Gelsolin Modulates Platelet Dense Granule Secretion and Hemostasis via the Actin Cytoskeleton

Abstract

Background and Objective The mechanisms underlying platelet granule release are not fully understood. The actin cytoskeleton serves as the platelet's structural framework that is remodeled upon platelet activation. Gelsolin is a calcium-dependent protein that severs and caps existing actin filaments although its role in modulating platelet granule exocytosis is unknown.

Methods The hemostatic function of wild-type (WT) and gelsolin null (Gsn^−/− ) mice was measured ex vivo by rotational thromboelastometry analysis of whole blood. Platelets were purified from WT and Gsn^−/− mouse blood and activated with thrombin. Platelet aggregation was assessed by light-transmission aggregometry. Clot retraction was measured to assess outside-in integrin signaling. Adenosine triphosphate (ATP) release and surface P-selectin were measured as markers of dense- and α-granule secretion, respectively.

Results The kinetics of agonist-induced aggregation, clot retraction, and ATP release were accelerated in Gsn^−/− platelets relative to WT. However, levels of surface P-selectin were diminished in Gsn^−/− platelets. ATP release was also accelerated in WT platelets pretreated with the actin-depolymerizing drug cytochalasin D, thus mimicking the kinetics observed in Gsn^−/− platelets. Conversely, ATP release kinetics were normalized in Gsn^−/− platelets treated with the actin polymerization agonist jasplakinolide. Rab27b and Munc13–4 are vesicle-priming proteins known to promote dense granule secretion. Co-immunoprecipitation indicates that the association between Rab27b and Munc13–4 is enhanced in Gsn^−/− platelets.

Conclusions Gelsolin regulates the kinetics of hemostasis by modulating the platelet's actin cytoskeleton and the protein machinery of dense granule exocytosis.

