Response to Harper et al.
Authors: Dagmar Schumacher, Boris Strilic, Kishor Kumar Sivaraj, Nina Wettschureck, Stefan Offermanns
Journal: Cancer Cell (2013)

We thank Harper et al. for their comments on our publication. The authors of this correspondence point to some differences between our publication (Schumacher et al., 2013Schumacher D. Strilic B. Sivaraj K.K. Wettschureck N. Offermanns S. Cancer Cell. 2013; 24: 130-137Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar) and others with regard to the in vitro phenotype of Munc13-4-deficient platelets. Based on our experimental data, we think that Munc13-4 deficiency primarily blocks the release from dense granules, but not from α-granules of platelets. It is, however, well known that platelet activation involves multiple positive feedback loops and that the release of ATP/ADP from dense granules can promote platelet aggregation and α-granule release via the autocrine and paracrine activation of P2Y1 and P2Y12 receptors (Gachet, 2006Gachet C. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 277-300Crossref PubMed Scopus (237) Google Scholar, Kahner et al., 2006Kahner B.N. Shankar H. Murugappan S. Prasad G.L. Kunapuli S.P. J. Thromb. Haemost. 2006; 4: 2317-2326Crossref PubMed Scopus (170) Google Scholar). Based on this concept, it is indeed expected that loss of ATP/ADP release from platelet dense granules also affects, under certain experimental conditions, the release of α-granules from activated platelets, as well as the aggregation of platelets. Whether these secondary effects of a blocked dense-granule release are seen in vitro depends on the experimental conditions used to study platelet function, such as platelet concentration or the concentration and type of applied stimuli. At intermediate thrombin concentrations, we actually saw a tendency of reduced PF4 release and integrin αIIbβ3 activation which, however, was not significant (Figures 2A and S2C in Schumacher et al., 2013Schumacher D. Strilic B. Sivaraj K.K. Wettschureck N. Offermanns S. Cancer Cell. 2013; 24: 130-137Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). In the experiments mentioned by Harper et al., we used relatively low platelet concentrations, reducing the likelihood that sufficient amounts of ATP/ADP are released to induce secondary effects. Finally, at least in the case of tumor-cell-induced platelet activation, it is also possible that the small amount of ATP released from tumor cells (see Figure 1D in Schumacher et al., 2013Schumacher D. Strilic B. Sivaraj K.K. Wettschureck N. Offermanns S. Cancer Cell. 2013; 24: 130-137Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar) obscured a somewhat reduced α-granule release. Whether the secondary effects of dense-granule release are of relevance for platelet-dependent tumor cell metastasis is not known. Nevertheless, we agree that solely on the basis of our experiments performed with Munc13-4-deficient platelets and animals, we would not be able to exclude that defects other than reduced dense granule secretion also contribute to the observed defects in transendothelial tumor cell migration and in vivo metastasis. We therefore mentioned in the discussion of this publication the possibility that “in addition to ATP, other platelet-derived factors, such as transforming growth factor β, promote, in particular, later stages of tumor cell extravasation (Labelle et al., 2011Labelle M. Begum S. Hynes R.O. Cancer Cell. 2011; 20: 576-590Abstract Full Text Full Text PDF PubMed Scopus (1230) Google Scholar).” However, our study goes beyond the description of the role of dense-granule secretion in platelet-dependent tumor cell transendothelial migration and metastasis. We also identified a downstream mechanism by demonstrating that the endothelial ATP receptor P2Y2 mediates platelet-dependent tumor cell transmigration and that loss of P2Y2, which is not present on platelets, strongly reduces tumor cell metastasis from primary tumors, as well as after intravenous injection of tumor cells. If the above described secondary effects played a major role in platelet dense-granule secretion-dependent tumor cell transendothelial migration and metastasis, we would expect the phenotype of Munc13-4 deficiency to be more severe than the phenotype of P2Y2 deficiency. However, both platelet Munc13-4 deficiency and endothelial knockdown of P2Y2 abolished platelet-dependent transendothelial tumor cell migration. More importantly, tumor cell metastasis in vivo, when studied in bone-marrow chimeras, was reduced both in P2Y2-deficient mice transplanted with wild-type bone-marrow and in P2Y2-deficient mice transplanted with Munc13-4-deficient bone marrow to the same degree, and no additive effect could be seen. Thus, while we acknowledge that loss of dense-granule secretion in Munc13-4-deficient platelets can lead under certain experimental conditions to a secondary impairment of α-granule release, we think that our data show that dense-granule-derived ATP acting on P2Y2 receptors is the primary mechanism through which blockade of ATP release from Munc13-4-deficient platelets affects tumor cell transendothelial migration and metastasis. Comment on “Platelet-Derived Nucleotides Promote Tumor Cell Transendothelial Migration and Metastasis via P2Y2 Receptor” by Schumacher et al.Harper et al.Cancer CellSeptember 09, 2013In BriefWe were very interested to read in Cancer Cell the recent paper by Schumacher et al., “Platelet-Derived Nucleotides Promote Tumor Cell Transendothelial Migration and Metastasis via P2Y2 Receptor” (Schumacher et al., 2013). In this paper, the authors show the importance of platelet secretion in promoting tumor metastasis and convincingly demonstrate an important role for ATP from platelet dense granules acting on endothelial P2Y2. Part of the evidence for this depends on Munc13-4-deficient mice (Unc13dJinx), whose platelets cannot secrete their dense granules. Full-Text PDF Open ArchivePlatelet-Derived Nucleotides Promote Tumor-Cell Transendothelial Migration and Metastasis via P2Y2 ReceptorSchumacher et al.Cancer CellJune 27, 2013In BriefTumor cells can activate platelets, which in turn facilitate tumor cell survival and dissemination. The exact mechanisms by which platelets promote metastasis have remained unclear. Here, we show that adenine nucleotides released from tumor cell-activated platelets induce opening of the endothelial barrier to allow transendothelial migration of tumor cells and thereby promote cancer cell extravasation. We identified the endothelial P2Y2 receptor, which is activated by ATP, as the primary mediator of this effect. Full-Text PDF Open Archive