Platelets are vital to hemostasis and have a critical role in immunological and inflammatory processes within human circulation. Severe thrombocytopenia often leads to hemorrhage, creating a rationale for developing thrombopoietic drugs. On the other hand, continuous activation of platelets is a major contributor to chronic inflammatory vascular diseases such as atherosclerosis and type-2 diabetes [2
], creating the demand for new anti-platelet drug development. Either condition is detrimental, further exemplifying the delicate balance of adequate platelet numbers, and the risks of excessive platelet activation. We demonstrate here that parthenolide is a potential candidate agent for treatment of both conditions, as it increases platelet production from megakaryocytes and attenuates platelet activation during stimulation. Specific delivery mechanisms would need to be implemented, depending on the condition needed to be treated.
Two megakaryoblastic cell lines, Meg-01 and MO7e, can spontaneously produce platelet-like particles in culture [23
]. We demonstrated that parthenolide facilitated morphological changes indicative of thrombopoiesis, and increased production of platelet-like particles within 24 hours of treatment (). Similarly, parthenolide enhanced platelet production within primary in vitro
differentiated human megakaryocytes (). Compared to 15-deoxy-Δ12,14
, which we previously reported as an enhancer of platelet production [4
], parthenolide showed a weaker, but still significant enhancement of platelet production (comparison not shown). However, platelet production enhancement in a clinical setting by parthenolide and similar novel agents has not yet been assessed. It is worthy of noting that these primary megakaryocytes were first differentiated and matured with thrombopoietin (see Materials and Methods) before treatment of parthenolide. A bone marrow-directed conjunctive therapy may need to be considered before transition to an in vivo
ROS and other oxidative stressors haven been shown to increase after parthenolide treatment [5
]. The increase of ROS in some cell types was associated with a decrease in GSH [5
]. Contrary to those cell types, Meg-01 cells increased their total GSH levels (), indicating they may be counteracting oxidative stress in this way, as the GSSG/2GSH couple is considered to be the “cellular redox buffer” [26
]. Meg-01 cells are capable of GSH depletion, as we demonstrated using BSO, but parthenolide does not act in this manner. Increase of GSH and HO-1, another antioxidant enzyme [24
], along with no detectable ROS increase, suggests that the cells are capable of equilibrating to the induced oxidative stress caused by parthenolide.
We showed that H2
, which produces more ROS in megakaryocytes than parthenolide (), and therefore generating an imbalanced redox state, was unable to affect platelet production from Meg-01, MO7e, or PHM cells (). Additionally, pretreatment of megakaryocytes with antioxidants before parthenolide treatment did not alter the increase of platelet production (data not shown). These data support the likelihood that oxidative stress does not play a major role in the enhancement of parthenolide-induced platelet production. This led us to investigate another targeted pathway of parthenolide: NF-κB [10
]. We have previously shown NF-κB signaling to play a role in megakaryocyte activation after PMA stimulation, and Zhang et al. showed that a constitutive NF-κB signal within proliferating megakaryoblasts decreased at final stages of maturation [13
Using two different assays, we showed that parthenolide decreased NF-κB activity in megakaryocytes (). The rebound of NF-κB activity, as measured by luciferase at 24 hours, indicates that parthenolide is not toxic at this concentration to these cells, and also that the stability and duration of the effects of parthenolide may be short lived. The more stable NF-κB inhibitor positive control, BMS, showed further reduction of luciferase activity. Additionally, this NF-κB inhibitor was also able to increase the number of platelets produced by megakaryocytes. While we cannot exclude the possibility that additional signaling pathways are also affected, we conclude that NF-κB inhibition is almost certainly a leading cause for the thrombopoietic effects of parthenolide observed in megakaryocytes. However, the short duration of parthenolide effectiveness may need to be addressed before transitioning into an in vivo
model. More stable parthenolide analogs are already under investigation in the anti-cancer setting [31
]. However, short lived efficacy of parthenolide may be advantageous if direct bone marrow delivery is feasible, as it may be harmful give an anti-platelet drug intravenously to a thrombocytopenic patient.
We previously identified that the NF-κB pathway functions in normal human platelet activation [19
], which is why we chose to investigate whether NF-κB inhibition within parthenolide-treated megakaryocytes affects the function of daughter platelets. Had this been the case, platelets created from parthenolide-treated megakaryocytes would have decreased activation ability. To the contrary, the platelets derived from parthenolide-treated megakaryocytes appear to be fully functional cells capable of activation (). This again, could be due to the short-lived effectiveness of parthenolide, therefore, only inhibiting NF-κB activity in mature megakaryocytes while it is not stable enough to affect daughter platelets. Importantly, this demonstrates that parthenolide enhances the production of functional platelets, making it a potential thrombopoietic drug candidate if it can be targeted to the bone marrow. Parthenolide is currently being investigated as an anticancer agent, but it may also have the potential to mitigate thrombocytopenia resulting from current therapies.
Anti-platelet drug therapies are increasingly sought as platelets are recognized as contributors to local and systemic inflammatory conditions such as atherosclerosis [2
]. Anti-platelet therapies have also been shown to decrease platelets’ ability to mediate tumor cell invasiveness [32
]. We found that when used as a pretreatment on platelets ex vivo
, parthenolide decreased, but did not abolish the activation of stimulated peripheral blood platelets (). This evidence marks parthenolide as a potential drug candidate for anti-platelet therapy. Intravenous injections should be considered as a means of drug delivery for an in vivo
setting, allowing parthenolide to quickly contact and dampen activated platelets before losing effectiveness.
Oxidative stress alone cannot be the mechanism of decreased platelet activation after parthenolide treatment because H2
did not affect platelet activation (). We previously reported that NF-κB inhibitors can attenuate platelet activation [19
], and our data suggest that the inhibition of this pathway by parthenolide led to the decrease in platelet activity reported here. Another proposed mechanism of parthenolide attenuation of platelet activation may include alteration of sulfhydryl groups [17
], as it has been established that platelet activation involves sulfhydryl-dependent pathways, particularly through inhibition of the glycoprotein IIbIIIa [33
]. Additional mechanisms of parthenolide-mediated anti-platelet activity include changes in arachidonic acid metabolism and interactions with protein kinase C [16
]. Parthenolide is already used as an herbal medicine to treat inflammatory conditions such as arthritis and migraines [34
]. Our new evidence corroborates previously reported literature of parthenolide-driven reduction of platelet activity [16
], and likely acts through multiple pathways including NF-κB inhibition.