Keywords

platelet physiology - secretion - exocytosis - cytoskeleton

Publikationsverlauf

Eingereicht: 22. April 2022

Angenommen: 01. Oktober 2022

Artikel online veröffentlicht:
15. Dezember 2022

• References

• 1 Broos K, Feys HB, De Meyer SF, Vanhoorelbeke K, Deckmyn H. Platelets at work in primary hemostasis. Blood Rev 2011; 25 (04) 155-167
• 2 Gremmel T, Frelinger III AL, Michelson AD. Platelet physiology. Semin Thromb Hemost 2016; 42 (03) 191-204
• 3 van der Meijden PEJ, Heemskerk JWM. Platelet biology and functions: new concepts and clinical perspectives. Nat Rev Cardiol 2019; 16 (03) 166-179
• 4 Rendu F, Brohard-Bohn B. The platelet release reaction: granules' constituents, secretion and functions. Platelets 2001; 12 (05) 261-273
• 5 Sharda A, Flaumenhaft R. The life cycle of platelet granules. F1000 Res 2018; 7: 236
• 6 Chen Y, Yuan Y, Li W. Sorting machineries: how platelet-dense granules differ from α-granules. Biosci Rep 2018; 38 (05) BSR20180458
• 7 Ambrosio AL, Di Pietro SM. Storage pool diseases illuminate platelet dense granule biogenesis. Platelets 2017; 28 (02) 138-146
• 8 Meng R, Wang Y, Yao Y. et al. SLC35D3 delivery from megakaryocyte early endosomes is required for platelet dense granule biogenesis and is differentially defective in Hermansky-Pudlak syndrome models. Blood 2012; 120 (02) 404-414
• 9 Introne W, Boissy RE, Gahl WA. Clinical, molecular, and cell biological aspects of Chediak-Higashi syndrome. Mol Genet Metab 1999; 68 (02) 283-303
• 10 Koseoglu S, Flaumenhaft R. Advances in platelet granule biology. Curr Opin Hematol 2013; 20 (05) 464-471
• 11 Joshi S, Whiteheart SW. The nuts and bolts of the platelet release reaction. Platelets 2017; 28 (02) 129-137
• 12 Flaumenhaft R. Molecular basis of platelet granule secretion. Arterioscler Thromb Vasc Biol 2003; 23 (07) 1152-1160
• 13 Golebiewska EM, Poole AW. Secrets of platelet exocytosis - what do we really know about platelet secretion mechanisms?. Br J Haematol 2013; 165 (02) 204-216
• 14 Shirakawa R, Higashi T, Tabuchi A. et al. Munc13-4 is a GTP-Rab27-binding protein regulating dense core granule secretion in platelets. J Biol Chem 2004; 279 (11) 10730-10737
• 15 Shirakawa R, Higashi T, Kondo H, Yoshioka A, Kita T, Horiuchi H. Purification and functional analysis of a Rab27 effector munc 13-4 using a semi-intact platelet dense-granule secretion assay. Methods Enzymol 2005; 403: 778-788
• 16 Ren Q, Wimmer C, Chicka MC. et al. Munc13-4 is a limiting factor in the pathway required for platelet granule release and hemostasis. Blood 2010; 116 (06) 869-877
• 17 Flaumenhaft R, Dilks JR, Rozenvayn N, Monahan-Earley RA, Feng D, Dvorak AM. The actin cytoskeleton differentially regulates platelet alpha-granule and dense-granule secretion. Blood 2005; 105 (10) 3879-3887
• 18 Woronowicz K, Dilks JR, Rozenvayn N. et al. The platelet actin cytoskeleton associates with SNAREs and participates in alpha-granule secretion. Biochemistry 2010; 49 (21) 4533-4542
• 19 McCarty OJ, Larson MK, Auger JM. et al. Rac1 is essential for platelet lamellipodia formation and aggregate stability under flow. J Biol Chem 2005; 280 (47) 39474-39484
• 20 Aslan JE, McCarty OJ. Rho GTPases in platelet function. J Thromb Haemost 2013; 11 (01) 35-46
• 21 Senini V, Amara U, Paul M, Kim H. Porphyromonas gingivalis lipopolysaccharide activates platelet Cdc42 and promotes platelet spreading and thrombosis. J Periodontol 2019; 90 (11) 1336-1345
• 22 Nobes CD, Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 1995; 81 (01) 53-62
• 23 Pollard TD. Actin and actin-binding proteins. Cold Spring Harb Perspect Biol 2016; 8 (08) a018226
• 24 Romero S, Le Clainche C, Gautreau AM. Actin polymerization downstream of integrins: signaling pathways and mechanotransduction. Biochem J 2020; 477 (01) 1-21
• 25 Falet H, Hoffmeister KM, Neujahr R. et al. Importance of free actin filament barbed ends for Arp2/3 complex function in platelets and fibroblasts. Proc Natl Acad Sci U S A 2002; 99 (26) 16782-16787
• 26 Shekhar S, Pernier J, Carlier MF. Regulators of actin filament barbed ends at a glance. J Cell Sci 2016; 129 (06) 1085-1091
