Cyclic peptide analogs of 558-565 epitope of A2 subunit of Factor VIII prolong aPTT. Toward a novel synthesis of anticoagulants.

Novel anticoagulant therapies target specific clotting factors in blood coagulation cascade. Inhibition of the blood coagulation through Factor VIII-Factor IX interaction represents an attractive approach for the treatment and prevention of diseases caused by thrombosis. Our research efforts are continued by the synthesis and biological evaluation of cyclic, head to tail peptides, analogs of the 558-565 sequence of the A2 subunit of FVIII, aiming at the efficient inhibition of Factor VIIIa-Factor IXa interaction. The analogs were synthesized on solid phase using the acid labile 2-chlorotrityl chloride resin, while their anticoagulant activities were examined in vitro by monitoring activated partial thromboplastin time and the inhibition of Factor VIII activity. The results reveal that these peptides provide bases for the development of new anticoagulant agents.

A novel approach in potential anticoagulants from peptides epitope 558-565 of A2 subunit of factor VIII.

Factor VIII, a human blood plasma protein, plays an important role during the intrinsic pathway of blood coagulation cascade after its activation by thrombin. The activated form of FVIII acts as cofactor to the serine protease Factor IXa, in the conversion of the zymogen Factor X to the active enzyme Factor Xa. The Ser558-Gln565 region of the A2 subunit of Factor VIII has been shown to be crucial for FVIIIa-FIXa interaction. Based on this, a series of linear peptides, analogs of the 558-565 loop of the A2 subunit of the heavy chain of Factor VIII were synthesized using the acid labile 2-chlorotrityl chloride resin and biologically evaluated in vitro by measuring the chronic delay of activated partial thromboplastin time and the inhibition of Factor VIII activity, as potential anticoagulants.

Clinical applications of intravascular ultrasound (IVUS): experience from an academic high volume centre of Northern Greece.

BACKGROUND: Intravascular ultrasound (IVUS) has become a valuable tool adjunctive to coronary angiography due to its ability to directly image atheroma and the vessel wall. We aimed to evaluate the use of IVUS during diagnostic angiography and coronary interventions in a coronary intervention academic high volume center of northern Greece. PATIENTS AND METHODS: IVUS studies have been retrospectively retrieved from 2005 to 2008 from the archives of the catheterization laboratory of our department. IVUS was performed in 403 patients (294 male) of mean age 62±6 years. Indications for coronary angiography +/- intervention were acute coronary syndromes (49%), stable angina (46%) and previous coronary angioplasty evaluation (5%). RESULTS: Forty eight per cent of the IVUS studies were performed in left anterior descending artery (LAD), 25% in right coronary artery (RCA), 18% in left circumflex artery (LCx), and the rest (9%) in left main coronary artery (LMCA) or in coronary branches. Indications for performing an IVUS study were assessment of intermediate lesions (60%), evaluation of stent placement (36.5%), and determination of stent restenosis aetiology (3.5%). Among studies performed for assessment of intermediate lesions, 63% showed a non critical stenosis. IVUS after coronary stenting revealed a suboptimal stent placement in 77% of the cases, while in cases of stent restenosis, IVUS showed inadequate initial stent deployment in 43% of the patients. CONCLUSIONS: The use of IVUS in our department has contributed to the optimization of intervertional treatment of coronary lesions by means of evaluating borderline lesions, stenting placement and stent restenosis.

Brugada syndrome associated with supraventricular tachycardia: diagnostic and therapeutic strategies.

We report the case of a patient with Brugada syndrome and a history of palpitations who presented with an episode of syncope and developed supraventricular tachycardia in the electrophysiological study. The patient was treated with radiofrequency ablation for the supraventricular tachycardia and an implantable cardioverter defibrillator for the Brugada syndrome. At 18 months following implantation of the defibrillator an electrical storm with ventricular fibrillation episodes occurred followed by appropriate discharges of the defibrillator.

Numerical modelling of simulated blood flow in idealized composite arterial coronary grafts: transient flow.

