Rabbit Platelet-rich Plasma Research Articles

Anticoagulant and antithrombotic activity of a new peptide pENW (pGlu-Asn-Trp)

The aim was to test a newly discovered oligopeptide, pENW (pGlu-Asn-Trp), for its anticoagulant and antithrombotic activity in vivo, and try to investigate its underlying mechanisms. We measured coagulation time by the glass slide method and bleeding time by cutting of mice tails. The thrombosis models employed here included an arterio-venous shunt model and inferior vena ligation model. An ELISA (enzyme-linked immunosorbent assay) was used to analyse t-PA/PAI (tissue-type plasminogen activator/plasminogen activator inhibitor) in the blood drawn from the rats with thrombosis. The ultrastructural changes of the endothelium in the vessels developing thrombosis were observed under a transmission electron microscope. We found that pENW-treated mice exhibited a prolonged coagulation time in a dose-dependent manner, but not an extended haemorrhage time. On the other hand, pENW significantly inhibited thrombus formation in both arterio-venous shunt models and inferior vena ligation models. Plasma t-PA/PAI was significantly higher as measured by ELISA. Transmission electron microscope photos of pENW-treated groups also displayed a better condition than model controls, with less erythrocytes in the vascular lumens. In addition, pENW concentration-dependently inhibited aggregation of platelets induced by ADP (adenosine 5'-diphosphate sodium salt) in rabbit platelet-rich plasma. These findings support the suggestion that pENW possesses antithrombotic activity and could be a promising drug in the prevention and treatment of unwanted clot formation.

Licofelone, a Balanced Inhibitor of Cyclooxygenase and 5-Lipoxygenase, Reduces Inflammation in a Rabbit Model of Atherosclerosis

Licofelone, a dual anti-inflammatory drug that inhibits 5-lipoxygenase (LOX) and cyclooxygenase (COX) enzymes, may have a better cardiovascular profile that cycloxygenase-2 inhibitors due to cycloxygenase-1 blockade-mediated antithrombotic effect and a better gastrointestinal tolerability. We examined the anti-inflammatory effect of licofelone on atherosclerotic lesions as well as in isolated neutrophils from whole blood of rabbits compared with a selective inhibitor of COX-2, rofecoxib. We also assessed the antithrombotic effect of licofelone in rabbit platelet-rich plasma. For this purpose, 30 rabbits underwent injury of femoral arteries, and they were randomized to receive 10 mg/kg/day licofelone or 5 mg/kg/day rofecoxib or no treatment during 4 weeks with atherogenic diet in all cases. Ten healthy rabbits were used as controls. Neutrophils and platelets were isolated from peripheral blood of rabbits for ex vivo studies. Licofelone reduced intima/media ratio in injured arteries, the macrophages infiltration in the neointimal area, monocyte chemoattractant protein-1 (MCP-1) gene expression, and the activation of nuclear factor-kappaB in rabbit atheroma. Moreover, licofelone inhibited COX-2 and 5-LOX protein expression in vascular lesions. Rofecoxib only diminished COX-2 protein expression and MCP-1 gene expression in vascular atheroma. Prostaglandin E(2) in rabbit plasma was attenuated by both drugs. Licofelone almost abolished 5-LOX activity by inhibiting leukotriene B4 generation in rabbit neutrophils and prevented platelet thromboxane B2 production from whole blood. Licofelone reduces neointimal formation and inflammation in an atherosclerotic rabbit model more markedly than rofecoxib. This effect, together with the antiplatelet activity of licofelone, suggests that this drug may have a favorable cardiovascular profile.

Pharmacological Characterization of 2NTX-99 [4-Methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a Potential Antiatherothrombotic Agent with Antithromboxane and Nitric Oxide Donor Activity in Platelet and Vascular Preparations

