Pharmacological Inhibition of Glycoprotein VI- and Integrin α2β1-Induced Thrombus Formation Modulated by the Collagen Type

Abstract

Background In secondary cardiovascular disease prevention, treatments blocking platelet-derived secondary mediators pose a risk of bleeding. Pharmacological interference of the interaction of platelets with exposed vascular collagens is an attractive alternative, with clinical trials ongoing. Antagonists of the collagen receptors, glycoprotein VI (GPVI), and integrin α2β1, include recombinant GPVI-Fc dimer construct Revacept, 9O12 mAb based on the GPVI-blocking reagent Glenzocimab, Syk tyrosine-kinase inhibitor PRT-060318, and anti-α2β1 mAb 6F1. No direct comparison has been made of the antithrombic potential of these drugs.

Methods Using a multiparameter whole-blood microfluidic assay, we compared the effects of Revacept, 9O12-Fab, PRT-060318, or 6F1 mAb intervention with vascular collagens and collagen-related substrates with varying dependencies on GPVI and α2β1. To inform on Revacept binding to collagen, we used fluorescent-labelled anti-GPVI nanobody-28.

Results and Conclusion In this first comparison of four inhibitors of platelet–collagen interactions with antithrombotic potential, we find that at arterial shear rate: (1) the thrombus-inhibiting effect of Revacept was restricted to highly GPVI-activating surfaces; (2) 9O12-Fab consistently but partly inhibited thrombus size on all surfaces; (3) effects of GPVI-directed interventions were surpassed by Syk inhibition; and (4) α2β1-directed intervention with 6F1 mAb was strongest for collagens where Revacept and 9O12-Fab were limitedly effective. Our data hence reveal a distinct pharmacological profile for GPVI-binding competition (Revacept), GPVI receptor blockage (9O12-Fab), GPVI signaling (PRT-060318), and α2β1 blockage (6F1 mAb) in flow-dependent thrombus formation, depending on the platelet-activating potential of the collagen substrate. This work thus points to additive antithrombotic action mechanisms of the investigated drugs.

Keywords

collagen - glycoprotein VI - integrin α2β1 - protein tyrosine kinase - thrombus

Authors' Contributions

N.J.J. designed and performed experiments, analyzed data, prepared figures, and wrote the manuscript. M.P.G. provided Revacept and revised the manuscript. M.J.-P. supplied 9O12-Fab and revised the manuscript. Y.M.C.H., N.S.P., and S.P.W. contributed by funding and supervision, and revised the manuscript. J.W.M.H. designed experiments, provided supervision and funding, and wrote the manuscript. All authors have read and approved the manuscript.

