Fine-Tuning of Platelet Responses by Serine/Threonine Protein Kinases and Phosphatases—Just the Beginning

Abstract

Comprehensive proteomic analyses of human and murine platelets established an extraordinary intracellular repertoire of signaling components, which control crucial functions. The spectrum of platelet serine/threonine protein kinases (more than 100) includes the AGC family (protein kinase A, G, C [PKA, PKG, PKC]), the mitogen-activated protein kinases (MAPKs), and others. PKA and PKG have multiple significantly overlapping substrates in human platelets, which possibly affect functions with clear “signaling nodes” of regulation by multiple protein kinases/phosphatases. Signaling nodes are intracellular Ca²⁺ stores, the contractile system (myosin light chains), and other signaling components such as G-proteins, protein kinases, and protein phosphatases. An example for this fine-tuning is the tyrosine kinase Syk, a crucial component of platelet activation, which is controlled by several serine/threonine and tyrosine protein kinases as well as phosphatases. Other protein kinases including PKA/PKG modulate protein phosphatase 2A, which may be a master regulator of MAPK signaling in human platelets. Protein kinases and in particular MAPKs are targeted by an increasing number of clinically used inhibitors. However, the precise regulation and fine-tuning of these protein kinases and their effects on other signaling components in platelets are only superficially understood—just the beginning. However, promising future approaches are in sight.

Keywords

platelet signaling - protein kinase networks - protein phosphatases

Publication History

Received: 23 December 2020

Accepted: 06 April 2021

Article published online:
30 June 2021

• References

• 1 Born GV. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 1962; 194: 927-929
• 2 Svensson J, Hamberg M, Samuelsson B. On the formation and effects of thromboxane A2 in human platelets. Acta Physiol Scand 1976; 98 (03) 285-294
• 3 Moncada S, Higgs EA, Vane JR. Human arterial and venous tissues generate prostacyclin (prostaglandin x), a potent inhibitor of platelet aggregation. Lancet 1977; 1 (8001): 18-20
• 4 Tateson JE, Moncada S, Vane JR. Effects of prostacyclin (PGX) on cyclic AMP concentrations in human platelets. Prostaglandins 1977; 13 (03) 389-397
• 5 Jurk K, Kehrel BE. Platelets: physiology and biochemistry. Semin Thromb Hemost 2005; 31 (04) 381-392
• 6 Versteeg HH, Heemskerk JWM, Levi M, Reitsma PH. New fundamentals in hemostasis. Physiol Rev 2013; 93 (01) 327-358
• 7 Jurk K. Analysis of platelet function and dysfunction. Hamostaseologie 2015; 35 (01) 60-72
• 8 Senis YA, Mazharian A, Mori J. Src family kinases: at the forefront of platelet activation. Blood 2014; 124 (13) 2013-2024
• 9 Brass LF, Ma P, Tomaiuolo M. et al. A systems approach to the platelet signaling network and the hemostatic response to injury. In: Platelets in Thrombotic and Non-Thrombotic Disorders. Springer; 2017: 367-378
• 10 Makhoul S, Walter E, Pagel O. et al. Effects of the NO/soluble guanylate cyclase/cGMP system on the functions of human platelets. Nitric Oxide 2018; 76: 71-80
• 11 Sutherland EW, Robison GA. The role of cyclic-3′,5′-AMP in responses to catecholamines and other hormones. Pharmacol Rev 1966; 18 (01) 145-161
• 12 Sutherland EW. On the biological role of cyclic AMP. JAMA 1970; 214 (07) 1281-1288