Thromboxane (TX) A(2), prostacyclin (PGI(2)), and nitric oxide (NO) regulate platelet function and interaction with the vessel wall. Inhibition of TXA(2), implemented synthesis of PGI(2), and supply of exogenous NO may afford therapeutic benefit. 2NTX-99 [4-methoxy-N(1)-(4-trans-nitrooxycyclohexyl)-N(3)-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a new chemical entity related to picotamide, showed antithromboxane activity and NO donor properties. 2NTX-99 relaxed rabbit aortic rings precontracted with norepinephrine or U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic acid; EC(50), 7.9 and 17.1 microM, respectively), an effect abolished by 10 microM 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ). 2NTX-99 inhibited arachidonic acid (AA)-induced washed platelet aggregation (EC(50), 9.8 microM) and TXB(2) formation (-71% at 10 microM), and its potency increased in the presence of aortic rings (EC(50), 1.4 microM). In whole rabbit aorta incubated with homologous platelets, AA caused contraction and TXA(2) formation, reduced by 2NTX-99 (10-40 microM): contraction, -28 and -47%, TXA(2) formation, -37 and -75.4%, respectively, with concomitant increase in PGI(2). 2NTX-99 (20-40 microM) inhibited U46619-induced aggregation in rabbit platelet-rich plasma (PRP) (-74 +/- 6.7 and -96 +/- 2.4%, respectively) and inhibited collagen-induced aggregation in human PRP (-48.2 +/- 10 and -79.2 +/- 6%), whereas ozagrel was ineffective. In human embryonic kidney 293 cells transfected with the TXA(2) receptor isophorm alpha receptor, 2NTX-99 did not compete with the ligand, [(3)H]SQ29,548 ([(3)H][1S-[1alpha,2beta(5Z),3beta,4alpha]]-7-[3-[[2-(phenylamino)-carbonyl]hydrazino]methyl]-7-oxabicyclo[2,2,1]-hept-2-yl]-5-heptanoic acid), or prevent inositol phosphate accumulation. After oral administration (50-250 mg/kg), 2NTX-99 inhibited TXA(2) production in rat clotting blood (-71 and -91%); at 250 mg/kg, an area under the curve, 0 to 16 h, of 149.5 h/microg/ml and a t(1/2) of 6 h were calculated, with a C(max) value of 31.8 +/- 8.2 microg/ml. An excellent correlation between plasma concentrations and TXA(2) inhibition occurs. 2NTX-99 controls platelet function and vessel wall interaction by multifactorial mechanisms and possesses therapeutic potential.

Bile salts secretion in cirrhosis.

The bile salts secretion was studied in ten normal subjects and sixteen patients with alcoholic cirrhosis, in a basal period and during 60 minutes after Secretin injection. Total bile salts were measured by a modification of the enzymatic method of Iwata and Yamasaki and the individual bile salts were separated by silica gel thin-layer chromatography. During the 60 minutes after Secretin the mean concentration was 2.88 +/- 2.58 muM/ml in normals and 1.96 +/- 1.25 muM/ml in cirrhotics. The difference is not significant. During the first 20 minutes however the concentration was higher than 3 muM/ml in 8 out of 10 normals and lower than 2 muM/ml in 10 out 16 cirrhotics. The ratios of tri-to dihydroxy bile salts was similar in both groups. The ratios between bile salts conjugated with glycine and with taurine was higher in normals, and the ratio between free to conjugated bile salts was higher in cirrhotics. The lower concentration of total bile salts immediatly after Secretin, the higher proportion of taurin conjugates and of free bile salts could be important factors in the difficulties of fact digestion and absorption frequently found in patients with alcoholic cirrhosis.

Platelet aggregation induced by serotype polysaccharides from Streptococcus mutans.

Platelet aggregation plays an important role in the pathogenesis of infective endocarditis induced by viridans streptococci or staphylococci. Aggregation induced in vitro involves direct binding of bacteria to platelets through multiple surface components. Using platelet aggregometry, we demonstrated in this study that two Streptococcus mutans laboratory strains, GS-5 and Xc, and two clinical isolates could aggregate platelets in an irreversible manner in rabbit platelet-rich plasma preparations. The aggregation was partially inhibited by prostaglandin I(2) (PGI(2)) in a dose-dependent manner. Whole bacteria and heated bacterial cell wall extracts were able to induce aggregation. Cell wall polysaccharides extracted from the wild-type Xc strain, containing serotype-specific polysaccharides which are composed of rhamnose-glucose polymers (RGPs), could induce platelet aggregation in the presence of plasma. Aggregation induced by the serotype-specific RGP-deficient mutant Xc24R was reduced by 50% compared to the wild-type strain Xc. In addition, cell wall polysaccharides extracted from Xc24R failed to induce platelet aggregation. The Xc strain, but not the Xc24R mutant, could induce platelet aggregation when preincubated with plasma. Both Xc and Xc24R failed to induce platelets to aggregate in plasma depleted of immunoglobulin G (IgG), but aggregation was restored by replenishment of anti-serotype c IgG. Analysis by flow cytometry showed that S. mutans RGPs could bind directly to rabbit and human platelets. Furthermore, cell wall polysaccharides extracted from the Xc, but not the Xc24R, strain could induce pseudopod formation of both rabbit and human platelets in the absence of plasma. Distinct from the aggregation of rabbit platelets, bacterium-triggered aggregation of human platelets required a prolonged lag phase and could be blocked completely by PGI(2). RGPs also trigger aggregation of human platelets in a donor-dependent manner, either as a transient and reversible or a complete and irreversible response. These results indicated that serotype-specific RGPs, a soluble product of S. mutans, could directly bind to and activate platelets from both rabbit and human. In the presence of plasma containing IgG specific to RGPs, RGPs could trigger aggregation of both human and rabbit platelets, but the degree of aggregation in human platelets depends on the donors.

