Giuseppe Andrioli

The F2-Isoprostane 8-epiprostaglandin F2α increases platelet adhesion and reduces the antiadhesive and antiaggregatory effects of NO

F2-isoprostanes are prostaglandin (PG) isomers produced in vivo through free radical-catalyzed peroxidation of arachidonic acid, which may affect platelet function. The current study investigated the effects of 8-epiprostaglandin F2alpha (8-epi-PGF2alpha) on critical events of platelet activation. A dose-dependent increase in platelet adhesion to fibrinogen- and plasma-coated microwells by 8-epi-PGF2alpha (1 to 1000 nmol/L) was observed when resting platelets (plasma from 1.3+/-0.2% to 5.5+/-0.2%, EC50 of 48 nmol/L; fibrinogen from 3.3+/-0.3% to 6.4+/-0.2%, EC50 of 35 nmol/L; mean+/-SEM, n=8, P<0.001) and thrombin-stimulated human platelets were used. The expression of the adhesion molecule glycoprotein IIb/IIIa was increased by 10 to 1000 nmol/L 8-epi-PGF2alpha in resting platelets (from 64.8+/-2.1% to 83.9+/-1.3%; n=5, P<0.01) and in stimulated platelets. The secretion of the glycoprotein GMP-140 increased only in the presence of both thrombin and 10 to 1000 nmol/L 8-epi-PGF2alpha (from 48.5+/-3.1% to 63.1+/-2.0%, P<0.05). The antiaggregatory effects of both the NO donor NOR-3 (basal, 21.4+/-4.6%; with 8-epi-PGF2alpha, 30.8+/-6.9%; n=14, P<0.05) and endothelial cells that release NO (basal, 18.5+/-4.6%; with 8-epi-PGF2alpha, 30.7+/-5.3%; n=15, P<0.001) were also reduced. All of these effects were prevented by the thromboxane receptor antagonist GR32191 but not affected by acetylsalicylic acid. An increase in free intracellular calcium concentration, measured with the use of fura 2, was observed with 8-epi-PGF2alpha. In conclusion, F2-isoprostanes may participate in oxidative injury by inducing platelet activation and by reducing the antiplatelet activity of NO: increased platelet adhesiveness and expression of the fibrinogen receptor are induced by nanomolar amounts of 8-epi-PG-F2alpha. Platelet secretion and aggregation can also be induced in the presence of platelet agonists.

Defective platelet response to arachidonic acid and thromboxane A2 in subjects with PlA2 polymorphism of β3 subunit (glycoprotein IIIa)

The membrane complex αIIbβ3 is the major receptor for fibrinogen and is involved in platelet adhesion and aggregation. Evidence has been presented that the PlA2 allele of the β3 PlA1/A2 gene polymorphism might be an independent risk factor for coronary thrombosis, but the matter is still controversial. We investigated the relationship between this polymorphism and possible alterations of platelet functions in vitro. The platelet adhesion to fibrinogen‐coated microplate wells and the aggregation induced by several different agonists were tested in 63 healthy volunteers, among them, 49 subjects with PlA1/A1 polymorphism, 12 subjects with PlA1/A2 polymorphism and two subjects with PlA2/A2 polymorphism. Subjects with PlA1/A2 polymorphism or with PlA2/A2 polymorphism showed significantly lower platelet responses as compared with PlA1/A1 subjects when either arachidonic acid or the thromboxane A2 analogue, U46619, were used as agonists. In resting condition and after thrombin or ADP stimulation, platelet function was normal in all the subjects. An increased sensitivity to the anti‐aggregatory effect of acetylsalicylic acid was observed in platelets from subjects with the PlA2 allele. Finally, using a flow‐cytometric evaluation and determining the β‐thromboglobulin plasma levels, we did not find any evidence of a PlA2 platelet hyper‐reactivity ex vivo. Our findings are not consistent with the hypothesis that the purported increase of cardiovascular risk in these subjects may be as a result of platelet hyperactivation. On the contrary, the PlA2 allele is associated with a platelet functional deficiency, specifically linked to the activation of the fibrinogen receptor by thromboxane A2.

Study of platelet adhesion in patients with uncomplicated hypertension.

Objective To evaluate platelet function in patients with essential hypertension by sensitive methods investigating platelet adhesion and expression of some platelet glycoproteins (GP), namely GPIIb/llla (CD41/α2β3) and GMP- 140 (CD62/P-selectin/PADGEM). Other markers of platelet (β-thromboglobulin) and endothelium activation (von Willebrand factor) were also measured. Methods We studied 21 uncomplicated essential hypertensive patients and 20 healthy normotensive control subjects, non-smokers, matched for age and sex. Resting and stimulated platelet adhesion was performed with a colorimetric method using the activity of platelet acid phosphatase for the determination of the number of platelets adhering to human plasma- or fibrinogen-coated microwells. Platelet activation was characterized by flow cytometric measurement of GPIIb/llla and GMP-140 in whole blood and washed platelets suspensions, with antihuman fluorescent monoclonal antibodies. Results Thrombin-stimulated platelet adhesion to human plasma-coated microwells was significantly higher in hypertensive patients than in control subjects (0.05 U/ml thrombin: 13.4 ± 1.0 versus 7.7 ± 0.6% adhesion; 0.1 U/ml thrombin: 19.4 ±2.3 versus 12.6 ± 1.8%; means ± SEM), whereas platelet adhesion to fibrinogen-coated wells did not differ in the two groups. Flow-cytometry analysis of whole blood demonstrated a significantly increased expression of GMP-140 in hypertensive patients compared with normal subjects (percentage of CD62+ platelets: 7.3 ± 1.2 versus 3.7 ± 1; means ± SEM), whereas the expression of GPIIb/llla did not differ in the two groups (percentage of CD41a+ platelets: 72.5 ± 4.5 versus 70.4 ± 3.9). Moreover, flow cytometry showed an increased size of platelets in hypertensive patients compared with that in control subjects (forwards scattering: 46.5 ± 1.5 versus 38.9 ±1.1; means ± SEM). Flow-cytometric evaluation of washed platelet suspensions showed no statistically significant differences between the expression of GMP-140 and GPIIb/llla in the two groups. β-Thromboglobulin plasma levels were higher in hypertensive patients than they were in normal subjects (36.3 ± 2.0 versus 28.2 ± 1.3 ng/ml; means ± SEM). Von Willebrand factor plasma levels were not significantly different in the two groups (101.2 ± 10.3 versus 86.3 ± 5.6 U/dl). Conclusions These findings provide further evidence that there is a significant, albeit weak, platelet activation in hypertensive patients compared with normal subjects.

