PubMed Health⌕ Search

Biomedical subjects

P Guzzo

Publications and source records attributed to P Guzzo.

12 recordsLinked to original sources
PubMed1998

Decrease of platelet intracellular pH and adhesion by ticlopidine in patients with vascular disease.

BACKGROUND: Ticlopidine inhibits platelet aggregation by preventing the binding of fibrinogen to its platelet receptor. We examined whether this inhibition involved platelet transduction system such as Na+/H+ pump and platelet intracellular calcium. METHODS: Platelet adhesion in 13 patients with peripheral vascular disease treated with ticlopidine, 250 mg b.i.d for 30 days, was measured in culture microplates before and after therapy. The microplate wells were coated with human plasma, fibrinogen or collagen, and platelet adhesion was studied in the resting condition and after stimulation with 1 and 10 microM ADP. At the same time, platelet intracellular calcium and ADP-induced calcium increases were measured with the fluorescent indicator Fura 2. In addition, intracellular pH and thrombin-induced pH variations were measured with the fluorescent probe BCECF. RESULTS: Platelet adhesion to plasma and fibrinogen was significantly reduced (about 50%) after treatment with ticlopidine, while adhesion to collagen was not modified. Basal calcium and ADP-induced calcium increase were not significantly different before and after ticlopidine. Platelet basal intracellular pH was reduced (from 7.44+/-0.009 to 7.41+/-0.017, p<0.05), but agonist-induced alkalinisation was not significantly different. Early acidification, not dependent on Na+/H+ exchange, was also reduced (p<0.05). CONCLUSIONS: These data do not seem to support the hypothesis that ticlopidine-induced reduction of platelet adhesion depends on alteration of the mechanisms determining signal transduction, at least as far as basal and post-stimulation intracellular calcium is concerned. On the contrary, the possibility that ticlopidine inhibits the Na+/H+ antiport remains open to consideration.

Aged↗
PubMed1994

A colorimetric method for the measurement of platelet adhesion in microtiter plates.

A procedure for the determination of the adhesion of human platelets to protein-coated culture microwells was developed. The number of platelets was quantitated by measuring the activity of acid phosphatase, a platelet enzyme whose activity is stable independently of platelet stimulation and is not released. Isolated and washed platelets were incubated in 96-well microtiter plates with flat-bottom wells that had been precoated with various compounds, including collagen, fibrinogen, human plasma, and human albumin. At the end of incubation (optimal time: 40-60 min), nonadherent platelets were washed out, adherent platelets were solubilized with Triton X-100, and the acid phosphatase activity was measured by using the substrate p-nitrophenyl phosphate. The p-nitrophenol produced was measured with a microplate reader at 405 nm and the percentage of adhesion was calculated with reference to known platelet standards. ADP and thrombin stimulated platelet adhesion in a dose-dependent manner to fibrinogen and human plasma, but not to human albumin. Platelets adhered to collagen even in the absence of stimulants. Simultaneous evaluation of adhesion and aggregation demonstrated that with ADP as stimulus, but not with thrombin, the two platelet responses were dissociated. Microscopic examination of culture wells showed that most of platelets adhered as single cells and not as aggregates. The sensitivity of this method allowed the assay of platelet adhesion by using only 2.5 x 10(5) platelets/well.

Acid Phosphatase↗
PubMed1994

Early agonist-induced intracellular acidification is increased in platelets from patients with essential hypertension.

Enhanced Na+/H+ exchange has been reported to be increased in patients with essential hypertension. However, early variations of intracellular pH, although influenced by the antiport, are only partially dependent on the exchange. In this study, we measured the initial platelet pH response to agonists in a group of untreated subjects with essential hypertension (EH, n = 24) and in a group of age- and sex-matched normotensive control subjects (CS, n = 24). Intracellular pH was measured with the specific fluorescence indicator 2'7'bis(carboxyethyl)-5,6-carboxyfluorescein. Measurements were performed on platelets in the presence or absence of extracellular calcium, in a carbonate-free medium. Intracellular calcium was measured by the Fura 2 method. Mean pH values were slightly higher in the platelets of EH (7.469 +/- 0.017 U) compared with CS (7.423 +/- 0.012 U, P < .05), although there was a substantial overlap. When stimulated with physiologic agonists ADP and thrombin and with the calcium ionophore ionomycin, a biphasic response consisting of early acidification followed by alkalinization was observed, the second phase not being detectable with ADP. The initial acidification was greater in EH, particularly with ADP (EH, -0.046 +/- 0.002 U; CS, -0.036 +/- 0.002 U, P < .001) and with ionomycin (EH, -0.074 +/- 0.007 U; CS, -0.051 +/- 0.005 U, P < .05). This acidification proved in some way calcium dependent, as it was reduced in the absence of extracellular calcium (EGTA) in both EH and CS. After incubation with amiloride a further decrease in intracellular pH, more marked in EH, was observed. Alkalinization induced by thrombin was increased in EH (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1993

Homologous priming in chemotactic peptide-stimulated neutrophils.