Surface mobility of polymers having phosphorylcholine groups connected with various bridging units and their protein adsorption-resistance properties

The effects of the surface properties of phospolipid polymers having various bridging units between the phosphorylcholine and the backbone on their blood compatibility were studied. Three methacrylates having a phosphorylcholine group, i.e. 2-methacryloyloxyethyl phosphorylcholine (MPC), 2-methacryloyloxyethoxyethyl phosphorylcholine (MEO2PC), and 6-methacryloyloxyhexyl phosphorylcholine (MHPC) were synthesized. These monomers were copolymerized with n-butyl methacrylate (BMA). Surface analysis with X-ray photoelectron spectroscopy showed the surface density of phosphorylcholine groups on the polymer surface, and the density of poly(MHPC-co-BMA) was remarkably higher than its theoretical value even in the dry state. The water contact angle of the polymer surfaces was measured to determine the hydration kinetics of the polymer surface in contact with water. The surface of poly(MEO2PC-co-BMA) was equilibrated rapidly. When human plasma and rabbit platelet-rich plasma were in contact with polymer surfaces without prehydration, the surface density of adsorbed proteins and adherent platelets on the poly(MPC-co-BMA) was similar to that on poly(BMA). On the other hand, poly(MEO2PC-co-BMA) and poly(MHPC-co-BMA) effectively reduced the biofouling compared with poly(MPC-co-BMA). It was clarified that phospholipid polymer surfaces, which could easily form a phosphorylcholine-enriched surface when the blood was contact with the surfaces, showed excellent blood compatibility without prehydration.

Antiplatelet and Antithrombotic Activity of SL65.0472, a Mixed 5-HT1B/5-HT2A Receptor Antagonist

The antiplatelet and antithrombotic activity of SL65.0472 (7-fluoro-2-oxo-4-[2-[4-(thieno [3,2-c]pyrin-4-yl) piperazin-1-yl]ethyl]-1,2-di-hydroquinoline-acetamide), a mixed 5-HT1B/5-HT2A receptor antagonist was investigated on 5HT-induced human platelet activation in vitro, and in rat, rabbit and canine platelet dependent thrombosis models. SL65.0472 inhibited 5-HT-induced platelet shape change in the presence of EDTA (IC50 values = 35, 69 and 225 nM in rabbit, rat and human platelet rich plasma (PRP)), and also inhibited aggregation induced in human PRP by 3-5 microM 5-HT + threshold concentrations of ADP (0.5-1 microM) or collagen (0.3 microg/ml) with mean IC50 values of 49 +/- 13 and 48 +/- 6 nM respectively. SL65.0472 inhibited thrombus formation when given both intravenously 5 min and orally 2 h prior to assembly of an arterio-venous (A-V) shunt in rats as from 0.1 and 0.3 mg/kg respectively. It was active in a rabbit A-V shunt model with significant decreases in thrombus weight as from 0.1 mg/kg i. v. and at 10 mg/kg p.o. The delay to occlusion in an electric current-induced rabbit femoral artery thrombosis model was increased by 251% (p <0.05) after 20 mg/kg p.o. SL65.0472 (30 microg/kg i.v.) virtually abolished coronary cyclic flow variations (7.2 +/- 1.0/h to 0.6 +/- 0.6/h, p <0.05) and increased minimum coronary blood flow (1.2 +/- 0.8 ml/min to 31.8 +/- 8.4 ml/min, p <0.05) in a coronary artery thrombosis model in the anaesthetised dog. Finally, SL65.0472 significantly increased the amount of blood lost after rat tail transection at 3 mg/kg p.o. Thus the anti-5-HT2A component of SL65.0472 is reflected by its ability to inhibit 5-HT-induced platelet activation, and platelet-rich thrombus formation.

