Platelet Activation Pathways Controlling Reversible Integrin αIIbβ3 Activation

Abstract

Background Agonist-induced platelet activation, with the integrin αIIbβ3 conformational change, is required for fibrinogen binding. This is considered reversible under specific conditions, allowing a second phase of platelet aggregation. The signaling pathways that differentiate between a permanent or transient activation state of platelets are poorly elucidated.

Objective To explore platelet signaling mechanisms induced by the collagen receptor glycoprotein VI (GPVI) or by protease-activated receptors (PAR) for thrombin that regulate time-dependent αIIbβ3 activation.

Methods Platelets were activated with collagen-related peptide (CRP, stimulating GPVI), thrombin receptor-activating peptides, or thrombin (stimulating PAR1 and/or 4). Integrin αIIbβ3 activation and P-selectin expression was assessed by two-color flow cytometry. Signaling pathway inhibitors were applied before or after agonist addition. Reversibility of platelet spreading was studied by microscopy.

Results Platelet pretreatment with pharmacological inhibitors decreased GPVI- and PAR-induced integrin αIIbβ3 activation and P-selectin expression in the target order of protein kinase C (PKC) > glycogen synthase kinase 3 > β-arrestin > phosphatidylinositol-3-kinase. Posttreatment revealed secondary αIIbβ3 inactivation (not P-selectin expression), in the same order, but this reversibility was confined to CRP and PAR1 agonist. Combined inhibition of conventional and novel PKC isoforms was most effective for integrin closure. Pre- and posttreatment with ticagrelor, blocking the P2Y₁₂ adenosine diphosphate (ADP) receptor, enhanced αIIbβ3 inactivation. Spreading assays showed that PKC or P2Y₁₂ inhibition provoked a partial conversion from filopodia to a more discoid platelet shape.

Conclusion PKC and autocrine ADP signaling contribute to persistent integrin αIIbβ3 activation in the order of PAR1/GPVI > PAR4 stimulation and hence to stabilized platelet aggregation. These findings are relevant for optimization of effective antiplatelet treatment.

Keywords

integrin αIIbβ3 - glycoprotein VI - P-selectin - protease-activated receptors - protein kinase C

