inhibition of platelet aggregation: possible mechanisms, and potential use in health promotion and disease prevention

Correction of uremic platelet serotonin (5-HT) storage pool deficiency is one of the very early hemostatic effects of erythropoietin (Epo) therapy. In this work, platelet 5-HT with relation to primary hemostasis was studied in 15 hemodialysis patients treated with Epo for 8 months. Moreover, effects of ketanserin, a blocker of platelet and vascular smooth muscle cell 5-HT2A receptors, in these patients were followed. The parameters studied were compared with relevant values in healthy controls and in hemodialysis patients not treated with Epo, and remeasured in the long-term Epo patients after a 14-day oral ketanserin trial. Platelet 5-HT content in the eighth month of Epo therapy was not different from the one in untreated patients. Ristocetin- and collagen-induced platelet aggregation were enhanced in comparison with both control groups, as opposed to unaltered response to ADP and arachidonic acid. Fibrinogen concentration was lower than in the untreated group. An inverse correlation between ADP-induced platelet aggregation and the skin bleeding time (r=-0.536, p<0.05) and a positive one between the former and platelet 5-HT (r=0.644, p<0.01) were found. Platelet count correlated positively with both platelet 5-HT (r=0.823, p<0.0002) and ADP-induced platelet aggregation (r=0.596, p<0.02). Ketanserin produced a decrease in ristocetin-induced platelet aggregation, fibrinogen, and prolongation of the bleeding time. The first two of the changes correlated positively with their pre-ketanserin values (r=0.923, p<0.00001 and r=0.839, p< 0.0001, respectively). Post-ketanserin, positive correlations between depressed ristocetin- and arachidonic acid-induced platelet aggregation (r=0.760, p<0.005), and between collagen- and corresponding values of arachidonic acid- (r=0.622, p<0.02), ADP-induced platelet aggregation (r=0.396, p<0.01), and platelet 5-HT (r=0.654, p<0.05) were found. Efficient hemostasis in hemodialysis patients on protracted Epo therapy is, in part, dependent on enhanced platelet aggregability. Correction of platelet 5-HT storage pool deficiency is not evident in this stage but 5-HT still influences complex mechanisms of primary hemostasis. Ketanserin is of anticoagulant value in these patients but its effects must be weighted against possible exacerbation of the anemia.