Publikationsverlauf

Eingereicht: 22. Mai 2022

Angenommen: 20. Dezember 2022

Artikel online veröffentlicht:
17. Februar 2023

• References

• 1 Nieswandt B, Pleines I, Bender M. Platelet adhesion and activation mechanisms in arterial thrombosis and ischaemic stroke. J Thromb Haemost 2011; 9 (Suppl. 01) 92-104
• 2 van der Meijden PEJ, Heemskerk JWM. Platelet biology and functions: new concepts and clinical perspectives. Nat Rev Cardiol 2019; 16 (03) 166-179
• 3 Soehnlein O, Libby P. Targeting inflammation in atherosclerosis - from experimental insights to the clinic. Nat Rev Drug Discov 2021; 20 (08) 589-610
• 4 Borst O, Gawaz M. Glycoprotein VI - novel target in antiplatelet medication. Pharmacol Ther 2021; 217: 107630
• 5 Boulaftali Y, Mawhin MA, Jandrot-Perrus M, Ho-Tin-Noé B. Glycoprotein VI in securing vascular integrity in inflamed vessels. Res Pract Thromb Haemost 2018; 2 (02) 228-239
• 6 Rayes J, Watson SP, Nieswandt B. Functional significance of the platelet immune receptors GPVI and CLEC-2. J Clin Invest 2019; 129 (01) 12-23
• 7 Baaten CCFMJ, Meacham S, de Witt SM. et al. A synthesis approach of mouse studies to identify genes and proteins in arterial thrombosis and bleeding. Blood 2018; 132 (24) e35-e46
• 8 Mackman N, Bergmeier W, Stouffer GA, Weitz JI. Therapeutic strategies for thrombosis: new targets and approaches. Nat Rev Drug Discov 2020; 19 (05) 333-352
• 9 Andrews RK, Arthur JF, Gardiner EE. Targeting GPVI as a novel antithrombotic strategy. J Blood Med 2014; 5: 59-68
• 10 Jandrot-Perrus M, Hermans C, Mezzano D. Platelet glycoprotein VI genetic quantitative and qualitative defects. Platelets 2019; 30 (06) 708-713
• 11 Nagy M, Perrella G, Dalby A. et al. Flow studies on human GPVI-deficient blood under coagulating and noncoagulating conditions. Blood Adv 2020; 4 (13) 2953-2961
• 12 Nieswandt B, Watson SP. Platelet-collagen interaction: is GPVI the central receptor?. Blood 2003; 102 (02) 449-461
• 13 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
• 14 Agbani EO, van den Bosch MT, Brown E. et al. Coordinated membrane ballooning and procoagulant spreading in human platelets. Circulation 2015; 132 (15) 1414-1424
• 15 Pugh N, Simpson AM, Smethurst PA, de Groot PG, Raynal N, Farndale RW. Synergism between platelet collagen receptors defined using receptor-specific collagen-mimetic peptide substrata in flowing blood. Blood 2010; 115 (24) 5069-5079
• 16 Jooss NJ, De Simone I, Provenzale I. et al. Role of platelet glycoprotein VI and tyrosine kinase Syk in thrombus formation on collagen-like surfaces. Int J Mol Sci 2019; 20 (11) 2788
• 17 Perrella G, Huang J, Provenzale I. et al. Nonredundant roles of platelet glycoprotein VI and integrin αIIbβ3 in fibrin-mediated microthrombus formation. Arterioscler Thromb Vasc Biol 2021; 41 (02) e97-e111
• 18 Nieuwenhuis HK, Akkerman JW, Houdijk WP, Sixma JJ. Human blood platelets showing no response to collagen fail to express surface glycoprotein Ia. Nature 1985; 318 (6045): 470-472
• 19 Holtkötter O, Nieswandt B, Smyth N. et al. Integrin α 2-deficient mice develop normally, are fertile, but display partially defective platelet interaction with collagen. J Biol Chem 2002; 277 (13) 10789-10794
• 20 Auger JM, Kuijpers MJ, Senis YA, Watson SP, Heemskerk JW. Adhesion of human and mouse platelets to collagen under shear: a unifying model. FASEB J 2005; 19 (07) 825-827
• 21 Sarratt KL, Chen H, Zutter MM, Santoro SA, Hammer DA, Kahn ML. GPVI and α2β1 play independent critical roles during platelet adhesion and aggregate formation to collagen under flow. Blood 2005; 106 (04) 1268-1277
• 22 Lecut C, Schoolmeester A, Kuijpers MJ. et al. Principal role of glycoprotein VI in α2β1 and alphaIIbbeta3 activation during collagen-induced thrombus formation. Arterioscler Thromb Vasc Biol 2004; 24 (09) 1727-1733
