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R Landolfi

Publications and source records attributed to R Landolfi.

At least 55 records · Page 3Linked to original sources
PubMed1995

Increased thromboxane biosynthesis in essential thrombocythemia.

In order to investigate the in vivo thromboxane (TX) biosynthesis in essential thrombocythemia (ET), we measured the urinary excretion of the major enzymatic metabolites of TXB2, 11-dehydro-TXB2 and 2,3-dinor-TXB2 in 40 ET patients as well as in 26 gender- and age-matched controls. Urinary 11-dehydro-TXB2 was significantly higher (p < 0.001) in thrombocythemic patients (4,063 +/- 3,408 pg/mg creatinine; mean +/- SD) than in controls (504 +/- 267 pg/mg creatinine), with 34 patients (85%) having 11-dehydro-TXB2 > 2 SD above the control mean. Patients with platelet number < 1,000 x 10 (9)/1 (n = 25) had significantly higher (p < 0.05) 11-dehydro-TXB2 excretion than patients with higher platelet count (4,765 +/- 3,870 pg/mg creatinine, n = 25, versus 2,279 +/- 1,874 pg/mg creatinine, n = 15). Average excretion values of patients aging > 55 was significantly higher than in the younger group (4,784 +/- 3,948 pg/mg creatinine, n = 24, versus 2,405 +/- 1,885 pg/mg creatinine, n = 16, p < 0.05). Low-dose aspirin (50 mg/d for 7 days) largely suppressed 11-dehydro-TXB2 excretion in 7 thrombocythemic patients, thus suggesting that platelets were the main source of enhanced TXA2 biosynthesis. The platelet count-corrected 11-dehydro-TXB2 excretion was positively correlated with age (r = 0.325, n = 40, p < 0.05) and inversely correlated with platelet count (r = -0.381, n = 40, p < 0.05). In addition 11 out of 13 (85%) patients having increased count-corrected 11-dehydro-TXB2 excretion, belonged to the subgroup with age > 55 and platelet count < 1,000 x 10(9)/1. We conclude that in essential thrombocythemia: 1) enhanced 11-dehydro-TXB2 excretion largely reflects platelet activation in vivo; 2) age as well as platelet count appear to influence the determinants of platelet activation in this setting, and can help in assessing the thrombotic risk and therapeutic strategy in individual patients.

Adult↗
PubMed1994

Thermodynamics of substrates and reversible inhibitors binding to the active site cleft of human alpha-thrombin.

The study of the temperature effect on the binding to the active site of human alpha-thrombin for ten different ligands, i.e. nine peptide substrates and the tight binding inhibitor N alpha -(naphthalene-sulphonyl-glycyl)-4-amidino-DL-phenyl-alanine-piperidine (alpha-NAPAP), showed that the enthalpy is constant over the temperature range spanning from 10 to 40 degrees C. It was found that the values of the binding enthalpy are linearly correlated to those of entropy, and that this correlation arises from a real phenomenon of chemical compensation. On the other hand, no compensatory chemical effect was found for the process of thrombin acylation. Additional experiments showed that binding to thrombin of two competitive thrombin inhibitors, i.e. proflavin and p-aminobenzamidine, is characterized by a change in the standard heat capacity change (delta Cp), approximately equal to -1 kcal/mol K. By analogy with model compound transfer studies and protein folding investigations, it is proposed that a burial of a large surface area of non-polar residues, roughly equal to 3000 A2, brings about the observed heat capacity change. Altogether, the observed phenomena of the chemical compensation and heat capacity change, although qualitatively different, are interpreted as expressions of the same property of the enzyme, i.e. the capacity to undergo conformational transitions upon ligation of the catalytic domain. These structural transitions are strictly ligand-linked and could play a central role for setting the rules which regulate the specificity of substrates and inhibitors binding to the catalytic groove of human alpha-thrombin.

Amino Acid Sequence↗
PubMed1994

Effects of protons on the thrombin-fibrinogen interaction.

Amidase activity of human alpha-thrombin toward the synthetic substrate Tosyl-Gly-Pro-Arg-NH-Ph and fibrinogen has been studied as a function of pH at t = 25 degrees C, under steady-state conditions. A viscosity-perturbation method allowed us to compute the equilibrium binding constant along with the rate constants for the acylation and deacylation reactions. The ionization constants for the groups affecting binding and hydrolysis of the synthetic substrate were measured by application of linkage thermodynamics principles. The binding of the synthetic substrate is controlled by two ionizable groups having pKa values of 7.5 and 8.7 in the free enzyme and 6.3 and 9.8 in the Michaelis adduct. These two groups were found to control the acylation process as well. Thrombin-fibrinogen interaction has been studied by measurements of steady-state hydrolysis of the synthetic substrate Phe-pipecolyl-Arg-NH-Ph in the presence of fibrinogen, used as a competitive inhibitor. This method allowed us to measure the Km of thrombin-fibrinogen interaction. The values of Km computed at different solution viscosities were used in order to calculate the equilibrium dissociation constant and both k2/k3 and k2/k-1 ratios. The same residues that were found to control binding of Tosyl-Gly-Pro-Arg-NH-Ph to alpha-thrombin, do modulate binding of fibrinogen as well. These residues shift their pKa values upon the formation of the Michaelis adduct from 7.5 to 5.7 and from 8.7 to 9.7, respectively. Furthermore the ratio kcat/Km as a function of pH has been obtained by HPLC measurements of fibrinopeptides release. The kcat/Km values along with the ratio k2/k-1, derived from viscometric experiments, allowed us to calculate the forward-rate constant, k+1, for the thrombin-fibrinogen interaction. The association process was found to depend on pH, namely in the alkaline region. The results for Tosyl-Gly-Pro-Arg-NH-Ph and fibrinogen are compared and discussed on the basis of the structural elements which differentiate the interactions of these substrates with human alpha-thrombin.

