We appreciate the letter from Dr. Pravin Patel and colleagues[1] regarding our recent studies to determine how phosphoinositide 3-kinase (PDK1) regulates thromboxane generation[2], a mechanism that had not previously been known. We also examined the downstream signaling cascades, reporting that PDK1-regulated thromboxane generation is controlled through the Raf1-ERK1/2 pathway.
In the current studies from Patel and colleagues, they now elegantly extend our work. Using 2MesADP, as we did in our studies, to stimulate platelets from ASK1 null or WT mice these investigators demonstrate that p38 is exclusively phosphorylated by ASK1 downstream of ADP receptors. Patel et al further demonstrate convincingly that convulxin activated ASK1, p38, cPLA2, and ERK1/2. Pharmacologic inhibition of ASK1 with GS-4997 attenuated ASK1, p38, and cPLA2 but not ERK1/2. These findings indicate that p38 predominantly drives the phosphorylation of cPLA2 in platelets. In support of this, a number of studies highlight the presence of other pathways that drive thromboxane production in a p38, ERK1/2, and Pyk2 mutually dependent manner [2] [3, 4].
These findings by Patel and colleagues provide conclusive evidence helping us understand how ASK1 regulates p38 activation downstream of ADP. We did not observe p38 inhibition when we pharmacologically blocked ASK1 in human platelets with MSC2032964A. These results are in contrast to those of Patel and colleagues in the current report. Reasons for this may include the use of human versus murine platelets (including genetically manipulated models), concentrations of 2MesADP, and potential pharmacologic differences between ASK1 inhibitors. Given the important role of PDK1 in platelet physiology and remaining unanswered questions regarding downstream activation pathways, this area warrants further investigation.
In conclusion, we appreciate the new findings provided by Patel and colleagues demonstrating that p38 is exclusively phosphorylated downstream of ASK1 in platelets and that the phosphorylation of cPLA2 is primarily regulated by p38[1]. Based on these new and previously published findings [2] [3, 4] [1], we propose the following model incorporating pathways regulating thromboxane generation by all three protein kinases (ERK1/2, p38, Pyk2), upon 2MesADP stimulation of platelets.
Figure: Model representation of different pathways regulating thromboxane generation.
Acknowledgements
This work was supported by the NHLBI and NIA (HL112311, HL126547, and AG04802 to M.T.R.). This material is the result of work supported with resources and the use of facilities at the George E. Wahlen VA Medical Center, Salt Lake City, Utah. The sponsor had no role in the design or preparation of paper. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.
Footnotes
Addendum
Bhanu Kanth Manne wrote the manuscript. Matthew T Rondina wrote the manuscript.
Disclosure of Conflict of Interest
The authors state that they have no conflict of interest.
References
- 1.Naik MU, Patel P, Derstine R, Turaga R, Chen X, Golla K, Neeves KB, Ichijo H, Naik UP. Ask1 regulates murine platelet granule secretion, thromboxane A2 generation, and thrombus formation. Blood. 2017; 129: 1197–209. 10.1182/blood-2016-07-729780. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Manne BK, Munzer P, Badolia R, Walker-Allgaier B, Campbell RA, Middleton E, Weyrich AS, Kunapuli SP, Borst O, Rondina MT. PDK1 governs thromboxane generation and thrombosis in platelets by regulating activation of Raf1 in the MAPK pathway. J Thromb Haemost. 2018; 16: 1211–25. 10.1111/jth.14005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Kim S, Cipolla L, Guidetti G, Okigaki M, Jin J, Torti M, Kunapuli SP. Distinct role of Pyk2 in mediating thromboxane generation downstream of both G12/13 and integrin alphaIIbbeta3 in platelets. J Biol Chem. 2013; 288: 18194–203. 10.1074/jbc.M113.461087. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Canobbio I, Cipolla L, Consonni A, Momi S, Guidetti G, Oliviero B, Falasca M, Okigaki M, Balduini C, Gresele P, Torti M. Impaired thrombin-induced platelet activation and thrombus formation in mice lacking the Ca(2+)-dependent tyrosine kinase Pyk2. Blood. 2013; 121: 648–57. 10.1182/blood-2012-06-438762. [DOI] [PubMed] [Google Scholar]