In composite arterial coronary grafts (CACGs), transport phenomena and geometry may considerably alter blood flow dynamics. CACGs aim at revascularizing pathological arteries according to the human anatomy. However, the exact mechanisms causing the failure of coronary bypass grafting are not yet well elucidated. In the present study, computational fluid dynamics (CFD) techniques are applied for the simulation of multi-branched CACGs under physiologically realistic inflow waveforms. The numerical solution is obtained by a finite-volume method formulated in non-orthogonal, curvilinear coordinates and a multi-grid approach. The geometrical models, consisting of idealized and rigid vessels, include the typical T- and a rather new pi-graft configuration. The stenotic effect is also investigated by comparing computational results for three different degrees of area constriction, namely 25%, 50% and 75%, as well as the case without stenosis. Different grafting distances and various inflow rate ratios are imposed, to give an insight into haemodynamical alterations of CACGs and to study the process of restenosis. The results focus on the interaction between the grafts and coronary flows in terms of spatial and temporal variations of velocity and wall shear stress (WSS) distribution. Prominent variations among the different geometries, concerning the velocity profiles and secondary flow motion, are shown. Moreover, the residual flow emerging from different degrees of area constriction shows that low and oscillating shear stresses may arise for even moderate stenotic fields.

Effect of activated carbons modification on porosity, surface structure and phenol adsorption.

The primary objective of this work was the examination of modified activated carbons with tailored adsorption capacity properties. Production of activated carbons with desired properties was accomplished by modification of surface functional groups and introduction of acidic/basic properties. Modification of an activated carbon was performed using partial oxygen gasification, nitric acid treatment, urea impregnation followed by pyrolysis and pyrolysis in a urea saturated stream. The surface properties of the produced samples were estimated by the multibasic titration method of Boehm and by the CO/CO2 gas evolution profiles, while pore structure development was measured by the N2 and CO2 gas adsorption isotherms. Oxygen gasification resulted in samples with surface area slightly lower that the raw activated carbon; the introduction of surface functional groups depended upon the severity of the treatment: carbonylic and phenolic type groups were introduced in all partially gasified samples, while low temperatures and short reaction times enhanced the basic character of the carbon. However, nitric acid treatment resulted in the introduction of high nitrogen amounts in the samples, the reduction of surface area and the development of a surface containing carboxylic, lactonic, phenolic and carbonylic groups with negligible HCl neutralization capacity. Treatment of activated carbon by urea supported the formation of basic groups and carbonyls. The presence of surface functional groups affected the adsorption capacity of the produced samples for the removal of specific pollutants such as phenols. Urea treated samples with a basic character and high nitrogen content presented the highest phenol uptake capacity; nitric acid treated carbons and oxygen gasified samples presented an acidic surface functionality and a low phenol adsorption capacity. The beneficial role of nitrogen on phenol adsorption was attributed to adsorbate-adsorbent interactions.

Activated carbon from olive kernels in a two-stage process: industrial improvement.

Activated carbons have been prepared from olive kernels and their adsorptive characteristics were investigated. A two stage process of pyrolysis-activation has been tested in two scales: (a) laboratory scale pyrolysis and chemical activation with KOH and (b) pilot/bench scale pyrolysis and physical activation with H(2)O-CO(2). In the second case, olive kernels were first pyrolysed at 800 degrees C, during 45 min under an inert atmosphere in an industrial pyrolyser with a throughput of 1t/h (Compact Power Ltd., Bristol, UK). The resulting chars were subsequently activated with steam and carbon dioxide mixtures at 970 degrees C in a batch pilot monohearth reactor at NESA facility (Louvain-la Neuve, Belgium). The active carbons obtained from both scales were characterized by N(2) adsorption at 77 K, methyl-blue adsorption (MB adsorption) at room temperature and SEM analysis. Surface area and MB adsorption were found to increase with the degree of burn-off. The maximum BET surface area was found to be around 1000-1200 m(2)/g for active carbons produced at industrial scale with physical activation, and 3049 m(2)/g for active carbons produced at laboratory with KOH activation. The pores of the produced carbons were composed of micropores at the early stages of activation and both micropores and mesopores at the late stages. Methylene blue removal capacity appeared to be comparable to that of commercial carbons and even higher at high degrees of activation.