Amidohydrolases↗
PubMed1994

Effects of unfractionated and low molecular weight heparins on platelet thromboxane biosynthesis "in vivo".

Several "in vitro" and "in vivo" studies indicate that heparin administration may affect platelet function. In this study we investigated the effects of prophylactic heparin on thromboxane (Tx)A2 biosynthesis "in vivo", as assessed by the urinary excretion of major enzymatic metabolites 11-dehydro-TxB2 and 2,3-dinor-TxB2. Twenty-four patients who were candidates for cholecystectomy because of uncomplicated lithiasis were randomly assigned to receive placebo, unfractionated heparin, low molecular weight heparin or unfractionated heparin plus 100 mg aspirin. Measurements of daily excretion of Tx metabolites were performed before and during the treatment. In the groups assigned to placebo and to low molecular weight heparin there was no statistically significant modification of Tx metabolite excretion while patients receiving unfractionated heparin had a significant increase of both metabolites (11-dehydro-TxB2: 3844 +/- 1388 vs 2092 +/- 777, p < 0.05; 2,3-dinor-TxB2: 2737 +/- 808 vs 1535 +/- 771 pg/mg creatinine, p < 0.05). In patients randomized to receive low-dose aspirin plus unfractionated heparin the excretion of the two metabolites was largely suppressed thus suggesting that platelets are the primary source of enhanced thromboxane biosynthesis associated with heparin administration. These data indicate that unfractionated heparin causes platelet activation "in vivo" and suggest that the use of low molecular weight heparin may avoid this complication.

Adult↗
PubMed1993

The linkage between binding of the C-terminal domain of hirudin and amidase activity in human alpha-thrombin.

A method derived from the analysis of viscosity effects on the hydrolysis of the amide substrates D-phenylalanylpipecolyl-arginine-p-nitroaniline, tosylglycylprolylarginine-p-nitroanaline and cyclohexylglycylalanylarginine-p-nitroalanine by human alpha-thrombin was developed to dissect the Michaelis-Menten parameters Km and kcat into the individual rate constants of the binding, acylation and deacylation reactions. This method was used to analyse the effect of the C-terminal hirudin (residues 54-65) [hir-(54-65)] domain on the binding and hydrolysis of the three substrates. The results showed that the C-terminal hir-(54-65) fragment affects only the acylation rate, which is increased approx. 1.2-fold for all the substrates. Analysis of the dependence of acylation rate constants on hirudin-fragment concentration, allowed the determination of the equilibrium binding constant of C-terminal hir-(54-65) (Kd approximately 0.7 microM). In addition this peptide was found to competitively inhibit thrombin-fibrinogen interaction with a Ki which is in excellent agreement with the equilibrium constant derived from viscosity experiments. These results demonstrate that binding of hir-(54-65) to the fibrinogen recognition site of thrombin does not affect the equilibrium binding of amide substrates, but induces only a small increase in the acylation rate of the hydrolysis reaction.

Amidohydrolases↗
PubMed1993

Fechtner syndrome: report of a third family and literature review.

We observed macrothrombocytopenia with leucocyte inclusions in 10 out of 14 members of a four-generation family. Morphological features of leucocyte inclusions and the presence of Alport-related symptoms supported the diagnosis of Fechtner syndrome. Compared to the two previously reported Fechtner families, our kindred showed reduced expression of Alport manifestations. These, in members aged less than 50, were represented by clinically silent ocular abnormalities. Due to the frequent non-recognition of macrothrombocytopenia. Fechtner variants with low penetrance might be difficult to diagnose. In addition, Sebastian syndrome, recently distinguished from Fechtner disease in lacking Alport manifestations, might be one of these variants.

Adolescent↗
PubMed1992

Increased thromboxane biosynthesis in patients with polycythemia vera: evidence for aspirin-suppressible platelet activation in vivo.