• 13 Mellion BT, Ignarro LJ, Ohlstein EH, Pontecorvo EG, Hyman AL, Kadowitz PJ. Evidence for the inhibitory role of guanosine 3′, 5′-monophosphate in ADP-induced human platelet aggregation in the presence of nitric oxide and related vasodilators. Blood 1981; 57 (05) 946-955
• 14 Eigenthaler M, Ullrich H, Geiger J. et al. Defective nitrovasodilator-stimulated protein phosphorylation and calcium regulation in cGMP-dependent protein kinase-deficient human platelets of chronic myelocytic leukemia. J Biol Chem 1993; 268 (18) 13526-13531
• 15 Waldmann R, Bauer S, Göbel C, Hofmann F, Jakobs KH, Walter U. Demonstration of cGMP-dependent protein kinase and cGMP-dependent phosphorylation in cell-free extracts of platelets. Eur J Biochem 1986; 158 (01) 203-210
• 16 Smolenski A. Novel roles of cAMP/cGMP-dependent signaling in platelets. J Thromb Haemost 2012; 10 (02) 167-176
• 17 Nagy Z, Smolenski A. Cyclic nucleotide-dependent inhibitory signaling interweaves with activating pathways to determine platelet responses. Res Pract Thromb Haemost 2018; 2 (03) 558-571
• 18 Kunapuli SP, Bhavanasi D, Kostyak JC. et al. Platelet signaling: protein phosphorylation. In: Platelets in Thrombotic and Non-Thrombotic Disorders: Pathophysiology, Pharmacology and Therapeutics: An Update. Springer International Publishing; 2017: 297-308
• 19 Golden A, Nemeth SP, Brugge JS. Blood platelets express high levels of the pp60c-src-specific tyrosine kinase activity. Proc Natl Acad Sci U S A 1986; 83 (04) 852-856
• 20 Séverin S, Nash CA, Mori J. et al. Distinct and overlapping functional roles of Src family kinases in mouse platelets. J Thromb Haemost 2012; 10 (08) 1631-1645
• 21 Patel P, Naik UP. Platelet MAPKs-a 20+ year history: What do we really know?. J Thromb Haemost 2020; 18 (09) 2087-2102
• 22 Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. The protein kinase complement of the human genome. Science 2002; 298 (5600): 1912-1934
• 23 Chen MJ, Dixon JE, Manning G. Genomics and evolution of protein phosphatases. Sci Signal 2017; 10 (474) 10
• 24 Burkhart JM, Vaudel M, Gambaryan S. et al. The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways. Blood 2012; 120 (15) e73-e82
• 25 Zeiler M, Moser M, Mann M. Copy number analysis of the murine platelet proteome spanning the complete abundance range. Mol Cell Proteomics 2014; 13 (12) 3435-3445
• 26 Jurk K, Walter U. New Insights into platelet signalling pathways by functional and proteomic approaches. Hamostaseologie 2019; 39 (02) 140-151
• 27 Miranda-Saavedra D, Barton GJ. Classification and functional annotation of eukaryotic protein kinases. Proteins 2007; 68 (04) 893-914
• 28 Omar MH, Scott JD. AKAP signaling islands: venues for precision pharmacology. Trends Pharmacol Sci 2020; 41 (12) 933-946
• 29 Burgers PP, Ma Y, Margarucci L. et al. A small novel A-kinase anchoring protein (AKAP) that localizes specifically protein kinase A-regulatory subunit I (PKA-RI) to the plasma membrane. J Biol Chem 2012; 287 (52) 43789-43797
• 30 Raslan Z, Aburima A, Naseem KM. The spatiotemporal regulation of cAMP signaling in blood platelets—old friends and new players. Front Pharmacol 2015; 6: 266
• 31 Margarucci L, Roest M, Preisinger C. et al. Collagen stimulation of platelets induces a rapid spatial response of cAMP and cGMP signaling scaffolds. Mol Biosyst 2011; 7 (07) 2311-2319