Inhibition of Platelet Aggregation by Putrescine, Spermidine, and Spermine in Hypercholesterolemic Rabbits

Background Hypercholesterolemia causes alterations in platelet function. Platelet hyperaggregation is considered a predisposing factor for atherosclerosis. In this paper, the antiaggregating effect of the polyamines putrescine, spermidine, and spermine was studied on platelets of normal and hypercholesterolemic rabbits. Methods New Zealand rabbits were fed with a cholesterol-enriched diet for 10 weeks. Lipids and glucose were determined in serum. The assays of platelet aggregation were carried out using platelet-rich plasma (PRP) obtained from both control and cholesterol-fed rabbits. We used 2.5 μmol /mL ADP and 2 μg/mL collagen as inductors of platelet aggregation. In addition, arginase activity and L-arginine content were determined in PRP. Results Serum total cholesterol and LDL-cholesterol concentrations were increased from 26.3 ± 8.1 to 1,485.0 ± 26.8 mg/dL and from 15.9 ± 5.9 to 1,383.8 ± 58.9 mg/dL, respectively, whereas triglyceride concentration increased from 88.3 ± 35.6 to 411.0 ± 154.5 mg/dL upon cholesterol feeding. Seventy-five percent of platelet aggregation inhibition was observed with 10 μM of polyamines in PRP of normal rabbits. Spermine inhibited platelet aggregation by 54% in PRP of hypercholesterolemic rabbits when ADP was used as agonist. The order of polyamine action was spermine > spermidine > putrescine. In addition, we found that platelet arginase activity and L-arginine content were unaltered upon hypercholesterolemia. Conclusions These results show that the polyamines putrescine, spermidine, and spermine have antagonist action in platelet aggregation and suggest a key role of polyamines in platelet aggregation under normal and hypercholesterolemic conditions.

Synthesis and antiplatelet evaluation of novel aryl-sulfonamide derivatives, from natural safrole.

In the scope of a research program aiming at the synthesis and pharmacological evaluation of novel possible antiplatelet prototype compounds, exploring bioisosterism principles for molecular design, we describe in this paper the synthesis of new aryl-sulfonamides derivatives, structurally similar to known thromboxane A2 receptor antagonists. The synthetic route used to access the new compounds described herein starts from safrole, an abundant Brazilian natural product, which occurs in Sassafras oil (Ocotea pretiosa). The results from preliminary evaluation of these novel aryl-sulfonamide compounds by the platelet aggregation inhibitory test, using rabbit PRP, induced by ADP, collagen, arachidonic acid, and U46619, identified the N-[2-(4-carboxymethoxyphenyl)ethyl]-6-methyl-3,4-methylenedioxyphe nyl- sulfonamido derivative as the most active among them, presenting in IC50 value for the U-46619-induced platelet aggregation in rabbit platelet-rich plasma: 329 microM.

Synthesis and pharmacological evaluation of a new class of bicyclic phospholipids, designed as platelet activating factor antagonists

(±)-3-Alkoxymethyl-(2-oxabicyclo[3.3.0]octane)-5-yl-methyl-phosphoryl-ethyl-pyridinium [alkyl chain=methyl ( 5a) and ( 5b), allyl ( 6a) and ( 6b), n-propyl ( 7a) and ( 7b) and n-hexyl ( 8a) and (8b)] derivatives, structurally designed as conformationally restricted platelet activating factor (PAF) antagonists were synthesized in 12–26% overall yield, using ethyl (±)-3-hydroxymethyl-5-(2-oxabicyclo[3.3.0] octane) carboxylate ( 13a,b) as key intermediate. The anti-platelet profile of the new derivatives was evaluated in a PAF-induced aggregation model in rabbit platelet-rich plasma; only compound 8a exhibited a modest activity.

Anti-platelet action of nitric oxide and selective phosphodiesterase inhibitors.

