In this study, we used A. purpurea leaves collected in Maracay, Venezuela and human platelets. We found that the A. purpurea EE inhibited the aggregation of human platelets induced by ADP, collagen and thrombin; and that these anti-aggregating activities were retained by the alkaloid fraction. Similar results were reported by Chang et al. [6, 7] for A. purpurea extract (leaves collected in Chia-Yi, Taiwan) using rabbit platelets. Those authors identified nine  and five  alkaloids with anti-platelet actions.
In this work, we identified two of the five known alkaloids isolated by Chang et al. : norpurpureine and purpureine (thalicsimidine). Norpurpureine was found to be the major anti-platelet compound of A. purpurea leaves, showing activity against ADP, collagen and thrombin in human platelets. Purpureine did not inhibit human platelet activation.
Chang et al.  reported that 100 μg/ml (269.2 μM) norpurpureine completely inhibited the actions of arachidonic acid, collagen and platelet-activating factor (PAF) but only partially inhibited (30%) the action of thrombin. They also found that 100 μg/ml (259.4 μM) purpureine had variable inhibitory potency against arachidonic acid (85%), collagen (63%) and PAF (40%) and no effect against thrombin in rabbit platelets. Thus far, it seems that norpurpureine (at 250 μM) is a nonselective inhibitor of human and rabbit platelets with better a platelet anti-aggregating profile than purpureine. It is noteworthy that purpureine inhibits rabbit platelets with greater effect than human platelets, suggesting that significant differences may exist between rabbit and human platelets at the level of its unknown molecular target.
The anti-platelet effects of norpurpureine and purpureine analyzed in terms of structure–activity relationships indicate the lack of a methyl group at the nitrogen in norpurpureine as the key feature by which these aporphine alkaloids interact with their molecular targets. This agrees with Chia et al. , who found that a small change in the structure of different sub-types of isoquinoline alkaloids caused significant changes in anti-platelet aggregation activity. On the other hand, by sharing the majority of their molecular structure, these alkaloids should also share most of their non-specific interactions, which makes the anti-platelet actions of norpurpureine less likely to be mediated by the induction of non-specific interactions in membrane fluidity, as suggested for several bioactive natural products .
As an anti-platelet agent, norpurpureine proved pharmacologically active from 20 to 220 μM, with a potency of 80 μM, and an IC50 value lower than that of aspirin (140 μM) and ticlopide (510 μM) obtained under similar in vitro conditions . Importantly, norpurpureine was pharmacologically effective (220 μM) in all 30 human platelet samples tested, which is evidence of its effectiveness and reveals that, at least 10 min prior to and during the 10 min of the aggregation response, it does not seem to be affected by the variability in oxidation and lipid state of these 30 PRP samples. Moreover, norpurpureine also gradually inhibited platelet granule secretion and adhesion of activated platelets to adhesive proteins like fibrinogen, suggesting that beyond hemostasis and thrombosis, this alkaloid could also modulate inflammatory and immunomodulatory activities, where these platelet functions have essential roles, particularly mediating intercellular communication .
Importantly, the cytotoxicity assessment of norpurpureine (100 μg/ml for 48 h) using the sulforhodamine B assay (available as Additional file 3) was promising. The compound reduced the initial cell populations of rhesus monkey kidney cell line MA104, human colon adenocarcinoma cell line HT29 and breast cancer mouse cell line 4 T1 by less than 10%. Additionally, the cytotoxicity assessment of norpurpureine (for 72 h) using alamar blue assay reports an IC50 value of 48.18 μM for peripheral blood mononuclear cells (PBMCs) . Thus, it is likely that the anti-platelet effects of norpurpureine, exerted in 10 min, correspond to pharmacological rather than toxicological effects.
