Amifostine, a reactive oxigen species scavenger with radiation- and chemo-protective properties, inhibits in vitro platelet activation induced by ADP, collagen or PAF.
OBJECTIVE
Reactive oxygen species (ROS) generation has been suggested to represent an important regulatory mechanism of platelet reactivity in both physiologic and pathologic conditions; consistent with this hypothesis is the observation that free-radical scavengers may inhibit platelet activation, thus contributing to the regulation of their reactivity. The purpose of the present study is to study the in vitro effects of amifostine (WR-2721, ethyol ), a selective cytoprotective agent for normal tissues against the toxicities of chemotherapy and radiation, on platelet activation induced by the physiologic agonists ADP, collagen and PAF.


METHODS
The effect of amifostine, added to the experimental system at final concentrations ranging from 10(-7) M to 10(-5) M, was studied on platelet aggregation induced by the following physiologic agonists at the given concentrations: ADP (1 microM), collagen (2 microg/mL), and PAF (0.1 microg/mL). Platelet aggregation was investigated using a platelet ionized calcium aggregometer and was expressed as the percentage change in light transmission. Furthermore, thromboxane B((2)) (TxB((2))) levels and nitric oxide (NO) production were determined by radioimmunoassay and by evaluating the total nitrite/nitrate concentration using a commercially available colorimetric kit, respectively, both in the control system and after the addition of amifostine.


RESULTS
Amifostine inhibited both platelet aggregation and TxB((2)) production induced by ADP, collagen and PAF, in a dose-dependent manner. Amifostine proved to be an effective inhibitor of platelet function and the effect was more pronounced if platelets were stimulated with ADP, intermediate when collagen was the chosen agonist, and less evident, though present, when PAF was used. Platelets stimulated with ADP, collagen or PAF produced significant amounts of NO over the baseline. When amifostine was added at a final concentration of 5 microM, it significantly increased ADP, collagen and PAF-induced NO production, which suggests that NO release by activated platelets was involved in the inhibitory effect of amifostine.


CONCLUSIONS
Amifostine proved to be an effective inhibitor of platelet activation induced in vitro by physiologic inducers. This previously unrecognized effect was more evident with the weak agonist ADP and was related to reduced NO consumption by free radicals generated during platelet activation. Amifostine proved to be not only a powerful cytoprotectant, but, more generally, a therapeutic agent endowed with several relevant, though largely unknown, biological effects. Finally, our data once again support the concept that oxidative balance is of crucial importance in regulating platelet reactivity in both health and disease.
Porta
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C
Medicina Interna ed Oncologia Medica, Università degli Studi di Pavia, IRCCS Policlinico San Matteo, 27100 Pavia, Italy. c.porta@smatteo.pv.it
Maiolo
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A
Tua
A
A
Grignani
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G
D000244:Adenosine Diphosphate / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D000328:Adult; D004999:Amifostine / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D003094:Collagen / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D016166:Free Radical Scavengers / Q000494:pharmacology*; D006801:Humans; D009569:Nitric Oxide / Q000096:biosynthesis; D018384:Oxidative Stress; D010972:Platelet Activating Factor / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D011837:Radiation-Protective Agents / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013929:Thromboxane B2 / Q000096:biosynthesis
D000975:Antioxidants; D016166:Free Radical Scavengers; D010972:Platelet Activating Factor; D010975:Platelet Aggregation Inhibitors; D011837:Radiation-Protective Agents; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; D013929:Thromboxane B2; D000244:Adenosine Diphosphate; D003094:Collagen; D004999:Amifostine