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Thromb Haemost 2023; 123(06): 597-612
DOI: 10.1055/s-0043-1761463
Cellular Haemostasis and Platelets

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

Natalie J. Jooss 
1   Department of Biochemistry, Cardiovascular Research Institute, Maastricht University, Maastricht, The Netherlands
2   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom
,
Yvonne M.C. Henskens
3   Central Diagnostic Laboratory, Maastricht University Medical Centre, Maastricht, The Netherlands
,
Steve P. Watson
2   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom
4   Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, Midlands, United Kingdom
,
Richard W. Farndale 
5   Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
6   CambCol Laboratories, Ely, United Kingdom
,
Meinrad P. Gawaz
7   Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany
,
Martine Jandrot-Perrus
8   UMR_S1148, Laboratory for Vascular Translational Science, INSERM, University Paris Cité, Paris, France
,
Natalie S. Poulter
2   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom
4   Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, Midlands, United Kingdom
,
Johan W. M. Heemskerk 
1   Department of Biochemistry, Cardiovascular Research Institute, Maastricht University, Maastricht, The Netherlands
9   Synapse Research Institute, Maastricht, The Netherlands
› Author Affiliations Funding This work has received funding form the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 766118 (TAPAS). This project was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—project number 374031971-TRR 240 (M.P.G.). N.J.J. is enrolled in a joint PhD program of the Universities of Maastricht and Birmingham (United Kingdom).
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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.


Supplementary Material



Publication History

Received: 22 May 2022

Accepted: 20 December 2022

Article published online:
17 February 2023

© 2023. Thieme. All rights reserved.

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  • 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
    CrossrefPubMedSearch in Google Scholar
  • 2 van der Meijden PEJ, Heemskerk JWM. Platelet biology and functions: new concepts and clinical perspectives. Nat Rev Cardiol 2019; 16 (03) 166-179
    CrossrefPubMedSearch in Google Scholar
  • 3 Soehnlein O, Libby P. Targeting inflammation in atherosclerosis - from experimental insights to the clinic. Nat Rev Drug Discov 2021; 20 (08) 589-610
    CrossrefPubMedSearch in Google Scholar
  • 4 Borst O, Gawaz M. Glycoprotein VI - novel target in antiplatelet medication. Pharmacol Ther 2021; 217: 107630
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 9 Andrews RK, Arthur JF, Gardiner EE. Targeting GPVI as a novel antithrombotic strategy. J Blood Med 2014; 5: 59-68
    PubMedSearch in Google Scholar
  • 10 Jandrot-Perrus M, Hermans C, Mezzano D. Platelet glycoprotein VI genetic quantitative and qualitative defects. Platelets 2019; 30 (06) 708-713
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 12 Nieswandt B, Watson SP. Platelet-collagen interaction: is GPVI the central receptor?. Blood 2003; 102 (02) 449-461
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    Thieme ConnectPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    Thieme ConnectPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    Thieme ConnectPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 31 Accessed April 2022 at: https://clinicaltrials.gov
    PubMed
  • 32 Mangin PH, Onselaer MB, Receveur N. et al. Immobilized fibrinogen activates human platelets through glycoprotein VI. Haematologica 2018; 103 (05) 898-907
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 37 De Witt S, Swieringa F, Cosemans JM, Heemkerk JW. Thrombus formation on microspotted arrays of thrombogenic surfaces. Nat Protocol Exchange. 2014;2014:3309#
    PubMed
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar