Huma Rasheed

Co-activation of Gi and Gq proteins exerts synergistic effect on human platelet aggregation through activation of phospholipase C and Ca2+ signalling pathways.

Our previous studies have shown that subthreshold concentrations of two platelet agonists exert synergistic effects on platelet aggregation. Here we studied the mechanism of synergistic interaction of 5-hydroxytryptamine (5-HT) and epinephrine mediated platelet aggregation. We show that 5-HT had no or little effect on aggregation but it did potentiate the aggregation response of epinephrine. The synergistic interaction of 5-HT (1-5 µM) and epinephrine (0.5-2 µM) was inhibited by α2-adrenoceptor blocker (yohimbine; IC50= 0.4 µM), calcium channel blockers (verapamil and diltiazem with IC50 of 10 and 48 mM, respectively), PLC inhibitor (U73122; IC50=6 µM) and nitric oxide (NO) donor, SNAP (IC50=1.6 µM)). The data suggest that synergistic effects of platelet agonists are receptor-mediated and occur through multiple signalling pathways including the activation PLC/Ca2+ signalling cascades.

Cardiovascular and airway relaxant activities of peony root extract

Paeonia emodi (peony) is a well known plant used medicinally to treat hypertension, palpitations, and asthma. Despite its popularity, there are few reports in the scientific literature examining its use in such conditions. We prepared a 70% ethanolic extract of peony root (Pe.Cr) and applied it to segments of guinea pig atria and trachea and rat aorta suspended separately in tissue baths. Activity against arachidonic acid (AA)-induced platelet aggregation was measured in human platelet-rich plasma. Airway relaxant effect was evaluated against acetylcholine (ACh)-induced airway contraction in mouse lung slices loaded with fluo-4. Pe.Cr (0.3-10 mg/mL) showed an atropine-resistant negative inotropic effect in atria. In rat aorta, an endothelium-independent vasodilatory effect (0.3-10 mg/mL) was seen in phenylephrine- and high-K+-induced contractions. Pe.Cr (0.01-1 mg/mL) also inhibited AA-induced platelet aggregation. In isolated trachea, Pe.Cr (0.3-10 mg/mL) relaxed carbachol- and histamine-induced contractions independently of beta-adrenergic receptors. In mouse lung slices, Pe.Cr (0.3-1 mg/mL) inhibited ACh-induced airway narrowing and oscillations of intracellular Ca2+ in airway smooth muscle cells. The results showed cardiosuppressant, vasodilatory, antiplatelet, and tracheal and airway relaxant activities of peony, providing potential justification for its medicinal use in different hyperactive cardiovascular and respiratory disorders.

Involvement of thromboxane A2 and tyrosine kinase in the synergistic interaction of platelet activating factor and calcium ionophore A23187 in human platelet aggregation.

The present study was carried out to examine the mechanisms of the synergistic interaction of PAF and A23187 mediated platelet aggregation. We found that platelet aggregation mediated by subthreshold concentrations of PAF (5 nM) and A23187 (1 µM) was inhibited by PAF receptor blocker (WEB 2086, IC50=0.65 µM) and calcium channel blockers, diltiazem (IC50=13 µM) and verapamil (IC50=18 µM). Pretreatment of platelets with PAF and A23187 induced rise in intracellular calcium and this effect was also blocked by verapamil. While examining the role of the down stream signaling pathways, we found that platelet aggregation induced by the co-addition of PAF and A23187 was also inhibited by low concentrations of phospholipase C (PLC) inhibitor (U73122; IC50 = 10 µM), a cyclooxygenase inhibitor (indomethacin; IC50=0.2 µM) and inhibitor of TLCK, herbimycin A with IC50 value of 5 µM. The effect was also inhibited by a specific TXA2 receptor antagonist, SQ 29548 with very low IC50 value of 0.05 µM. However, the inhibitors of MAP kinase, PD98059 and protein kinase C, chelerythrine had no effect on PAF and A23187-induced platelet aggregation. These data suggest that the synergism between PAF and A23187 in platelet aggregation involves activation of thromboxane and tyrosine kinase pathways.

Molecular mechanisms involved in human platelet aggregation by synergistic interaction of platelet-activating factor and 5-hydroxytryptamine.