• References

• 1 Huang J, Li X, Shi X. et al. Platelet integrin αIIbβ3: signal transduction, regulation, and its therapeutic targeting. J Hematol Oncol 2019; 12: 1-22
• 2 Versteeg HH, Heemskerk JW, Levi M, Reitsma PH. New fundamentals in hemostasis. Physiol Rev 2013; 93 (01) 327-358
• 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 Zou J, Swieringa F, de Laat B, de Groot PG, Roest M, Heemskerk JWM. Reversible platelet integrin αIIbβ3 activation and thrombus instability. Int J Mol Sci 2022; 23 (20) 12512
• 5 Kauskot A, Di Michele M, Loyen S, Freson K, Verhamme P, Hoylaerts MF. A novel mechanism of sustained platelet αIIbβ3 activation via PEAR1. Blood 2012; 119 (17) 4056-4065
• 6 Nergiz-Unal R, Cosemans JM, Feijge MA. et al. Stabilizing role of platelet P2Y(12) receptors in shear-dependent thrombus formation on ruptured plaques. PLoS One 2010; 5 (04) e10130
• 7 Trumel C, Payrastre B, Plantavid M. et al. A key role of adenosine diphosphate in the irreversible platelet aggregation induced by the PAR1-activating peptide through the late activation of phosphoinositide 3-kinase. Blood 1999; 94 (12) 4156-4165
• 8 Ahmed MU, Kaneva V, Loyau S. et al. Pharmacological blockade of glycoprotein VI promotes thrombus disaggregation in the absence of thrombin. Arterioscler Thromb Vasc Biol 2020; 40 (09) 2127-2142
• 9 Perrella G, Montague SJ, Brown HC. et al. Role of tyrosine kinase Syk in thrombus stabilisation at high shear. Int J Mol Sci 2022; 23 (01) 493
• 10 De Simone I, Baaten CCFMJ, Gibbins JM. et al. Repeated platelet activation and the potential of previously activated platelets to contribute to thrombus formation. J Thromb Haemost 2023; 21 (05) 1289-1306
• 11 van Willigen G, Akkerman JW. Protein kinase C and cyclic AMP regulate reversible exposure of binding sites for fibrinogen on the glycoprotein IIB-IIIA complex of human platelets. Biochem J 1991; 273 (Pt 1): 115-120
• 12 Siess W, Lapetina EG. Platelet aggregation induced by α 2-adrenoceptor and protein kinase C activation. A novel synergism. Biochem J 1989; 263 (02) 377-385
• 13 Mattheij NJ, Gilio K, van Kruchten R. et al. Dual mechanism of integrin αIIbβ3 closure in procoagulant platelets. J Biol Chem 2013; 288 (19) 13325-13336
• 14 Quinton TM, Kim S, Dangelmaier C. et al. Protein kinase C- and calcium-regulated pathways independently synergize with GI pathways in agonist-induced fibrinogen receptor activation. Biochem J 2002; 368 (Pt 2): 535-543
• 15 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
• 16 Heemskerk JW, Harper MT, Cosemans JM, Poole AW. Unravelling the different functions of protein kinase C isoforms in platelets. FEBS Lett 2011; 585 (12) 1711-1716
• 17 Konopatskaya O, Gilio K, Harper MT. et al. PKCalpha regulates platelet granule secretion and thrombus formation in mice. J Clin Invest 2009; 119 (02) 399-407
• 18 Gilio K, Munnix IC, Mangin P. et al. Non-redundant roles of phosphoinositide 3-kinase isoforms α and β in glycoprotein VI-induced platelet signaling and thrombus formation. J Biol Chem 2009; 284 (49) 33750-33762
• 19 Kauffenstein G, Bergmeier W, Eckly A. et al. The P2Y(12) receptor induces platelet aggregation through weak activation of the α(IIb)β(3) integrin–a phosphoinositide 3-kinase-dependent mechanism. FEBS Lett 2001; 505 (02) 281-290
• 20 Hechler B, Dupuis A, Mangin PH, Gachet C. Platelet preparation for function testing in the laboratory and clinic: historical and practical aspects. Res Pract Thromb Haemost 2019; 3 (04) 615-625
• 21 Veninga A, Baaten CCFMJ, De Simone I. et al. Effects of platelet agonists and priming on the formation of platelet populations. Thromb Haemost 2022; 122 (05) 726-738
• 22 Tengah A, Syed NI, Talip STA, Bujang SNB, Kennedy C. Comparison of signalling mechanisms underlying UTP-evoked vasoconstriction of rat pulmonary and tail arteries. Eur J Pharmacol 2018; 837: 45-52
• 23 van Kruchten R, Mattheij NJ, Saunders C. et al. Both TMEM16F-dependent and TMEM16F-independent pathways contribute to phosphatidylserine exposure in platelet apoptosis and platelet activation. Blood 2013; 121 (10) 1850-1857