The three agonists used in this study act through different receptors and signal transduction mechanisms: ADP acts via Gαq-mediated P2Y1 and Gαi-mediated P2Y12 receptors; collagen acts mainly through tyrosine kinase-mediated immunoglobulin GP VI; and thrombin through Gα(q,12 and io)-mediated PAR1 and Gα(q,12)-mediated PAR4 receptors . Activation of these receptors triggers different signaling pathways that converge into common signaling events to stimulate platelet shape change, granule secretion and aggregation to support platelet function. Thus, the observation that norpurpureine inhibits the actions of three different agonists with similar potency (IC50 around 80 μM), strongly suggests that its molecular target should be a common downstream effector of the signaling pathways activated by these agonists.
Since norpurpureine gradually affected the amplitude of transient elevation in [Ca2+]i induced by thrombin, its mechanism of action likely involves the negative regulation of the agonist-stimulated raise in [Ca2+]i. This correlates well with its potency to inhibit the second wave of platelet aggregation and granule secretion, and the adhesion of activated platelets to fibrinogen. In platelets, as in other non-excitable cells, increments in [Ca2+]i involve the release of Ca2+ sequestered in the dense tubular system (DTS, the equivalent of the endoplasmic reticulum in platelets), followed by Ca2+ influx through the plasma membrane, a process referred as store-operated calcium entry (SOCE) . Thus, norpurpureine actions probably involve the negative regulation of Ca2+ release from the DTS.
Activation of platelets by ADP and thrombin (G protein-coupled receptors) is via phospholipase C beta (PLCβ), while collagen (protein-tyrosine kinase receptor, GPVI) acts via PLCγ(2) . PLC activation generates inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (PIP2), IP3 activates its receptors (IP3-R) on the DTS to release Ca2+ into cytosol. DAG, together with Ca2+, activates PKC allowing downstream PKC-dependent events that regulate different steps during platelet activation . It is interesting that the PKC activator PMA, a DAG analog, fully rescued the aggregation response inhibited by norpurpureine in platelets stimulated by ADP and collagen but only partially rescued that response in platelets stimulated by thrombin. Human platelets express at least seven of the 12 PKC isoforms, namely conventional PKCα, PKCβI, PKCβII (regulated by both DAG and Ca2+) and novel PKCθ, PKCη’, PKCδ and PKCε (regulated only by DAG) . Thus, a specific PKC isoform (or may be upstream of PKC, at the PLC level) could be the molecular target of norpurpureine. However, additional detailed studies will be required, since the specific PKC isoforms activated downstream of each receptor are not clearly understood and PKC play isoform-specific inhibitory and stimulatory roles in platelet activation .
Agonist-induced reduction in cAMP is a key signaling step to remove the negative regulation of cAMP-dependent protein kinase (PKA) on calcium-related signaling elements, such as PLC-β3  and IP3 receptors . Under our experimental conditions, norpurpureine did not significantly modify intra-platelet cAMP in resting platelets, but significantly prevented the reduction in cAMP levels induced by the agonists used. Similar results were observed for IBMX, which strongly suggests the ability of norpurpureine to prevent the activation of PDEs in platelets. Human platelets express three PDE isoenzymes (PDE2, PDE3 and PDE5) and cAMP is hydrolyzed by PDE2 and PDE3 . PDE3A is the most abundant isoform in platelets and has a ~ 250-fold lower Km for cAMP than PDE2 . Different platelet agonists, including thrombin, significantly enhance the activity of PDE3A in a phosphorylation-dependent manner, actions that require the activation of PKC . Further examination is needed to determine whether norpurpureine targets a PDE isoform to potentiate the negative regulation of cAMP on Ca2+ homeostasis or regulates cAMP levels via PKC.
Beyond platelets, anti-plasmodial activity  and in vitro cytotoxic activity toward the tumor cell lines  have been reported for norpurpureine. So far, no other types of biological activities have been reported for purpureine. Based on our results, in future studies it will be interesting to explore the effect of these alkaloids on the activity of different PLC, PKC and PDE isoforms in human and rabbit platelets, to have additional evidence on their structure–activity relationships and their molecular mechanisms as anti-platelet agents.