3,4-Methylenedioxy-beta-nitrostyrene及其衍生物抗血小板作用機轉的探討

Abstract

In the search of new anti- platelet agents, several natural components and chemical synthetic compounds are tested for their anti-platelet activities. Among them, we found a small chemical compound, 3,4-methylenedioxy-??-nitrostyrene (MNS) showed potent and broad-spectrum inhibitory effects on platelet aggregation caused by various inducers. Moreover, addition of MNS to human platelets that had been aggregated by ADP caused a rapid disaggregation. We demonstrated that the anti-aggregatory activity of MNS is due to inhibition of GPIIb/IIIa activation by measuring the binding amount of PAC-1 in platelets. To determine whether MNS directly interferes with fibrinogen binding to platelet GPIIb/IIIa, the effect of MNS on fibrinogen- induced aggregation in ADP-treated fixed platelets was tested. The results showed that MNS is not a direct antagonist of GPIIb/IIIa, since MNS did not affect fibrinogen binding to fixed ADP-stimulated platelets. To investigate how does MNS inhibit GPIIb/IIIa activation, we found that MNS potently inhibited the activity of tyrosine kinases (Src and Syk), prevented protein tyrosine phosphorylation and cytoskeletal association of GPIIb/IIIa and talin, but had no direct effects on protein kinase C, Ca2+ mobilization, Ca2+-dependent enzymes (myosin light chain kinase and calpain) and arachidonic acid metabolism, and did not affect the cellular levels of cyclic nucleotides. Therefore, MNS represents a new class of tyrosine kinase inhibitor that potently prevents GPIIb/IIIa activation and platelet aggregation caused by various stimulators without directly affecting other signaling pathways required for platelet activation. In order to further examine the structural determinants required for the actions of MNS and to develop more potent tyrosine kinase inhibitors and antiplatelet agents, a new series of β-nitrostyrene derivatives have been synthesized and pharmacologically characterized. Compounds 3、4、5、6、7、8、9、10 inhibited thrombin- or collagen- induced human platelet aggregation, ATP secretion, GPIIb/IIIa activation and protein tyrosine phosphorylation. In recombinant enzyme assay, some β-nitrostyrene derivatives also demonstrated potent inhibition of Src and/or Syk kinase activity. Furthermore, there was a good correlation between the inhibitory potency of these compounds on tyrosine kinases and on platelet activation/aggregation. Among them, a benzoyl ester derivative (compound 10) possess up to 8-fold greater potency than MNS and over two orders of magnitude greater potency than genistein or tyrphostin A47 in inhibiting platelet responses to thrombin. Our data suggest that β-nitrostyrenes may represent a new class of tyrosine kinase inhibitors with potent antiplatelet activity. In conclusion, MNS and β-nitrostyrenes derivatives inhibit GPIIb/IIIa functions in a manner different from GPIIb/IIIa antagonists and cyclic nucleotide-elevating agents. Therefore, the study on the antiplatelet effects of MNS and β-nitrostyrenes derivatives may provide a new strategy for treatment of artery thrombosis.