Nitric oxide gas is a potent inhibitor of platelet aggregation, with an IC50 of 3.6 microM for rabbit platelets. Since the NO effect is mediated via increased cGMP, this in vitro study was undertaken to test the hypothesis that selective phosphodiesterase (PDE) inhibitors might enhance aggregation inhibition at lower NO concentrations. Because the cAMP-selective PDE III and the cGMP-selective PDE V are prominent in platelets, milrinone, a PDE III inhibitor, and zaprinast, a PDE V inhibitor, were tested alone and in the presence of NO for their effect on aggregation. Aggregometry was performed on rabbit platelet-rich plasma following addition of ADP as agonist. Milrinone alone gave an IC50 of 12.4 microM. With each agent set to give suboptimal inhibition of aggregation, the combination of milrinone (3-16 microM) and NO (2-10 microM) produced a greater effect than either agent alone. Zaprinast exhibited no effect on aggregation in concentrations up to 160 microM. However, adding zaprinast to 2 microM NO, which alone reduced aggregation approximately 30%, produced a marked synergism in the inhibitory effect up to and including no observable aggregation. These results indicate that elevation of either cAMP or cGMP is sufficient to inhibit platelet function. The platelet cAMP concentration appears high enough to be inhibitory when degradation is suppressed by milrinone. However, basal cGMP levels must be increased by NO before the zaprinast effect is observed.

Modulatory effect of 8-iso-PGE 2 on platelets

1. 1. 8-Iso-PGE 2 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 EC 50 for irreversible aggregation in PRP and WP were 4 and 2 μM, respectively. 2. 2. In rabbit PRP, 8-iso-PGE 2 (0.1-100 μM) itself did not induce or induced only reversible aggregation. 3. 3. 8-Iso-PGE 2 (0.1-20μM) 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. 4. The lower concentrations (0.2-0.5 μM) of 8-iso-PGE 2 decreased, and higher concentrations (1–2 μM) increased platelet aggregating factor- (PAF) induced aggregation in human PRP. In rabbit PRP, 8-iso-PGE 2 (0.02–200 μM) had only a decreasing effect on PAF-induced aggregation. 5. 5. The results suggest that low concentrations of 8-iso-PGE 2 can amplify or weaken platelet aggregation induced by various aggregatory agents.

Protein adsorption and platelet adhesion on polymer surfaces having phospholipid polar group connected with oxyethylene chain

We evaluated the blood compatibility of various amphiphilic polymers, that is, n-butyl methacrylate (BMA) copolymers with methacrylates having a phosphorylcholine (PC), hydroxy (OH) or methoxy (MeO) group as an end polar group in the oxyethylene side chain. The amount of proteins adsorbed on the PC-polymer from human plasma was smaller than that on not only the poly(2-hydroxyethyl methacrylate) and poly(methyl methacrylate) but also the OH-polymer and MeO-polymer. The PC group could weaken the interaction between plasma proteins and polymer surfaces. The amount of adsorbed proteins on the PC-polymer decreased with an increase in the mole fraction of the PC units in the polymers. We could observe an effect of the oxyethylene chain length (n is the number of repeating units of oxyethylene) on protein adsorption between n = 2 and n = 3. The platelet adhesion on these polymer surfaces was evaluated using rabbit platelet-rich plasma. On the polymers without the PC group, that is, poly(BMA), OH-polymer, and MeO-polymer, many platelets adhered and a considerable shape change in the adherent platelets occurred. On the other hand, the PC-polymers could effectively suppress platelet adhesion. The platelet adhesion behavior on the polymers was strongly dependent on the adsorbed proteins. Platelet adhesion was completely inhibited on all of the PC-polymers studied having a 0.3 PC unit mole fraction. However, it was observed that the oxyethylene chains on the PC-polymers with a 0.1 PC unit mole fraction affected platelet adhesion.

Dose-responses to inducers and inhibitors of platelet aggregation analysed via a micro-method.

