Munc13-4 is critical for thrombosis through regulating release of ADP from platelets

Abstract

Effective hemostasis and pathological arterial thrombosis rely upon the rapid development of a platelet aggregate at the site of blood vessel damage. In order to achieve this, the initial platelet response to a wide array of different soluble and insoluble agonists is amplified and sustained by positive feedback mechanisms. Secretion of ADP from platelet dense granules forms a major positive feedback loop that is essential for thrombus growth and stability 1.Andre P. Delaney S.M. LaRocca T. Vincent D. DeGuzman F. Jurek M. Koller B. Phillips D.R. Conley P.B. P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries.J Clin Invest. 2003; 112: 398-406Crossref PubMed Google Scholar, 2.Storey R.F. Biology and pharmacology of the platelet P2Y12 receptor.Curr Pharm Des. 2006; 12: 1255-9Crossref PubMed Scopus (0) Google Scholar, 3.Storey R.F. Sanderson H.M. White A.E. May J.A. Cameron K.E. Heptinstall S. 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One member of the family, Munc13-4, is prominently expressed in hematopoietic cells and a lack of Munc13-4 in Unc13dJinx mice results in severe secretion defects in neutrophils, natural killer (NK) cells and cytotoxic T cells (CTLs) 19.Crozat K. Hoebe K. Ugolini S. Hong N.A. Janssen E. Rutschmann S. Mudd S. Sovath S. Vivier E. Beutler B. Jinx, an MCMV susceptibility phenotype caused by disruption of Unc13d: a mouse model of type 3 familial hemophagocytic lymphohistiocytosis.J Exp Med. 2007; 204: 853-63Crossref PubMed Scopus (125) Google Scholar. Munc13-4 dysfunction in humans is associated with familial hemophagocytic lymphohistiocytosis subset 3 (FHL3). In this disease, the lytic granules of NK cells and CTLs fail to fuse with the plasma membrane to release their cargo 20.Feldmann J. Callebaut I. Raposo G. Certain S. Bacq D. Dumont C. Lambert N. Ouachée-Chardin M. Chedeville G. Tamary H. Minard-Colin V. Vilmer E. Blanche S. Le Deist F. Fischer A. de Saint Basile G. Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3).Cell. 2003; 115: 461-73Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar, 21.Gholam C. Grigoriadou S. Gilmour K.C. Gaspar H.B. Familial haemophagocytic lymphohistiocytosis: advances in the genetic basis, diagnosis and management.Clin Exp Immunol. 2011; 163: 271-83Crossref PubMed Scopus (110) Google Scholar, 22.Janka G.E. Familial and acquired hemophagocytic lymphohistiocytosis.Annu Rev Med. 2012; 63: 233-46Crossref PubMed Scopus (399) Google Scholar. Munc13-4 is also an important regulator of platelet granule secretion. Platelets from Unc13dJinx mice showed no dense granule secretion (Fig. 1A,B), demonstrating an essential role for Munc13-4 that is consistent with previous reports 23.Shirakawa R. Higashi T. Tabuchi A. Yoshioka A. Nishioka H. Fukuda M. Kita T. Horiuchi H. Munc13-4 is a GTP-Rab27-binding protein regulating dense core granule secretion in platelets.J Biol Chem. 2004; 279: 10730-7Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 24.Ren Q. Wimmer C. Chicka M.C. Ye S. Ren Y. Hughson F.M. Whiteheart S.W. Munc13-4 is a limiting factor in the pathway required for platelet granule release and hemostasis.Blood. 2010; 116: 869-77Crossref PubMed Scopus (96) Google Scholar. Platelet count and mean platelet volume (Table S1) were normal in Unc13dJinx mice. A panel of key platelet signaling proteins was also screened, which showed no change in expression compared with wild-type (WT) mouse platelets (Fig. S1). We hypothesized that any subsequent functional defects in Unc13dJinx platelet activation are a result of the lost ability to secrete ADP, which forms a major positive feedback pathway. Importantly, re-addition of ADP, to replace that normally secreted from dense granules, was shown to rescue the functional deficits seen. Unc13dJinx mice may therefore provide a useful tool to investigate the role of secreted ADP in platelet function. Unc13dJinx platelets showed reduced aggregation and thrombus formation. Figure 1C shows that platelet aggregation stimulated by either PAR-4-activating peptide (PAR4-AP) or collagen-related peptide (CRP) was impaired in these mice compared with WT. Co-stimulation with ADP was able fully to rescue responses, however, resulting in aggregation that was indistinguishable from WT platelets in both rate and extent of aggregation. This was reflected also in rescue of the deficit seen in activation of integrin αIIbβ3, as assessed by binding of the antibody Jon/A (Fig. 1D), which specifically recognizes the activated form of the integrin. A P2Y12 antagonist (ARC69931MX) also inhibited αIIbβ3 activation (Fig. 1D), whereas a P2Y1 antagonist (MRS2279) had no effect, either on WT platelets or on the ability of ADP to rescue αIIbβ3 activation in Unc13Jinx platelets. 