Numerical modeling of simulated blood flow in idealized composite arterial coronary grafts: steady state simulations.

This paper presents a comparative study of simulated blood flow in different configurations of simplified composite arterial coronary grafts (CACGs). Even though the composite arterial grafting is increasingly used in cardiac surgery, it is still questionable whether or not the blood flow in such grafts can adequately meet the demands of the native myocardial circulation. A computational fluid dynamics (CFD) model was developed to conduct computer-based studies of simulated blood flow in four different geometric configurations of CACGs, corresponding to routinely used networks in cardiac surgery coronary grafts (T, Y, Pi and sequential). The flow was assumed three-dimensional, laminar and steady and the fluid as Newtonian, while the vessel walls were considered as inelastic and impermeable. It was concluded that local haemodynamics, practically described by velocity, pressure drop, wall shear stress (WSS) and flow rates, may be strongly influenced by the local geometry, especially at the anastomotic sites. The computations were made at mean flow rates of 37.5, 75 and 150ml/min. The side-branch outflow rates, computed for each bypass graft, showed noticeable differences. The results, which were found both qualitatively and quantitatively consistent with other studies, indicate that the Pi-graft exhibits significantly less uniform distribution of outflow rates than the other geometric configurations. Moreover, prominent variations in WSS and velocity distribution among the assessed CACGs were predicted, showing remarkable flow interactions among the arterial branches. The lowest shear stress regions were found on the lateral walls of bifurcations, which are predominantly susceptible to the occurrence of coronary artery disease (CAD). In contrast, the highest WSS were observed at the turn of the arterial branches.

Conjugation of resveratrol with RGD and KGD derivatives.

The reaction between Arg-Gly-Asp (RGD) and Lys-Gly-Asp (KGD) derivatives with 3,4',5-trihydroxy-trans-stilbene (resveratrol) was investigated. Knowing that resveratrol, RGD as well as KGD analogues inhibit human platelet aggregation in vitro, it was tempting for us to examine whether their coupling products present enhanced biological activity. Here, we report on the synthesis and identification of these coupling products. The N-protected peptides were synthesized by solid phase technique, using the 2-chlorotrityl-chloride resin, by the method of carbodiimides. Coupling reactions with resveratrol took place in solution using N,N-dicyclohexylcarbodiimide (DCC) as coupling reagent and 4-dimethylaminopyridine (DMAP) as catalyst. The reaction products were purified by reversed phase HPLC and identified by ESI-MS. The mono-esterified resveratrol derivative was the main (or only) reaction product, whereas the di- and the tri-ester (to a less extent) formation was noticed in some cases.

Assessment of cortisol and ACTH responses to the desmopressin test in patients with Cushing's syndrome and simple obesity.