• 32 Lohmann SM, Vaandrager AB, Smolenski A, Walter U, De Jonge HR. Distinct and specific functions of cGMP-dependent protein kinases. Trends Biochem Sci 1997; 22 (08) 307-312
• 33 Massberg S, Sausbier M, Klatt P. et al. Increased adhesion and aggregation of platelets lacking cyclic guanosine 3′,5′-monophosphate kinase I. J Exp Med 1999; 189 (08) 1255-1264
• 34 Gambaryan S, Butt E, Kobsar A. et al. The oligopeptide DT-2 is a specific PKG I inhibitor only in vitro, not in living cells. Br J Pharmacol 2012; 167 (04) 826-838
• 35 Beck F, Geiger J, Gambaryan S. et al. Time-resolved characterization of cAMP/PKA-dependent signaling reveals that platelet inhibition is a concerted process involving multiple signaling pathways. Blood 2014; 123 (05) e1-e10
• 36 Beck F, Geiger J, Gambaryan S. et al. Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition. Blood 2017; 129 (02) e1-e12
• 37 Swieringa F, Solari FA, Pagel O. et al. Impaired iloprost-induced platelet inhibition and phosphoproteome changes in patients with confirmed pseudohypoparathyroidism type Ia, linked to genetic mutations in GNAS. Sci Rep 2020; 10 (01) 11389
• 38 Comer S, Nagy Z, Bolado A. et al. The RhoA regulators Myo9b and GEF-H1 are targets of cyclic nucleotide-dependent kinases in platelets. J Thromb Haemost 2020; 18 (11) 3002-3012
• 39 Daniel JL, Adelstein RS. Isolation and properties of platelet myosin light chain kinase. Biochemistry 1976; 15 (11) 2370-2377
• 40 Hathaway DR, Adelstein RS. Human platelet myosin light chain kinase requires the calcium-binding protein calmodulin for activity. Proc Natl Acad Sci U S A 1979; 76 (04) 1653-1657
• 41 Nishikawa M, de Lanerolle P, Lincoln TM, Adelstein RS. Phosphorylation of mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase. J Biol Chem 1984; 259 (13) 8429-8436
• 42 Kawahara Y, Takai Y, Minakuchi R, Sano K, Nishizuka Y. Phospholipid turnover as a possible transmembrane signal for protein phosphorylation during human platelet activation by thrombin. Biochem Biophys Res Commun 1980; 97 (01) 309-317
• 43 Kikkawa U, Nishizuka Y. The role of protein kinase C in transmembrane signalling. Annu Rev Cell Biol 1986; 2: 149-178
• 44 Kajikawa N, Kikkawa U, Itoh K, Nishizuka Y. Membrane-permeable diacylglycerol, its application to platelet secretion, and regulation of platelet protein kinase C. Methods Enzymol 1989; 169: 430-442
• 45 Khapchaev AY, Shirinsky VP. Myosin light chain kinase MYLK1: anatomy, interactions, functions, and regulation. Biochemistry (Mosc) 2016; 81 (13) 1676-1697
• 46 Takai A, Eto M, Hirano K, Takeya K, Wakimoto T, Watanabe M. Protein phosphatases 1 and 2A and their naturally occurring inhibitors: current topics in smooth muscle physiology and chemical biology. J Physiol Sci 2018; 68 (01) 1-17
• 47 Aburima A, Naseem KM. Platelet myosin light chain phosphatase: keeping it together. Biochem Soc Trans 2014; 42 (02) 279-283
• 48 Aburima A, Walladbegi K, Wake JD, Naseem KM. cGMP signaling inhibits platelet shape change through regulation of the RhoA-Rho kinase-MLC phosphatase signaling pathway. J Thromb Haemost 2017; 15 (08) 1668-1678
• 49 Newton AC. Protein kinase C: perfectly balanced. Crit Rev Biochem Mol Biol 2018; 53 (02) 208-230
• 50 Heemskerk JWM, Harper MT, Cosemans JM, Poole AW. Unravelling the different functions of protein kinase C isoforms in platelets. FEBS Lett 2011; 585 (12) 1711-1716