• 23 Pugh N, Maddox BD, Bihan D, Taylor KA, Mahaut-Smith MP, Farndale RW. Differential integrin activity mediated by platelet collagen receptor engagement under flow conditions. Thromb Haemost 2017; 117 (08) 1588-1600
• 24 Jamasbi J, Megens RT, Bianchini M. et al. Cross-linking GPVI-Fc by anti-Fc antibodies potentiates its inhibition of atherosclerotic plaque- and collagen-induced platelet activation. JACC Basic Transl Sci 2016; 1 (03) 131-142
• 25 Mojica Muñoz AK, Jamasbi J, Uhland K. et al. Recombinant GPVI-Fc added to single or dual antiplatelet therapy in vitro prevents plaque-induced platelet thrombus formation. Thromb Haemost 2017; 117 (08) 1651-1659
• 26 Massberg S, Konrad I, Bültmann A. et al. Soluble glycoprotein VI dimer inhibits platelet adhesion and aggregation to the injured vessel wall in vivo. FASEB J 2004; 18 (02) 397-399
• 27 Gröschel K, Uphaus T, Loftus I. et al. Revacept, an inhibitor of platelet adhesion in symptomatic carotid artery stenosis: design and rationale of a randomized phase II clinical trial. TH Open 2020; 4 (04) e393-e399
• 28 Mayer K, Hein-Rothweiler R, Schüpke S. et al. Efficacy and safety of revacept, a novel lesion-directed competitive antagonist to platelet glycoprotein VI, in patients undergoing elective percutaneous coronary intervention for stable ischemic heart disease: the randomized, double-blind, placebo-controlled ISAR-PLASTER phase 2 trial. JAMA Cardiol 2021; 6 (07) 753-761
• 29 Voors-Pette C, Lebozec K, Dogterom P. et al. Safety and tolerability, pharmacokinetics, and pharmacodynamics of ACT017, an antiplatelet GPVI (glycoprotein VI) Fab. Arterioscler Thromb Vasc Biol 2019; 39 (05) 956-964
• 30 Jadoui S, Le Chapelain O, Ollivier V. et al. Glenzocimab does not impact glycoprotein VI-dependent inflammatory haemostasis. Haematologica 2021; 106 (07) 2000-2003
• 31 Accessed April 2022 at: https://clinicaltrials.gov
• 32 Mangin PH, Onselaer MB, Receveur N. et al. Immobilized fibrinogen activates human platelets through glycoprotein VI. Haematologica 2018; 103 (05) 898-907
• 33 Alenazy FO, Harbi MH, Kavanagh DP. et al. GPVI inhibition by glenzocimab synergistically inhibits atherosclerotic plaque-induced platelet activation when combined with conventional dual antiplatelet therapy. Eur Heart J 2021; 42: ehab724.1425
• 34 Penz S, Reininger AJ, Brandl R. et al. Human atheromatous plaques stimulate thrombus formation by activating platelet glycoprotein VI. FASEB J 2005; 19 (08) 898-909
• 35 van Geffen JP, Brouns SLN, Batista J. et al. High-throughput elucidation of thrombus formation reveals sources of platelet function variability. Haematologica 2019; 104 (06) 1256-1267
• 36 Smethurst PA, Joutsi-Korhonen L, O'Connor MN. et al. Identification of the primary collagen-binding surface on human glycoprotein VI by site-directed mutagenesis and by a blocking phage antibody. Blood 2004; 103 (03) 903-911
• 37 De Witt S, Swieringa F, Cosemans JM, Heemkerk JW. Thrombus formation on microspotted arrays of thrombogenic surfaces. Nat Protocol Exchange. 2014;2014:3309#
• 38 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
• 39 Slater A, Di Y, Clark JC. et al. Structural characterization of a novel GPVI-nanobody complex reveals a biologically active domain-swapped GPVI dimer. Blood 2021; 137 (24) 3443-3453
• 40 de Witt SM, Swieringa F, Cavill R. et al. Identification of platelet function defects by multi-parameter assessment of thrombus formation. Nat Commun 2014; 5: 4257
• 41 Huang J, Jooss NJ, Fernández DI. et al. Roles of focal adhesion kinase PTK2 and integrin αIIbβ3 signaling in collagen- and GPVI-dependent thrombus formation under shear. Int J Mol Sci 2022; 23 (15) 8688