OBJECTIVE: The desmopressin test has recently been introduced in clinical practice as an adjunctive tool in the differential diagnosis of ACTH-dependent Cushing's syndrome (CS). It has been reported that the majority of patients with pituitary-dependent CS (Cushing's disease, CD) respond to desmopressin, while no such response is usually observed in other forms of this syndrome. In the present study, the responsiveness of the HPA axis to desmopressin was studied in a group of obese subjects. In addition, the ability of desmopressin administration to differentiate between patients with obesity and the various forms of Cushing's syndrome was investigated. DESIGN AND SUBJECTS: Cortisol and ACTH responses to the administration of desmopressin (10 microg bolus i.v.) were examined in 20 consecutive patients with obesity (14 women and six men; BMI range: 34.5-66.7 kg/m2). Obese subjects had no clinical stigmata of CS. In all obese patients, either an overnight (dex 1 mg at 2300 h) (n = 8) or a formal low-dose (dex 0.5 mg 6-hourly for 2 days) (n = 12) dexamethasone suppression test was performed for the exclusion of Cushing's syndrome. Three of eight subjects showed failure of cortisol suppression (i.e. F > 28 nmol/l) to the overnight dexamethasone suppression test, but they had undetectable cortisol levels (< 28 nmol/l) on further testing with the formal 2-day test. All but two of the remaining subjects had undetectable cortisol levels (< 28 nmol/l) following the formal 2-day, low-dose, dexamethasone suppression test. For comparison, desmopressin responses were also tested in 33 patients with CS of varied aetiologies (25 patients with pituitary-dependent CS, three patients with occult ectopic ACTH secretion and five patients with primary adrenal CS). A positive response was considered to be an increment greater than 20% and 50% from baseline levels of cortisol and ACTH, respectively. RESULTS: Mean cortisol (F) and ACTH levels did not differ from the baseline at any time point following desmopressin administration in the obese group (basal F: 417 +/- 41, peak F: 389 +/- 32 nmol/l, P > 0.05; basal ACTH: 33.5 +/- 4.3, peak ACTH: 50.6 +/- 16.6 ng/l, P > 0.05), or in patients with occult ectopic or primary adrenal CS. In contrast, in the group of patients with CD, there was a significant rise in the mean ACTH and F levels from baseline (basal F: 725 +/- 50, peak F: 1010 +/- 64 nmol/l, P < 0.01; basal ACTH: 88.6 +/- 11.8, peak ACTH: 351 +/- 64 ng/l, P < 0.01). Cortisol responses greater than 20% from baseline were observed in 21/25 (84%) patients with CD, but in only 3/20 (15%) of the obese patients. With regard to ACTH, increments greater than 50% over baseline were observed in 23/25 (92%) of patients with CD, and in only 3/20 (15%) of the obese patients. As previously reported, none of the patients with occult ectopic ACTH secretion or primary adrenal CS had a positive response. CONCLUSIONS: The prevalence of subjects who met the criteria adopted to define positive cortisol and ACTH responses to the desmopressin test was significantly higher in the group of patients with Cushing's disease than in the group of patients with obesity. It is therefore suggested that this test may be occasionally useful in the differentiation between simple obesity and the pituitary-dependent form (but not other forms) of Cushing's syndrome.

Spectral analysis of a series of partially protected and deprotected tetrapeptides, analogues of AS-I phytotoxin.

A series of six tetrapeptides, analogues of AS-I phytotoxin, pathogenic to sunflower, have been synthesized either in solution and/or by solid phase methods and have been tested for phytotoxic activity in various plants and cytotoxic activity in three cancer cell lines. These peptides were identified as model compounds by fast atom bombardment (FAB), plasma desorption (PD), electrospray ionization (ESI) mass spectrometry and by 1H, 1H-1H, 13C and 1H-13C NMR. The data presented show that in protected tetrapeptides the molecular ion was easily identified whereas some difficulties appeared with the fully deprotected peptides. NMR spectra are given.

Substance P C-terminal octapeptide analogues augment tumor necrosis factor-alpha release by human blood monocytes and macrophages.

We have investigated the effects of the substance P C-terminal octapeptide analogues [Pro4, Glu (OBzl)11] SP4-11, [Hyp4, Glu(OBzl)11] SP4-11, [cHyp4, Glu(OBzl)11] SP4-11 and [kPro4, Glu(OBzl)11] SP4-11 on the constitutive and/or lipopolysaccharide (LPS)-induced expression of tumor necrosis factor (TNF-alpha) in both freshly isolated human blood monocytes (FIBM) and monocyte-derived macrophages (MDM). The cells were treated with substance P and the substance P analogues at various concentrations (10-14 to 10-6 M) in the presence or absence of LPS and culture supernatants were analyzed for TNF-alpha as measured by an enzyme immunosorbent assay (ELISA). Monocytes and macrophages treated with the substance P analogues alone increased TNF-alpha secretion at a magnitude similar to the effect of entire undecapeptide substance P. The stimulatory effects of the substance P analogues on TNF-alpha secretion are inhibited by substance P antagonists, spantide ([D-Arg-1-D-Trp-7-D-Trp-9-Leu-11]-SP) and CP-96,345 (a nonpeptide antagonist of the substance P receptor), indicating that these effects are specific and substance P receptor-mediated. Treatment of monocytes and macrophages with the substance P analogues in combination with LPS, however, showed no synergistic interaction in upregulation of TNF-alpha. These data indicate that the biological effect of substance P on TNF-alpha production by human monocytes and macrophages depends mainly on the sequence of the C-terminal region of the molecule.