The ex vivo study of platelet function in small experimental animals treated with anti-platelet drugs is restricted by the limited availability of platelet-rich plasma (PRP). To circumvent this obstacle, a micro-method for platelet aggregation was developed that enables ED50 measurements for various inducers of platelet aggregation. Fifty microliters of human, hamster, or rabbit PRP was mixed with agonist in each well of a microtiter plate, and shaken at 900 rpm at 37 degrees C for intervals up to 5 min. After stopping the aggregation with formaldehyde in PBS, light scattering was measured vs platelet-poor plasma (PPP) at 620 nm. Thus, aggregation in human PRP by ADP (t = 2 min) occurred with an ED50 = 1.8 microM, whereas the collagen (t = 3 min; ED50 = 2 micrograms/ml) and AA (t = 1 min; ED50 = 0.3 microM) induced aggregation occurred at those concentrations that induce aggregation in classical aggregometry. Likewise, aggregation was inhibited by the anti-GPIIb/IIIa antibody MA-16N7C2 and by the GPIIb/IIIa antagonists G4120 or MK-852. In comparison with human PRP, hamster (ED50 = 0.8 microM at t = 2 min) and rabbit (ED50 = 5 microM at t = 4 min) platelet aggregation by ADP occurred with comparable sensitivities, whereas the aggregation of rabbit platelets by collagen (ED50 = 15 micrograms/ml at t = 3 min) appeared to be slightly less sensitive and subject to large interindividual variations. The method was applied to measure plasma levels of a GPIIb/IIIa antagonist following injection into hamsters.

Inhibition of arachidonic acid induced-aggregation of rabbit platelets with CV-4151 (isbogrel), a selective thromboxane A 2 (TXA 2) synthase inhibitor: modulation of the antiplatelet action and prostanoid metabolism by rat aortic rings

In rabbit platelet-rich plasma, a specific TXA 2 synthase inhibitor, CV-4151 (isbogrel) alone modestly inhibited the platelet aggregation induced by arachidonic acid (AA); even at 10 −4 M the inhibition was less than 50% (IC 40 value: 8.1 X 10 −5 M). Aspirin-treated rat aortic rings alone inhibited the AA-induced platelet aggregation by 7%. However, CV-4151 exhibited a potent antiplatelet action in the presence of the aortic rings and its IC 40 value was 8.3 X 10 −8 M. Thus, the aortic rings caused 1000-fold enhancement of the antiplatelet action of CV-4151. Next, we investigated the effect of CV-4151 on the production of two highly biologically active prostanoids, TXA 2 and PGI 2, and the contribution of these prostanoids to the antiplatelet action of CV-4151. TXA 2 and PGI 2 were measured by a radioimmunoassay of their stable metabolites, TXB 2 and 6-keto-PGF 1α, respectively. Under the experimental conditions, CV-4151 simultaneously inhibited TXA 2 generation and enhanced PGI 2 generation. There was a positive linear relationship ( r = 0.975, p < 0.01) between % inhibition of platelet aggregation and PGI 2 generation and a significant negative linear relationship ( r = 0.994, p < 0.001) between % inhibition of platelet aggregation and TXA 2 generation. CV-4151 exhibits a potent antiplatelet action in the presence of PGI 2 synthase, which is the in vivo situation, by simultaneously inhibiting TXA 2 generation and enhancing PGI 2 generation.

Effect of methylene chain length in phospholipid moiety on blood compatibility of phospholipid polymers.

To investigate the effects of the methylene chain length between the phospholipid polar group and the backbone on blood compatibility of a phospholipid polymer, copolymers of omega-methacryloyloxyalkyl phosphorylcholine (MAPC) with n-butyl methacrylate (BMA) were synthesized. The methylene chains were ethylene (n = 2), tetramethylene (n = 4), and hexamethylene (n = 6). Every MAPC copolymer with an MAPC mole fraction in the range of 0.1-0.3 was soluble in ethanol but only swelled in water, and the equilibrium water fraction of the water-swollen MAPC copolymer membrane decreased with the length of the methylene chain. When a rabbit platelet-rich plasma was applied on the MAPC copolymer surface with an 0.1 MAPC mol fraction for 180 min, the number of adhered platelets depended on the length of the methylene chain in the MAPC moiety of the copolymer. The amount of phospholipid adsorbed on the MAPC copolymer from human plasma was larger than that on hydrophobic poly(BMA) and increased with the length of the methylene chain in the MAPC moiety. That is, the reduction of platelet adhesion corresponded to the increase in the amount of phospholipid adsorbed on the MAPC copolymer.