Our recent studies have shown that co-activation of Gq and Gi proteins by 5-hydroxytryptamine (5-HT) and adrenaline show synergism in human platelet aggregation. This study was conducted to examine the mechanism(s) of synergistic interaction of 5-HT and platelet activating factor (PAF) in human platelets. We show that PAF, but not 5-HT, increased platelet aggregation in a concentration-dependent manner. However, low concentrations of 5-HT (2 µM) potentiated platelet aggregation induced by subthreshold concentration of PAF (40 nM) indicating a synergistic interaction between the two agonists and this synergism was blocked by receptor antagonists to either 5-HT or PAF. 5-HT also potentiated the effect of PAF on thromboxane A2 (TXA2) formation and phosphorylation of extracellularly regulated mitogen-activated protein kinases (ERK1/2). The synergism of 5-HT and PAF in platelet aggregation was inhibited by calcium (Ca2+) channel blockers, verapamil and diltiazem, phospholipase C (PLC) inhibitor, U73122, cyclooxygenase (COX) inhibitor, indomethacin, and MEK inhibitor, PD98059. These data suggest that synergistic effect of 5-HT and PAF on human platelet aggregation involves activation of PLC/Ca2+, COX and MAP kinase pathways.

Second messengers in platelet aggregation evoked by serotonin and A23187, a calcium ionophore

We investigated the combined effect of 5-hydroxytryptamine (5-HT, serotonin) and calcium ionophore (A23187) on human platelet aggregation. Aggregation, monitored at 37 degrees C using a Dual-channel Lumi-aggregometer, was recorded for 5 min after challenge by a change in light transmission as a function of time. 5-HT (2-200 microM) alone did not cause platelet aggregation, but markedly potentiated A23187 (low dose) induced aggregation. Inhibitory concentration (IC50) values for a number of compounds were calculated as means +/- SEM from dose-response determinations. Synergism between 5-HT (2-5 microM) and A23187 (0.5-2 microM) was inhibited by 5-HT receptor blockers, methysergide (IC50 = 18 microM) and cyproheptadine (IC50 = 20 microM), and calcium channel blockers (verapamil and diltiazem, IC50 = 20 microM and 40 microM respectively). Interpretation of the effects of these blockers is complicated by their lack of specificity. Similarly, U73122, an inhibitor of phospholipase C (PLC), blocked the synergistic effect at an IC50 value of 9.2 microM. Wortmannin, a phosphatidylinositide 3-kinase (PI 3-K) inhibitor, also blocked the response (IC50 = 2.6 microM). However, neither genistein, a tyrosine-specific protein kinase inhibitor, nor chelerythrine, a protein kinase C inhibitor, affected aggregation at concentrations up to 10 microM. We conclude that the synergistic interaction between 5-HT and ionophore may be mediated by activation of PLC/Ca2+ and PI 3-kinase signalling pathways, but definitive proof will require other enzyme inhibitors with greater specificity.

Involvement of cyclooxygenase, phospholipase C and MAP kinase pathways in human platelet aggregation mediated by the synergistic interaction of adrenaline and PAF

This study was conducted to examine the mechanism(s) of synergistic interaction of adrenaline and platelet-activating factor (PAF) mediated human platelet aggregation. We found that platelet aggregation mediated by subthreshold concentrations of PAF (5-8 nM) plus adrenaline (0.5-2 μM) was inhibited by both α2-adrenoceptor blocker (yohimbine) and PAF receptor antagonist (WEB2086). While examining the role of the downstream signalling pathways, we found that this synergism was inhibited by calcium channel blockers, verapamil, and diltiazem. In addition, platelet aggregation by co-addition of adrenaline and PAF was also inhibited by very low concentrations of phospholipase C (PLC) inhibitor (U73122; IC50 = 0.2 μM), the MAP kinase inhibitor, PD98059 (IC50 = 3 μM) and cyclooxygenase (COX-1) inhibitors including indomethacin (IC50= 0.25 μM), flurbiprofen (IC50 = 0.7 μM) and piroxicam (IC50 = 7 μM). However, the COX-2 inhibitor, nimesulide, was also effective in inhibiting the aggregation. The inhibitors of tyrosine kinase (genistien) and phosphatidylinositol 3-kinase inhibitor (wortmannin) had no significant effects on platelet aggregation. These data suggest that the synergistic effect of adrenaline and PAF on human platelet aggregation is receptor-mediated and involves the activation of PLC/calcium, COX and MAP kinase signalling pathways.