Antiproliferative activity of synthetic tetrapeptides, analogs of AS-I phytotoxin, towards cancer cell lines.

The in vitro chemosensitivity of three cancer cell lines [HT29 (colon), HeLa (cervical) and T47D (breast)] to eight synthetic tetrapeptides, analogs of AS-I toxin, with phytotoxic effect on a series of plants was studied. Mouse fibroblast L929 cell line was also tested for chemosensitivity to these peptides. All cell lines were especially sensitive to Cys-Val-Gly-Glu tetrapeptide with IC50 values of 0.18, 0.3 and 0.63 mM for HT29, HeLa and T47D cells, respectively, whereas the IC50 value for the L929 cells was higher than 1 mM. Antiproliferative activity was also observed with peptides Tyr-Val-Gly-Glu and His-Val-Gly-Glu with IC50 values higher than those obtained for Cys-Val-Gly-Glu. For the rest of the peptides tested the IC50 values were found close to or higher than 3 mM.

Synthesis of potent agonists of substance P by replacement of Met11 with Glu(OBzl) and N-terminal glutamine with Glp of the C-terminal hexapeptide and heptapeptide of substance P.

The analogues [Glp6,Glu(OBzl)11]SP(6-11) and [Glp5,Glu(OBzl)11]SP(5-11) of the C-terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the N-terminal glutamine has been replaced by pyroglutamic acid, while the COOCH2C6H5 ester group has replaced the SCH3 group of the Met11 side chain. The in vitro activity of both analogues has been determined on three biological preparations: guinea pig ileum (GPI), rat vas deferens (RVD) and rat portal vein (RPV). The results showed that both analogues are highly potent and selective agonists on GPI through the NK-1 receptor. They are more potent than SP itself, with 1.54 and 1.25 respective values of relative potency on GPI. Their selectivity has been studied by utilizing atropine-treated guinea pig ileum (GPI+At). The analogues showed low activity on RVD and RPV tissues, which represent NK-2 and NK-3 monoreceptor assay, respectively.

Severe superior vena caval syndrome after the Mustard repair in a patient with persistent left superior vena cava.

This report describes our experience with a 6-year-old girl with dextro-transposition of the great arteries and persistent left superior vena cava, who manifested severe superior vena caval syndrome following a Mustard repair. A direct end-to-end anastomosis of the left superior vena cava to the left atrial appendage, which formed part of the systemic venous atrium, was performed with excellent relief of the obstruction. The rationale for application of this method is discussed as an alternative to the use of cardiopulmonary bypass for intra-atrial baffle revision.

Synthesis of a potent antagonist of substance P by replacing the CH2SCH3 and the alpha-carboxamide groups of the methionine at [Orn6]-SP6-11 by benzyl ester groups.

Analogues of [Orn6]-SP6-11 have been synthesized in which the CH2SCH3 group of Met11 is replaced by a COOCH3 or a COOBzl group. These analogues, which were tested for agonist and antagonist activity in three in vitro preparations representative of NK-1, NK-2 and NK-3 receptor types, were full agonists at NK-1 receptors, showed very weak agonist activity at NK-2, receptors and were weak antagonists at NK-3 receptors. The above analogues were modified by substituting the alpha-carboxamide of residue 11 by a COOCH3 and a COOBzl group, respectively. The resulting analogues were found to be devoid of agonist activity in each of the functional assays. However, they showed weak antagonist activity at each receptor subtype, with the exception of the dibenzyl analogue, which was a potent and selective NK-1 receptor antagonist. It is concluded that appropriate modification of the side chain of Met11 and its alpha-carboxamide leads to a potent and selective at NK-1 receptor antagonist.