• 51 Gilio K, Harper MT, Cosemans JM. et al. Functional divergence of platelet protein kinase C (PKC) isoforms in thrombus formation on collagen. J Biol Chem 2010; 285 (30) 23410-23419
• 52 Yacoub D, Théorêt JF, Villeneuve L. et al. Essential role of protein kinase C delta in platelet signaling, alpha IIb beta 3 activation, and thromboxane A2 release. J Biol Chem 2006; 281 (40) 30024-30035
• 53 Mócsai A, Ruland J, Tybulewicz VLJ. The SYK tyrosine kinase: a crucial player in diverse biological functions. Nat Rev Immunol 2010; 10 (06) 387-402
• 54 Bradshaw JM. The Src, Syk, and Tec family kinases: distinct types of molecular switches. Cell Signal 2010; 22 (08) 1175-1184
• 55 Mansueto MS, Reens A, Rakhilina L, Chi A, Pan BS, Miller JR. A reevaluation of the spleen tyrosine kinase (SYK) activation mechanism. J Biol Chem 2019; 294 (19) 7658-7668
• 56 Grädler U, Schwarz D, Dresing V. et al. Structural and biophysical characterization of the Syk activation switch. J Mol Biol 2013; 425 (02) 309-333
• 57 Vang T, Torgersen KM, Sundvold V. et al. Activation of the COOH-terminal Src kinase (Csk) by cAMP-dependent protein kinase inhibits signaling through the T cell receptor. J Exp Med 2001; 193 (04) 497-507
• 58 Abrahamsen H, Vang T, Taskén K. Protein kinase A intersects SRC signaling in membrane microdomains. J Biol Chem 2003; 278 (19) 17170-17177
• 59 Obergfell A, Eto K, Mocsai A. et al. Coordinate interactions of Csk, Src, and Syk kinases with [alpha]IIb[beta]3 initiate integrin signaling to the cytoskeleton. J Cell Biol 2002; 157 (02) 265-275
• 60 Schmidt GJ, Reumiller CM, Ercan H. et al. Comparative proteomics reveals unexpected quantitative phosphorylation differences linked to platelet activation state. Sci Rep 2019; 9 (01) 19009
• 61 Makhoul S, Trabold K, Gambaryan S. et al. cAMP- and cGMP-elevating agents inhibit GPIbα-mediated aggregation but not GPIbα-stimulated Syk activation in human platelets. Cell Commun Signal 2019; 17 (01) 122
• 62 Makhoul S, Dorschel S, Gambaryan S, Walter U, Jurk K. Feedback regulation of Syk by protein kinase C in human platelets. Int J Mol Sci 2019; 21 (01) 21
• 63 Makhoul S, Kumm E, Zhang P, Walter U, Jurk K. The serine/threonine protein phosphatase 2A (PP2A) regulates Syk activity in human platelets. Int J Mol Sci 2020; 21 (23) 21
• 64 Bohnenberger H, Oellerich T, Engelke M, Hsiao HH, Urlaub H, Wienands J. Complex phosphorylation dynamics control the composition of the Syk interactome in B cells. Eur J Immunol 2011; 41 (06) 1550-1562
• 65 Buffard M, Naldi A, Freiss G. et al. Comparison of SYK signaling networks reveals the potential molecular determinants of its tumor-promoting and suppressing functions. Biomolecules 2021; 11 (02) 308
• 66 Krisenko MO, Geahlen RL. Calling in SYK: SYK's dual role as a tumor promoter and tumor suppressor in cancer. Biochim Biophys Acta 2015; 1853 (01) 254-263
• 67 Senis YA. Protein-tyrosine phosphatases: a new frontier in platelet signal transduction. J Thromb Haemost 2013; 11 (10) 1800-1813
• 68 Tautz L, Senis YA, Oury C, Rahmouni S. Perspective: tyrosine phosphatases as novel targets for antiplatelet therapy. Bioorg Med Chem 2015; 23 (12) 2786-2797
• 69 Kumm EJ, Pagel O, Gambaryan S. et al. The cell cycle checkpoint system MAST(L)-ENSA/ARPP19-PP2A is targeted by cAMP/PKA and cGMP/PKG in anucleate human platelets. Cells 2020; 9 (02) 9