The kinetics and dose-dependence of activation of human neutrophils exposed to sequential additions of the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (fMLP) have been investigated by multiwell microplate assays. Treatment of neutrophils with medium-high doses (from 10(-8) to 5 x 10(-7) M) of fMLP caused activation of superoxide anion (O2-) production, but prevented further activation by a subsequent addition of an optimal dose (from 10(-7) M to 5 x 10(-7) M) of fMLP. These findings represent an example of cell desensitization, or adaptation. However, neutrophils treated with low, sub-stimulatory doses (from 10(-10) to 5 x 10(-9) M) of the peptide and then treated with optimal doses of fMLP exhibited an O2- production that was two to three-fold higher than that induced by the same optimal doses on untreated cells. A similar phenomenon of homologous priming of the oxidative metabolism of neutrophil has not previously been described or characterized. Priming was maximal after about 30 min of incubation with fMLP, which differed from desensitization, which required only a few minutes. Homologous priming was not confined to O2- production, but was also observed with the release of the granule enzyme, lysozyme. Low doses of fMLP were also capable of triggering an increase of intracellular free Ca2+ and of fMLP membrane receptors, which are possible mechanisms responsible for priming.

Calcium↗
PubMed1993

Platelet intracellular pH and glucose metabolism in hypertensive patients.

Intracellular platelet pH was studied by the BCECF fluorescent pH-sensitive intracellular indicator method in 52 patients with essential hypertension and 42 control subjects of similar age and sex. In 40 hypertensive patients studied by oral glucose tolerance test (standard OGTT) 23 presented with pathological blood glucose and plasma insulin values. Intracellular pH was on average higher in hypertensives (7.415 +/- 0.114, mean +/- SD) than in controls (7.348 +/- 0.109, P < 0.05), although there was a considerable overlap in values. In the group of hypertensive patients, no difference was observed between those with normal and those with pathological OGTT. There was also no significant correlation between intracellular pH and blood glucose, plasma insulin after OGTT, plasma cholesterol and triglycerides, age, BP or body mass index. These data are consistent with an anomaly of intraplatelet pH, perhaps owing to alteration of Na+/H+ exchange in essential hypertension, but do not indicate that this is related to a condition of hyper-insulinaemia or insulin resistance.

Adolescent↗
PubMed1991

Effect of picotamide on platelet aggregation and on thromboxane A2 production in vivo.

Effects of picotamide (900 mg in 3 oral administrations for 7 days) on ex vivo and in vivo platelet TxA2 production and on platelet aggregation were evaluated in 8 patients with peripheral arteriopathy and in 8 normal subjects. Picotamide significantly reduced ADP-induced platelet aggregation, but had no effect on that induced by arachidonic acid or the thromboxane analogue U46619. Though ex vivo platelet TxA2 production (TxB2 concentration after arachidonic-acid-induced aggregation) was reduced from 946 +/- 141 (mean +/- SD) to 285 +/- 91 ng/ml in controls and from 1515 +/- 673 to 732 +/- 420 ng/ml in patients with arteriopathy, there was no effect on urinary excretion of 2,3-dinor-TXB2 (in vivo indicator of platelet TxA2 production), or on in vivo PGI2 production (urinary excretion of 6-keto-PGF1 alpha and 2,3-dinor-6-keto-PGF1 alpha). In the same subjects, single-dose aspirin reduced ex vivo TxB2 production by at least 98% and 2,3-dinor-TxB2 excretion from 116.7 +/- 61.4 to 32.6 +/- 17.0 ng/g creatinine in control subjects, and from 156.3 +/- 66.1 to 59.1 +/- 19.2 ng/g creatinine in patients with peripheral arteriopathy. Our data suggest that inhibition of platelet TxA2 production in vivo may not be picotamide's main mechanism of action.

Aged↗
PubMed1989

Increased platelet aggregation and intracellular calcium in hypertensive patients: effects of cyclo-oxygenase blockade.

Increased platelet aggregation induced by ADP and arachidonic acid was observed in 12 patients with essential hypertension compared with 12 control subjects, but not after pretreatment with acetylsalicylic acid. The increase in intracellular calcium induced by ADP was also greater in the hypertensive patients, and again this difference disappeared after cyclo-oxygenase blockade. Urinary excretion of 2,3-dinor-thromboxane B2, the main metabolite of platelet thromboxane A2, was slightly, but not significantly increased in the hypertensive patients. These data suggest that thromboxane system activity is increased in the platelets of hypertensive patients.

Adenosine Diphosphate↗
PubMed1989

Platelet intracellular free Ca2+ after incubation with plasma from hypertensive patients.

Variations in intracellular free calcium were measured in platelets from normal donors, incubated with plasma from hypertensive patients and from control subjects to test the hypothesis that a circulating factor might induce an increase in calcium concentration. Before incubation, plasma was heat-inactivated or ultrafiltered. Incubation both with heat-inactivated and ultrafiltered plasma failed to result in any significant modifications in intracellular free calcium. Similarly, no significant increase was observed after incubation with ouabain at concentrations ranging from 10(-7) to 10(-4) M. No significant differences were observed between platelets incubated with plasma from hypertensive as compared with control subjects. These findings are not consistent with the hypothesis that the increase in intracellular free calcium observed in platelets of hypertensive patients may be due to a plasma ouabain-like factor.

Adult↗