Synthesis of potent antagonists of substance P by modifying the methionyl and glutaminyl residues of its C-terminal hexapeptide and without using D-amino acids.

Analogues of [Orn6]-SP6-11 have been synthesized in which the Met11-NH2 residue is replaced by the alpha, gamma-dimethyl, alpha, gamma-dibenzyl and alpha, gamma-di-tert-butyl esters of glutamic acid. These analogues were tested in three in vitro preparations representative of NK-1, NK-2 and NK-3 receptor types for agonist and antagonist activity. The dimethyl analogue is a selective full agonist in the NK-1 receptor type and a weak antagonist in the other two receptor types, while the dibenzyl and the di-tert-butyl analogues are potent antagonists in the NK-1 receptor type and weak antagonists in the other two receptor types. It is concluded that appropriate modification at the alpha-carboxamide and the side chain of the methionine residue of substance P may induce antagonism without using D-amino acids.

Effect of X-Asp (or Asn)-Phe-Nh2 (where X; H, Met) peptides on human platelet aggregation in vitro.

A series of di- and tripeptides containing aspartic or asparagine as N-terminal or intermediate amino acid were synthesized and tested for their effect on human platelet aggregation in vitro. It was found that only Met-Asp(or Asn)-Phe-NH2 inhibited platelet aggregation induced by collagen, ADP or adrenaline. Asn-Phe-NH2 and to a small extent Asp-Phe-NH2 presented strong aggregatory activity at low concentrations (at 0.5 mM or lower than this). All the other peptides tested, did not show any effect on platelet aggregation even at the concentration of 10 mM.

Solid-phase synthesis and spectroscopic studies of TRH analogues incorporating cis- and trans-4-hydroxy-L-proline.

An efficient solid-phase synthesis of the TRH analogue Glp-His(Nim-Trt)-Hyp-OH is described. Na-Fmoc protected amino acids and DCC/HOBt activation were employed. The bulky and mild-acid-sensitive 2-chlorotrityl resin, utilised as the solid support, completely suppressed dioxopiperazine formation. The tripeptide is a key intermediate in the synthesis of TRH analogues incorporating cis- and trans-4-hydroxy-L-proline. The tripeptide was converted, with inversion of configuration at C-4 of the Hyp residue, to Glp-His(Nim-Trt)-cHyp lactone in the presence of triphenylphosphine-diethyl azodicarboxylate (TPP-DEAD). One-pot MeOH-TPP-DEAD transesterification of the lactone, followed by Nim-detritylation, provided Glp-His-cHyp-OMe. This ester gave the corresponding amide and acid on ammonolysis and saponification, respectively. A high-field 1H NMR investigation of Glp-His-cHyp-OH and its diastereomer Glp-His-Hyp-OH, obtained by Nim-detritylation of the key tripeptide, showed that the configuration at C-4 of the prolyl residues is critical for the determination of the preferred three-dimensional structure of the molecules.

Synthesis of a potent agonist of substance P by modifying the methionyl and glutaminyl residues of the C-terminal hexapeptide of substance P. Structure-activity relationships.

Analogues of [Orn6]-SP6-11 have been synthesized in which the Met11 residue is replaced by glutamate gamma-alkylesters. These analogues were tested in three in vitro preparations representative of NK-1, NK-2, and NK-3 receptor types. Substitution of the SCH3 group of the Met11 side chain by a COOR (R = methyl, ethyl, n-propyl, n-butyl, cyclohexyl) group results in analogues which are full agonists in NK-1 and NK-2 preparations but show little agonist activity in the NK-3 preparation. When the SCH3 group is replaced by a t-butyl ester group and the resulting analogue is a full agonist in all the above preparations and more active than the parent hexapeptide and SP-OCH3 at NK-1 receptors. It is concluded that for activity at NK-1 receptors methionine can be replaced by gamma-t-butyl glutamate without loss of activity, whilst at NK-2 and NK-3 receptors the above substitution increases the activity of [Orn6]-SP6-11. Other gamma-alkyl esters of the glutamic acid reduce its biological activity.