• 70 MacDonald JA, Walsh MP. Regulation of smooth muscle myosin light chain phosphatase by multisite phosphorylation of the myosin targeting subunit, MYPT1. Cardiovasc Hematol Disord Drug Targets 2018; 18 (01) 4-13
• 71 Reynhout S, Janssens V. Physiologic functions of PP2A: lessons from genetically modified mice. Biochim Biophys Acta Mol Cell Res 2019; 1866 (01) 31-50
• 72 Mochida S, Maslen SL, Skehel M, Hunt T. Greatwall phosphorylates an inhibitor of protein phosphatase 2A that is essential for mitosis. Science 2010; 330 (6011): 1670-1673
• 73 Gharbi-Ayachi A, Labbé JC, Burgess A. et al. The substrate of Greatwall kinase, Arpp19, controls mitosis by inhibiting protein phosphatase 2A. Science 2010; 330 (6011): 1673-1677
• 74 Castro A, Lorca T. Greatwall kinase at a glance. J Cell Sci 2018; 131 (20) 131
• 75 Meeusen B, Janssens V. Tumor suppressive protein phosphatases in human cancer: Emerging targets for therapeutic intervention and tumor stratification. Int J Biochem Cell Biol 2018; 96: 98-134
• 76 Morita K, He S, Nowak RP. et al. Allosteric activators of protein phosphatase 2A display broad antitumor activity mediated by dephosphorylation of MYBL2. Cell 2020; 181 (03) 702-715.e20
• 77 Leonard D, Huang W, Izadmehr S. et al. Selective PP2A enhancement through biased heterotrimer stabilization. Cell 2020; 181 (03) 688-701.e16
• 78 Westermarck J, Neel BG. Piecing together a broken tumor suppressor phosphatase for cancer therapy. Cell 2020; 181 (03) 514-517
• 79 Yue M, Luo D, Yu S. et al. Misshapen/NIK-related kinase (MINK1) is involved in platelet function, hemostasis, and thrombus formation. Blood 2016; 127 (07) 927-937
• 80 Fan X, Wang C, Shi P. et al. Platelet MEKK3 regulates arterial thrombosis and myocardial infarct expansion in mice. Blood Adv 2018; 2 (12) 1439-1448
• 81 Shankar H, Garcia A, Prabhakar J, Kim S, Kunapuli SP. P2Y12 receptor-mediated potentiation of thrombin-induced thromboxane A2 generation in platelets occurs through regulation of Erk1/2 activation. J Thromb Haemost 2006; 4 (03) 638-647
• 82 Sledz KM, Moore SF, Vijayaragavan V. et al. Redundant role of ASK1-mediated p38MAPK activation in human platelet function. Cell Signal 2020; 68: 109528
• 83 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
• 84 Kamiyama M, Shirai T, Tamura S. et al. ASK1 facilitates tumor metastasis through phosphorylation of an ADP receptor P2Y₁₂ in platelets. Cell Death Differ 2017; 24 (12) 2066-2076
• 85 Liu Q, Hofmann PA. Protein phosphatase 2A-mediated cross-talk between p38 MAPK and ERK in apoptosis of cardiac myocytes. Am J Physiol Heart Circ Physiol 2004; 286 (06) H2204-H2212
• 86 Tullemans BME, Heemskerk JWM, Kuijpers MJE. Acquired platelet antagonism: off-target antiplatelet effects of malignancy treatment with tyrosine kinase inhibitors. J Thromb Haemost 2018; 16 (09) 1686-1699
• 87 Busygina K, Denzinger V, Bernlochner I, Weber C, Lorenz R, Siess W. Btk inhibitors as first oral atherothrombosis-selective antiplatelet drugs?. Thromb Haemost 2019; 119 (08) 1212-1221
• 88 Series J, Ribes A, Garcia C. et al. Effects of novel Btk and Syk inhibitors on platelet functions alone and in combination in vitro and in vivo. J Thromb Haemost 2020; 18 (12) 3336-3351
• 89 Sangodkar J, Farrington CC, McClinch K, Galsky MD, Kastrinsky DB, Narla G. All roads lead to PP2A: exploiting the therapeutic potential of this phosphatase. FEBS J 2016; 283 (06) 1004-1024