• 42 Munnix IC, Gilio K, Siljander PR. et al. Collagen-mimetic peptides mediate flow-dependent thrombus formation by high- or low-affinity binding of integrin α2β1 and glycoprotein VI. J Thromb Haemost 2008; 6 (12) 2132-2142
• 43 Henrita van Zanten G, Saelman EU, Schut-Hese KM. et al. Platelet adhesion to collagen type IV under flow conditions. Blood 1996; 88 (10) 3862-3871
• 44 Morton LF, Peachey AR, Knight CG, Farndale RW, Barnes MJ. The platelet reactivity of synthetic peptides based on the collagen III fragment α1(III)CB4. Evidence for an integrin α2β1 recognition site involving residues 522-528 of the α1(III) collagen chain. J Biol Chem 1997; 272 (17) 11044-11048
• 45 Farndale RW, Sixma JJ, Barnes MJ, de Groot PG. The role of collagen in thrombosis and hemostasis. J Thromb Haemost 2004; 2 (04) 561-573
• 46 Savage B, Almus-Jacobs F, Ruggeri ZM. Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow. Cell 1998; 94 (05) 657-666
• 47 Jung SM, Takemura Y, Imamura Y, Hayashi T, Adachi E, Moroi M. Collagen-type specificity of glycoprotein VI as a determinant of platelet adhesion. Platelets 2008; 19 (01) 32-42
• 48 Poulter NS, Pollitt AY, Owen DM. et al. Clustering of glycoprotein VI (GPVI) dimers upon adhesion to collagen as a mechanism to regulate GPVI signaling in platelets. J Thromb Haemost 2017; 15 (03) 549-564
• 49 Jooss NJ, Smith CW, Slater A. et al. Anti-GPVI nanobody blocks collagen- and atherosclerotic plaque-induced GPVI clustering, signaling, and thrombus formation. J Thromb Haemost 2022; 20 (11) 2617-2631
• 50 Ungerer M, Rosport K, Bültmann A. et al. Novel antiplatelet drug revacept (Dimeric Glycoprotein VI-Fc) specifically and efficiently inhibited collagen-induced platelet aggregation without affecting general hemostasis in humans. Circulation 2011; 123 (17) 1891-1899
• 51 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
• 52 van der Meijden PE, Feijge MA, Swieringa F. et al. Key role of integrin αIIbβ3 signaling to Syk kinase in tissue factor-induced thrombin generation. Cell Mol Life Sci 2012; 69 (20) 3481-3492
• 53 Nieswandt B, Brakebusch C, Bergmeier W. et al. Glycoprotein VI but not α2β1 integrin is essential for platelet interaction with collagen. EMBO J 2001; 20 (09) 2120-2130
• 54 He L, Pappan LK, Grenache DG. et al. The contributions of the α 2 β 1 integrin to vascular thrombosis in vivo. Blood 2003; 102 (10) 3652-3657
• 55 Mita M, Kelly KR, Mita A. et al. Phase I study of E7820, an oral inhibitor of integrin α-2 expression with antiangiogenic properties, in patients with advanced malignancies. Clin Cancer Res 2011; 17 (01) 193-200
• 56 Schulz C, Penz S, Hoffmann C. et al. Platelet GPVI binds to collagenous structures in the core region of human atheromatous plaque and is critical for atheroprogression in vivo. Basic Res Cardiol 2008; 103 (04) 356-367
• 57 Busygina K, Jamasbi J, Seiler T. et al. Oral Bruton tyrosine kinase inhibitors selectively block atherosclerotic plaque-triggered thrombus formation in humans. Blood 2018; 131 (24) 2605-2616
• 58 Harbi MH, Smith CW, Nicolson PLR, Watson SP, Thomas MR. Novel antiplatelet strategies targeting GPVI, CLEC-2 and tyrosine kinases. Platelets 2021; 32 (01) 29-41
• 59 Hughes DM, Toste C, Nelson C, Escalon J, Blevins F, Shah B. Transitioning from thrombopoietin agonists to the novel Syk inhibitor fostamatinib: a multicenter, real-world case series. J Adv Pract Oncol 2021; 12 (05) 508-517
• 60 Renaud L, Lebozec K, Voors-Pette C. et al. Population pharmacokinetic/pharmacodynamic modeling of glenzocimab (ACT017) a glycoprotein VI inhibitor of collagen-induced platelet aggregation. J Clin Pharmacol 2020; 60 (09) 1198-1208