• 27 Marcu MG, Zhang L, Nau-Staudt K, Trifaró JM. Recombinant scinderin, an F-actin severing protein, increases calcium-induced release of serotonin from permeabilized platelets, an effect blocked by two scinderin-derived actin-binding peptides and phosphatidylinositol 4,5-bisphosphate. Blood 1996; 87 (01) 20-24
• 28 Sun HQ, Yamamoto M, Mejillano M, Yin HL. Gelsolin, a multifunctional actin regulatory protein. J Biol Chem 1999; 274 (47) 33179-33182
• 29 Lind SE, Janmey PA, Chaponnier C, Herbert TJ, Stossel TP. Reversible binding of actin to gelsolin and profilin in human platelet extracts. J Cell Biol 1987; 105 (02) 833-842
• 30 Eaton N, Subramaniam S, Schulte ML. et al. Bleeding diathesis in mice lacking JAK2 in platelets. Blood Adv 2021; 5 (15) 2969-2981
• 31 Patel VB, Zhabyeyev P, Chen X. et al. PI3Kα-regulated gelsolin activity is a critical determinant of cardiac cytoskeletal remodeling and heart disease. Nat Commun 2018; 9 (01) 5390
• 32 Naik MU, Patel P, Derstine R. et al. Ask1 regulates murine platelet granule secretion, thromboxane A₂ generation, and thrombus formation. Blood 2017; 129 (09) 1197-1209
• 33 Kim H, Falet H, Hoffmeister KM, Hartwig JH. Wiskott-Aldrich syndrome protein (WASp) controls the delivery of platelet transforming growth factor-β1. J Biol Chem 2013; 288 (48) 34352-34363
• 34 Cho JH, Wool GD, Tjota MY, Gutierrez J, Mikrut K, Miller JL. Functional assessment of platelet dense granule ATP release. Am J Clin Pathol 2021; 155 (06) 863-872
• 35 Varga-Szabo D, Braun A, Nieswandt B. Calcium signaling in platelets. J Thromb Haemost 2009; 7 (07) 1057-1066
• 36 Golebiewska EM, Poole AW. Platelet secretion: from haemostasis to wound healing and beyond. Blood Rev 2015; 29 (03) 153-162
• 37 Dolan AT, Diamond SL. Systems modeling of Ca(2+) homeostasis and mobilization in platelets mediated by IP3 and store-operated Ca(2+) entry. Biophys J 2014; 106 (09) 2049-2060
• 38 Jonnalagadda D, Izu LT, Whiteheart SW. Platelet secretion is kinetically heterogeneous in an agonist-responsive manner. Blood 2012; 120 (26) 5209-5216
• 39 Nurden AT, Nurden P. Should any genetic defect affecting α-granules in platelets be classified as gray platelet syndrome?. Am J Hematol 2016; 91 (07) 714-718
• 40 Tolmachova T, Abrink M, Futter CE, Authi KS, Seabra MC. Rab27b regulates number and secretion of platelet dense granules. Proc Natl Acad Sci U S A 2007; 104 (14) 5872-5877
• 41 Chicka MC, Ren Q, Richards D. et al. Role of Munc13-4 as a Ca2+-dependent tether during platelet secretion. Biochem J 2016; 473 (05) 627-639
• 42 Cerecedo D. Platelet cytoskeleton and its hemostatic role. Blood Coagul Fibrinolysis 2013; 24 (08) 798-808
• 43 Sorrentino S, Studt JD, Medalia O, Tanuj Sapra K. Roll, adhere, spread and contract: structural mechanics of platelet function. Eur J Cell Biol 2015; 94 (3–4): 129-138
• 44 Moskalensky AE, Litvinenko AL. The platelet shape change: biophysical basis and physiological consequences. Platelets 2019; 30 (05) 543-548
• 45 Stritt S, Beck S, Becker IC. et al. Twinfilin 2a regulates platelet reactivity and turnover in mice. Blood 2017; 130 (15) 1746-1756
• 46 Peters CG, Michelson AD, Flaumenhaft R. Granule exocytosis is required for platelet spreading: differential sorting of α-granules expressing VAMP-7. Blood 2012; 120 (01) 199-206
• 47 Koseoglu S, Peters CG, Fitch-Tewfik JL. et al. VAMP-7 links granule exocytosis to actin reorganization during platelet activation. Blood 2015; 126 (05) 651-660
• 48 Chou J, Stolz DB, Burke NA, Watkins SC, Wells A. Distribution of gelsolin and phosphoinositol 4,5-bisphosphate in lamellipodia during EGF-induced motility. Int J Biochem Cell Biol 2002; 34 (07) 776-790
• 49 Mazur AJ, Gremm D, Dansranjavin T. et al. Modulation of actin filament dynamics by actin-binding proteins residing in lamellipodia. Eur J Cell Biol 2010; 89 (05) 402-413
• 50 Azuma T, Witke W, Stossel TP, Hartwig JH, Kwiatkowski DJ. Gelsolin is a downstream effector of rac for fibroblast motility. EMBO J 1998; 17 (05) 1362-1370
• 51 Schurr Y, Sperr A, Volz J. et al. Platelet lamellipodium formation is not required for thrombus formation and stability. Blood 2019; 134 (25) 2318-2329