5-HT, which is also released from dense granules, did not affect integrin activation under these conditions. ARC69931MX inhibited aggregation of WT platelets to the level seen in Unc13dJinx platelets without ARC69931MX, but had no effect on aggregation of Unc13dJinx platelets themselves. ADP could not rescue the reduced aggregation when P2Y12 was blocked (Fig. 1E). Finally, it was important to show that ADP was able to rescue deficits in thrombus formation under flow, both in vitro and in vivo. In vitro flow of blood over a collagen-coated surface (Fig. 1F) showed a marked diminution of accumulation of thrombi in Unc13dJinx compared with WT. Addition of ADP (10 μm) into the flowing blood was able to enhance thrombus formation in WT and fully rescue the response in Unc13dJinx blood. Importantly, we also provide parallel evidence from an in vivo approach. Thrombus formation in FeCl3-injured carotid arteries was also markedly defective in Unc13dJinx mice (Fig. 1G), and when ADPβS was directly applied to the outside of the artery immediately after FeCl3 injury, thrombus formation was partially rescued in Unc13dJinx mice (Fig. 1G, second row). ADPβS applied to uninjured arteries did not induce thrombus formation by itself. This indicates that the thrombosis defect in vivo involves, at least in part, the lack of ADP secretion from platelets in these mice. Thrombus formation was not completely rescued in this model, which may reflect the difficulty of exogenous ADPβS reaching intravascular platelets. Alternatively, other factors released by platelets may be important for thrombosis in vivo. For example, polyphosphates, secreted from platelet dense granules, may be important in thrombin generation 25.Smith S.A. Mutch N.J. Baskar D. Rohloff P. Docampo R. Morrissey J.H. Polyphosphate modulates blood coagulation and fibrinolysis.Proc Natl Acad Sci USA. 2006; 103: 903-8Crossref PubMed Scopus (429) Google Scholar. In summary, Munc13-4 has been shown to be a critical gene regulating granule secretion in platelets 20.Feldmann J. Callebaut I. Raposo G. Certain S. Bacq D. Dumont C. Lambert N. Ouachée-Chardin M. Chedeville G. Tamary H. Minard-Colin V. Vilmer E. Blanche S. Le Deist F. Fischer A. de Saint Basile G. Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3).Cell. 2003; 115: 461-73Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar, 23.Shirakawa R. Higashi T. Tabuchi A. Yoshioka A. Nishioka H. Fukuda M. Kita T. Horiuchi H. Munc13-4 is a GTP-Rab27-binding protein regulating dense core granule secretion in platelets.J Biol Chem. 2004; 279: 10730-7Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar. We have extended the original observations made by Ren et al. 24.Ren Q. Wimmer C. Chicka M.C. Ye S. Ren Y. Hughson F.M. Whiteheart S.W. Munc13-4 is a limiting factor in the pathway required for platelet granule release and hemostasis.Blood. 2010; 116: 869-77Crossref PubMed Scopus (96) Google Scholar to show that Munc13-4 is also critical for development of thrombosis in vitro and in vivo. Unc13dJinx mice showed reduced αIIbβ3 activation, aggregation and thrombus formation, all of which could be rescued by exogenous addition of ADP or the non-hydrolyzable analog ADPβS. These observations demonstrate the important role played by Munc13-4 in regulating thrombosis, and indicate that the major mechanism involves its regulation of ADP secretion from dense granules. J. S. Savage performed research, collected data and contributed to discussion and co-wrote the manuscript. C. M. Williams performed research and collected, analyzed and interpreted the data. O. Konopatskaya collected data and helped design the research. I. Hers contributed to discussion and edited the manuscript. M. T. Harper designed research, performed research, collected data, analyzed and interpreted data, contributed to discussion, performed statistical analysis and co-wrote the manuscript. A. W. Poole designed research, interpreted data, contributed to discussion and co-wrote the manuscript. The authors state that they have no conflict of interest. We thank the healthy blood donors within the Medical Sciences building, University of Bristol, for their generous donations, and Elizabeth Aitken for expert technical support. We are grateful to Professor Sussan Nourshargh for valuable discussions on in vivo experiments. This work was supported by the British Heart Foundation (grants no RG/10/006/28299, PG/08/056/25325 and PG/10/100/28658).University of BristolBritish Heart FoundationRG/10/006/28299PG/08/056/25325PG/10/100/28658 Download .jpg (.2 MB) Help with files Data S1. Materials and methods.Figure S1. Normal expression of secretion-related proteins in Unc13dJinx platelets. Download .docx (.01 MB) Help with docx files Table S1. Haematological parameters and glycoprotein expression are normal in Unc13dJinx mice.