• 90 O'Connor CM, Perl A, Leonard D, Sangodkar J, Narla G. Therapeutic targeting of PP2A. Int J Biochem Cell Biol 2018; 96: 182-193
• 91 Séverin S, Ghevaert C, Mazharian A. The mitogen-activated protein kinase signaling pathways: role in megakaryocyte differentiation. J Thromb Haemost 2010; 8 (01) 17-26
• 92 Di Buduo CA, Currao M, Pecci A, Kaplan DL, Balduini CL, Balduini A. Revealing eltrombopag's promotion of human megakaryopoiesis through AKT/ERK-dependent pathway activation. Haematologica 2016; 101 (12) 1479-1488
• 93 Bluteau D, Balduini A, Balayn N. et al. Thrombocytopenia-associated mutations in the ANKRD26 regulatory region induce MAPK hyperactivation. J Clin Invest 2014; 124 (02) 580-591
• 94 Desterke C, Bilhou-Nabéra C, Guerton B. et al; French Intergroup of Myeloproliferative Disorders, French INSERM, European EUMNET Networks on Myelofibrosis. FLT3-mediated p38-MAPK activation participates in the control of megakaryopoiesis in primary myelofibrosis. Cancer Res 2011; 71 (08) 2901-2915
• 95 Hurtado B, Trakala M, Ximénez-Embún P. et al. Thrombocytopenia-associated mutations in Ser/Thr kinase MASTL deregulate actin cytoskeletal dynamics in platelets. J Clin Invest 2018; 128 (12) 5351-5367
• 96 Bekker-Jensen DB, Bernhardt OM, Hogrebe A. et al. Rapid and site-specific deep phosphoproteome profiling by data-independent acquisition without the need for spectral libraries. Nat Commun 2020; 11 (01) 787
• 97 Roskoski Jr R. Properties of FDA-approved small molecule protein kinase inhibitors: a 2020 update. Pharmacol Res 2020; 152: 104609
• 98 Babur Ö, Melrose AR, Cunliffe JM. et al. Phosphoproteomic quantitation and causal analysis reveal pathways in GPVI/ITAM-mediated platelet activation programs. Blood 2020; 136 (20) 2346-2358
• 99 Aslan JE. Platelet proteomes, pathways, and phenotypes as informants of vascular wellness and disease. Arterioscler Thromb Vasc Biol 2021; 41 (03) 999-1011
• 100 Stefanini L, Lee RH, Bergmeier W. GTPases. In: Platelets in Thrombotic and Non-Thrombotic Disorders: Pathophysiology, Pharmacology and Therapeutics: An Update. Springer International Publishing; 2017: 263-284
• 101 Lee RH, Stefanini L, Bergmeier W. Platelet signal transduction. In: Platelets. 4th ed.. Academic Press; 2019: 329-348
• 102 Hubertus K, Mischnik M, Timmer J. et al. Reciprocal regulation of human platelet function by endogenous prostanoids and through multiple prostanoid receptors. Eur J Pharmacol 2014; 740: 15-27
• 103 Yeung J, Li W, Holinstat M. Platelet signaling and disease: targeted therapy for thrombosis and other related diseases. Pharmacol Rev 2018; 70 (03) 526-548
• 104 Chatterjee M, Rath D, Gawaz M. Role of chemokine receptors CXCR4 and CXCR7 for platelet function. Biochem Soc Trans 2015; 43 (04) 720-726
• 105 Cheng Z, Gao W, Fan X. et al. Extracellular signal-regulated kinase 5 associates with casein kinase II to regulate GPIb-IX-mediated platelet activation via the PTEN/PI3K/Akt pathway. J Thromb Haemost 2017; 15 (08) 1679-1688
• 106 Yang M, Kholmukhamedov A, Schulte ML. et al. Platelet CD36 signaling through ERK5 promotes caspase-dependent procoagulant activity and fibrin deposition in vivo. Blood Adv 2018; 2 (21) 2848-2861
• 107 Manikanta K, Naveen Kumar SK, Hemshekhar M, Kemparaju K, Girish KS. ASK1 inhibition triggers platelet apoptosis via p38-MAPK-mediated mitochondrial dysfunction. Haematologica 2020; 105 (08) e419-e423