Increased thromboxane (TX) production and modified aspirin sensitivity has been detected in vitro in platelets isolated from patients with polycythemia vera. To verify the relevance of these capacity-related measurements to the actual rate of TXA2 biosynthesis in vivo and its suppression by oral aspirin, we have investigated the urinary excretion of major enzymatic metabolites of TXB2 in 17 patients with polycythemia vera and 23 gender- and age-matched controls. Urinary 11-dehydro-TXB2 and 2,3-dinor-TXB2 were measured by previously validated radioimmunoassays. In addition, urinary immunoreactive leukotriene (LT) E4 was measured to explore the 5-lipoxygenase pathway of arachidonate metabolism. Polycythemic patients had significantly (P < .001) higher excretion rates of both 11-dehydro-TXB2 (1,033 +/- 1,050 v 117 +/- 45 pmol/mmol creatinine; mean +/- SD) and 2,3-dinor-TXB2 (725 +/- 676 v 82 +/- 43 pmol/mmol creatinine) than controls. In contrast, urinary LTE4 was not significantly different. Enhanced metabolite excretion did not correlate with the platelet count or with the hematocrit value, and was not related to the current treatment or to a clinical history of thrombotic complications. Platelet TX receptor studies did not show any significant changes in the binding characteristics of two different ligands. A platelet-selective regimen of aspirin therapy (50 mg/d for 7 to 14 days) was associated with greater than 80% suppression in metabolite excretion in nine patients. These results are consistent with abnormal stimuli operating in polycythemia vera to induce a selective enhancement in the platelet biosynthesis of TXA2 without changes in receptor binding. This in vivo abnormality in platelet biochemistry can be largely suppressed by low doses of aspirin.

Adult↗
PubMed1991

Effect of fibrinogen concentration on the velocity of platelet aggregation.

The relationship between fibrinogen binding to its receptor and platelet aggregation has been investigated by comparing 125I-fibrinogen binding and aggregation velocities of gel-filtered platelets in the presence of adenosine diphosphate (ADP). Aggregometric responses at various fibrinogen concentrations are found to be bell-shaped and show a maximum at fibrinogen concentrations (Fmax) similar to the 125I-fibrinogen hemisaturating doses. At higher and lower fibrinogen concentrations, platelet aggregation velocities decrease in a parallel manner. Lowering ADP concentration increases Fmax, in agreement with the modulatory effect of ADP on fibrinogen binding to platelets. Variations of fibrinogen in the range of physiopathologic plasma concentrations affect platelet aggregation induced by any ADP dose. These results clarify the relationship between the fibrinogen binding process and aggregation and demonstrate that plasma fibrinogen concentration has a major influence on the velocity of platelet aggregation.

Adenosine Diphosphate↗
PubMed1990

The linkage between adenosine nucleotide binding and amidase activity in human alpha-thrombin.

The amidase activity of human alpha-thrombin has been studied in the presence of the adenosine nucleotides AMP, ADP and ATP. At low concentrations, adenosine nucleotides increase thrombin activity up to 30%, while at high concentrations (greater than 5 mM) inhibition takes place up to 20%. Inhibition is progressively reduced by increasing substrate concentration. A simple, phenomenological description of the linkage between adenosine nucleotide binding and amidase activity of human alpha-thrombin is proposed and the free energy changes for the underlying reactions involved in the linkage scheme are resolved by global analysis of the experimental data. The linkage scheme assumes that thrombin activation is determined by a conformational transition due to binding of adenosine nucleotides to a regulatory site. Inhibition, on the other hand, would be a consequence of competitive binding to the catalytic site.

Adenosine Diphosphate↗
PubMed1989

Inhibition of fibrinogen binding to human platelets by blockage of Na+/H+ exchange.

Binding of ADP to platelets enhances the binding of fibrinogen to Gp IIb-IIIa, the specific platelet receptor for adhesive proteins. The linkage between ADP and fibrinogen binding is indirect since ADP does not bind to the same receptor as fibrinogen. We have recently proposed that a third component, once affected by ADP binding, induces a conformational transition of the fibrinogen receptor from the low to the high affinity state, which is responsible for platelet aggregation [De Cristofaro, R., Landolfi, R., Castagnola, M., De Candia, E., Di Cera, E., & Wyman, J. (1988) Proc. Natl. Acad. Sci. USA 85, 8473-8476]. In the present study we provide evidence that this component should be identified with the platelet Na+/H+ antiport. Inhibition of the antiport by pharmacological agents such as amiloride, or else by decreasing extracellular Na+, results in a marked decrease of fibrinogen binding to platelets.

Adenosine Diphosphate↗
PubMed1989

Carbon monoxide and oxygen binding to human hemoglobin F0.

Differential binding curve measurements of carbon monoxide and oxygen binding to human hemoglobin F0 under near-physiological conditions (0.1 M NaCl and 15 mM 2,3-diphosphoglyceric acid, pH 7.35, and 37 degrees C) have allowed a detailed description of the binding and linkage between these two gaseous ligands. Comparison with human hemoglobin A0 under identical solution conditions shows that fetal hemoglobin F0 binds oxygen and carbon monoxide with higher affinity than human hemoglobin A0, but with the same cooperativity. Construction of the partition coefficient surface for carbon monoxide and oxygen binding reveals a failure of Haldane's laws for both hemoglobins. Linkage graphs are used to explore the phenomenological properties of the system. The graphs provide a quantitative description of the mechanism of carbon monoxide toxicity on oxygen transport by hemoglobin in vivo and demonstrate striking similarities between the functional properties of fetal and adult hemoglobins.

Carbon Monoxide↗