• 24 Acikgoz E, Güler G, Camlar M, Oktem G, Aktug H. Glycogen synthase kinase-3 inhibition in glioblastoma multiforme cells induces apoptosis, cell cycle arrest and changing biomolecular structure. Spectrochim Acta A Mol Biomol Spectrosc 2019; 209: 150-164
• 25 Barak LS, Bai Y, Peterson S. et al. ML314: a biased neurotensin receptor ligand for methamphetamine abuse. ACS Chem Biol 2016; 11 (07) 1880-1890
• 26 Peddibhotla S, Hedrick MP, Hershberger P. et al. Discovery of ML314, a brain penetrant nonpeptidic β-arrestin biased agonist of the neurotensin NTR1 receptor. ACS Med Chem Lett 2013; 4 (09) 846-851
• 27 Slosky LM, Caron MG, Barak LS. Biased allosteric modulators: new frontiers in GPCR drug discovery. Trends Pharmacol Sci 2021; 42 (04) 283-299
• 28 Bumbak F, Bower JB, Zemmer SC. et al. Stabilization of pre-existing neurotensin receptor conformational states by β-arrestin-1 and the biased allosteric modulator ML314. Nat Commun 2023; 14 (01) 3328
• 29 Fernández DI, Kuijpers MJE, Heemskerk JWM. Platelet calcium signaling by G-protein coupled and ITAM-linked receptors regulating anoctamin-6 and procoagulant activity. Platelets 2021; 32 (07) 863-871
• 30 Surviladze Z, Ursu O, Miscioscia F. et al. Three small molecule pan activator families of Ras-related GTPases. Probe Reports from the NIH Molecular Libraries Program. Bethesda (MD):: National Center for Biotechnology Information;; 2010
• 31 Baaten CC, Veenstra LF, Wetzels R. et al. Gradual increase in thrombogenicity of juvenile platelets formed upon offset of prasugrel medication. Haematologica 2015; 100 (09) 1131-1138
• 32 Grippi C, Izzi B, Gianfagna F. et al. Neuromedin U potentiates ADP- and epinephrine-induced human platelet activation. Thromb Res 2017; 159: 100-108
• 33 Cheung HYF, Zou J, Tantiwong C. et al. High-throughput assessment identifying major platelet Ca²⁺ entry pathways via tyrosine kinase-linked and G protein-coupled receptors. Cell Calcium 2023; 112: 102738
• 34 Navarro S, Stegner D, Nieswandt B, Heemskerk JWM, Kuijpers MJE. Temporal roles of platelet and coagulation pathways in collagen-and tissue factor-induced thrombus formation. Int J Mol Sci 2021; 23 (01) 358
• 35 Zou J, Wu J, Roest M, Heemskerk JWM. Long-term platelet priming after glycoprotein VI stimulation in comparison to protease-activating receptor (PAR) stimulation. PLoS One 2021; 16 (03) e0247425
• 36 Eckly A, Rinckel J-Y, Proamer F. et al. Respective contributions of single and compound granule fusion to secretion by activated platelets. Blood 2016; 128 (21) 2538-2549
• 37 Wu CC, Wu SY, Liao CY, Teng CM, Wu YC, Kuo SC. The roles and mechanisms of PAR4 and P2Y12/phosphatidylinositol 3-kinase pathway in maintaining thrombin-induced platelet aggregation. Br J Pharmacol 2010; 161 (03) 643-658
• 38 Chari R, Getz T, Nagy Jr B. et al. Protein kinase C[delta] differentially regulates platelet functional responses. Arterioscler Thromb Vasc Biol 2009; 29 (05) 699-705
• 39 Chari R, Kim S, Murugappan S, Sanjay A, Daniel JL, Kunapuli SP. Lyn, PKC-δ, SHIP-1 interactions regulate GPVI-mediated platelet-dense granule secretion. Blood 2009; 114 (14) 3056-3063
• 40 Moore SF, Agbani EO, Wersäll A. et al. Opposing roles of GSK3α and GSK3β phosphorylation in platelet function and thrombosis. Int J Mol Sci 2021; 22 (19) 10656
• 41 Chen CH, Paing MM, Trejo J. Termination of protease-activated receptor-1 signaling by beta-arrestins is independent of receptor phosphorylation. J Biol Chem 2004; 279 (11) 10020-10031
• 42 Willis Fox O, Preston RJS. Molecular basis of protease-activated receptor 1 signaling diversity. J Thromb Haemost 2020; 18 (01) 6-16
• 43 Anastassiadis T, Deacon SW, Devarajan K, Ma H, Peterson JR. Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 2011; 29 (11) 1039-1045
• 44 Aungraheeta R, Conibear A, Butler M. et al. Inverse agonism at the P2Y12 receptor and ENT1 transporter blockade contribute to platelet inhibition by ticagrelor. Blood 2016; 128 (23) 2717-2728