Synthesis and evaluation of novel fluorinated sulotroban-related sulfonamide derivatives as thromboxane A 2 receptor antagonists

Summary A series of sulotroban-related arylsulfonamide derivatives possessing a fluorinated phenoxyacetic acid moiety was synthesized and tested for TXA2 antagonizing ability on U-46619-induced platelet aggregation of rabbit platelet-rich plasma. Introduction of one or more fluorine atoms to the phenoxyacetic acid moiety increased this activity. The most potent compound among these compounds was 10c, which was 40-fold more potent (IC50 3.4 × 10−7 M) than sulotroban. 10c exhibited high activity (ID50 0.14 mg/kg) against a U-46619-induced acute thrombocytopenia model in mice when orally administrated. These findings and those of radioligand binding assays with various ligands showed 10c to be a potent and selective systemic TXA2 receptor antagonist.

Synthesis and NMR Studies of RGDSPASS-Containing Cystine Peptides

Three types of Arg–Gly–Asp–Ser–Pro–Ala–Ser–Ser (RGDSPASS)-containing cystine peptides, FR-1, FR-2, and FR-3, were designed and synthesized. These cyclic peptides exhibited high activities as a platelet aggregation inhibitor. Using rabbit platelet-rich plasma, the IC50 values of FR-1, FR-2, and FR-3 were found to be 67, 280, and 135 μM, respectively. In each peptide, the position of the Pro residue was either i + 1 or i + 2 in the turn, as observed by NMR (ROESY, NOESY, and H–D exchange of amide proton).

Human recombinant phospholipase A 2 inhibits platelet aggregation in vitro and in vivo in rat and guinea pig

Platelets contain a phospholipase A 2 in their granules which can be released in response to various activating stimuli in vitro as well as in vivo. Human recombinant phospholipase A 2 (1–10 μg/ml) had no direct effect on platelet aggregation in vitro using rabbit platelet rich plasma. In contrast human recombinant phospholipase A 2 (1–20 μg/ml) was able to inhibit aggregation of washed rabbit platelet in vitro induced by collagen (0.250–2.0 μg/ml). When rabbit platelet rich plasma was recalcified with CaCl 2 1 M in the presence of the thrombin inhibitor hirulog (10 μg/ml), human recombinant phospholipase A 2 (10–40 μg/ml) was able to inhibit platelet aggregation. The anti-aggregatory effect was removed by incubation of platelet rich plasma with a monoclonal anti-human recombinant phospholipase A 2 antibody. Human recombinant phospholipase A 2 (1–10 μg) inhibited 111In-labelled platelet accumulation within the thoracic region of rats and guinea pigs induced by i.v. administration of submaximal doses of collagen or adenosine diphosphate. Phospholipase A 2 (1–20 μg/ml) from Naja mocambique mocambique snake venom had no direct effect on platelet aggregation in vitro. However, Naja phospholipase A 2 administered i.v. to rats or guinea pigs was able to induce a dose related accumulation of 111In-labelled platelet within the thoracic region. The inhibitory effect of exogenously added human recombinant phospholipase A 2 on platelet aggregation in vivo suggests a possible pathophysiological role for extracellular form of phospholipase A 2 but this property is not a feature of all phospholipase A 2 preparations.

Purification and characterization of platelet aggregation inhibitors from snake venoms

Proteins that inhibit glycoprotein (GP) IIb/IIIa mediated platelet aggregation have been purified from the venom of two snake species. A small platelet aggregation inhibitor (pl.AI), multisquamatin (Mr=5,700), was purified from Echis multisquamatus venom by hydrophobic interaction HPLC and two steps on C18 reverse phase HPLC. A larger pl.AI, contortrostatin (Mr=15,000), was purified by a similar HPLC procedure from the venom of Agkistrodon contortrix contortrix. Both pl.AIs inhibit ADP-induced human, canine and rabbit platelet aggregation using platelet rich plasma (PRP). Multisquamatin has an IC 50 of 97 nM, 281 nM and 333 nM for human, canine and rabbit PRP, respectively. Contortrostatin has an IC 50 of 49 nM, 120 nM and 1,150 nM for human, canine and rabbit PRP, respectively. In a competitive binding assay using 125I-7E3 (a monoclonal antibody to GPIIb/IIIa that inhibits platelet aggregation) both contortrostatin and multisquamatin demonstrated GPIIb/IIIa specific binding to human and canine platelets. The IC 50 for contortrostatin displacement of 7E3 binding to human and canine GPIIb/IIIa is 27 nM and 16 nM, respectively and for multisquamatin it is 3 nM and 63 nM, respectively. Our results indicate that both pl.AIs inhibit platelet aggregation by binding with high affinity to GPIIb/IIIa.