Vascular adhesion molecule-1 and markers of platelet function before and after a treatment with iloprost or a supervised physical exercise program in patients with peripheral arterial disease

Platelet function and levels of vascular adhesion molecule-1 (VCAM-1) were investigated in 24 patients with peripheral arterial disease at Fontaine stage II undergoing a 2 weeks treatment with iloprost (0.5-2 ng/kg/h i.v. infused, 6 h/day) or a 2 weeks supervised physical training, randomly assigned. Patients were studied before (T0) and after (T14) treatments and 10 days later (T24). The adhesion of washed platelets to fibrinogen coated microwells was reduced after treatment both with iloprost (1.9+/-0.4 vs 6.8+/-0.7%; T24 vs T0; M+/-SEM; p<0.05) and physical exercise (3.0+/-1.0 vs 6.7+/-0.7; p<0.05) while adhesion to human plasma coated microwells was reduced only after treatment with iloprost (1.9+/-0.8 vs 5.8+/-0.9; p<0.05). The expression of fibrinogen receptor (glycoprotein IIb/IIIa) on platelets, measured by flow-cytometry was also reduced after iloprost treatment (17.1+/-1.5 vs 31.8+/-4.8 AU; p<0.05) and physical exercise (14.6+/-1.5 vs 34.0+/-3.3; p<0.05). Theurinaryexcretion of platelet thromboxane A2 metabolite 2,3-dinor-thromboxane B2 decreased only in patients treated with iloprost (154.7+/-97.9 vs 256.2+/-106.4 pg mg creatinine(-1); p<0.05). Similarly plasma VCAM-1 was lower in patients who were treated with iloprost (827.7+/-77.4 vs 999.0+/-83.8 ng ml(-1); p<0.05). In conclusion, both iloprost and physical exercise seem to act on reversible phenomena such as the expression of adhesion molecules or ex vivo adhesion, whereas only iloprost reduces thromboxane A2 biosynthesis in vivo. This anti-platelet activity seems to be extended in time and to be associated with an improvement in vascular function.

Study on Paradoxical Effects of NSAIDs on Platelet Activation

We recently described a stimulatory effect of high doses (>100 μmol/L) diclofenac on platelet adhesion. In this study we extend our research to the possible biochemical mechanisms of the observed effects, to other non steroidal anti-inflammatory drugs (NSAIDs) (flurbiprofen, indomethacin, acetylsalicylic acid, ibuprofen, nitrofenac and nitroflurbiprofen) and to the effect of high doses diclofenac and flurbiprofen on platelet aggreation. We observed that high doses of diclofenac and of flurbiprofen, but not of the other tested NSAIDs, increased platelet adhesion at doses ranging from 100 to 500 μmol/L, an effect completely removed by the 12-lipoxygenase-inhibitor nordihydroguaiaretic acid. Moreover, they had no pro-aggregating effect, inhibiting platelet aggregation induced by 10 μmol/L arachidonic acid and dose-dependently increasing the [Ca2+]i. Finally, whereas no basal nitric oxide release by washed platelets was detected, when platelets were incubated by 500 μmol/L diclofenac or flurbiprofen, the production of nitric oxide, as measured by amounts of nitrite released, was 4.4 ± 0.5 and 3.8 ± 0.4 pmol/5 × 108 platelets/min, respectively. Our data indicate that high doses diclofenac and flurbiprofen are promoters of the early phases of platelet activation, probably through the 12-lipoxygenase pathway.

Dose-Dependence of the Various Functional Responses of Neutrophils to Formylpeptides

The up- and down-regulation of the response to a specific stimulatory compound, according to its doses and to the sensitivity of the cells, is a widespread process in biology, immunology and endocrinology. Leukocytes are particularly suitable models for studying these changes of biological response as these cells may be easily isolated from blood and from inflammatory exudates and appear to be regulated by a large variety of mediators both in vitro and in vivo (disease states).

A computer model of the ‘five elements’ theory of traditional Chinese medicine

Summary In this article, a model is presented of a network whose structure was inspired by the ‘five elements law’ of Chinese medicine. Computer simulations illustrate the dynamic behavior of this system, that can be set in different attractors of Boolean type and can be differentially modified by delays and perturbations. We suggest that this model may contribute to the understanding of the ‘logic’ of the regulation of biological systems by means of small and carefully selected perturbations, a major line of thought of complementary therapies.