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

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.

[1]  A. Sickmann,et al.  Roles of Focal Adhesion Kinase PTK2 and Integrin αIIbβ3 Signaling in Collagen- and GPVI-Dependent Thrombus Formation under Shear , 2022, International journal of molecular sciences.

[2]  Natalie S. Poulter,et al.  Anti‐GPVI nanobody blocks collagen‐ and atherosclerotic plaque–induced GPVI clustering, signaling, and thrombus formation , 2022, Journal of thrombosis and haemostasis : JTH.

[3]  B. Nieswandt,et al.  Temporal Roles of Platelet and Coagulation Pathways in Collagen- and Tissue Factor-Induced Thrombus Formation , 2021, International journal of molecular sciences.

[4]  P. Kirchhof,et al.  GPVI inhibition by glenzocimab synergistically inhibits atherosclerotic plaque-induced platelet activation when combined with conventional dual antiplatelet therapy , 2021, European Heart Journal.

[5]  B. Shah,et al.  Transitioning From Thrombopoietin Agonists to the Novel SYK Inhibitor Fostamatinib: A Multicenter, Real-World Case Series , 2021, Journal of the advanced practitioner in oncology.

[6]  P. Libby,et al.  Targeting inflammation in atherosclerosis — from experimental insights to the clinic , 2021, Nature Reviews Drug Discovery.

[7]  H. Schunkert,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. , 2021, JAMA cardiology.

[8]  J. Heemskerk,et al.  Long-term platelet priming after glycoprotein VI stimulation in comparison to Protease-Activating Receptor (PAR) stimulation , 2021, PloS one.

[9]  Natalie S. Poulter,et al.  Structural characterisation of a novel GPVI nanobody-complex reveals a biologically active domain-swapped GPVI dimer. , 2021, Blood.

[10]  J. Desilles,et al.  Glenzocimab does not impact glycoprotein VI-dependent inflammatory hemostasis , 2020, Haematologica.

[11]  S. Watson,et al.  Novel antiplatelet strategies targeting GPVI, CLEC-2 and tyrosine kinases , 2020, Platelets.

[12]  S. Watson,et al.  Nonredundant Roles of Platelet Glycoprotein VI and Integrin αIIbβ3 in Fibrin-Mediated Microthrombus Formation. , 2020, Arteriosclerosis, thrombosis, and vascular biology.

[13]  H. Diener,et al.  Revacept, an Inhibitor of Platelet Adhesion in Symptomatic Carotid Artery Stenosis: Design and Rationale of a Randomized Phase II Clinical Trial , 2020, TH Open.

[14]  O. Borst,et al.  Glycoprotein VI - Novel target in antiplatelet medication. , 2020, Pharmacology & therapeutics.

[15]  S. Watson,et al.  Flow studies on human GPVI-deficient blood under coagulating and noncoagulating conditions. , 2020, Blood advances.

[16]  Y. Plétan,et al.  Population Pharmacokinetic/Pharmacodynamic Modeling of Glenzocimab (ACT017) a Glycoprotein VI Inhibitor of Collagen‐Induced Platelet Aggregation , 2020, Journal of clinical pharmacology.

[17]  N. Mackman,et al.  Therapeutic strategies for thrombosis: new targets and approaches , 2020, Nature Reviews Drug Discovery.

[18]  H. ten Cate,et al.  Role of Platelet Glycoprotein VI and Tyrosine Kinase Syk in Thrombus Formation on Collagen-Like Surfaces , 2019, International journal of molecular sciences.

[19]  C. Hermans,et al.  Platelet glycoprotein VI genetic quantitative and qualitative defects , 2019, Platelets.

[20]  Y. Plétan,et al.  Safety and Tolerability, Pharmacokinetics, and Pharmacodynamics of ACT017, an Antiplatelet GPVI (Glycoprotein VI) Fab: First-in-Human Healthy Volunteer Trial , 2019, Arteriosclerosis, thrombosis, and vascular biology.

[21]  S. Watson,et al.  Functional significance of the platelet immune receptors GPVI and CLEC-2. , 2019, The Journal of clinical investigation.

[22]  W. Ouwehand,et al.  High-throughput elucidation of thrombus formation reveals sources of platelet function variability , 2018, Haematologica.

[23]  Jacqueline K. White,et al.  A synthesis approach of mouse studies to identify genes and proteins in arterial thrombosis and bleeding. , 2018, Blood.

[24]  J. Heemskerk,et al.  Platelet biology and functions: new concepts and clinical perspectives , 2018, Nature Reviews Cardiology.

[25]  C. Weber,et al.  Oral Bruton tyrosine kinase inhibitors selectively block atherosclerotic plaque-triggered thrombus formation in humans. , 2018, Blood.

[26]  B. Ho-Tin-Noé,et al.  Glycoprotein VI in securing vascular integrity in inflamed vessels , 2018, Research and practice in thrombosis and haemostasis.

[27]  S. Watson,et al.  Immobilized fibrinogen activates human platelets through glycoprotein VI , 2018, Haematologica.

[28]  Martine,et al.  Immobilized fibrinogen activates human platelets through glycoprotein , 2018 .

[29]  D. Bihan,et al.  Differential integrin activity mediated by platelet collagen receptor engagement under flow conditions , 2017, Thrombosis and Haemostasis.

[30]  R. W. Farndale,et al.  Clustering of glycoprotein VI (GPVI) dimers upon adhesion to collagen as a mechanism to regulate GPVI signaling in platelets , 2017, Journal of thrombosis and haemostasis : JTH.

[31]  C. Weber,et al.  Recombinant GPVI-Fc added to single or dual antiplatelet therapy in vitro prevents plaque-induced platelet thrombus formation , 2017, Thrombosis and Haemostasis.

[32]  C. Weber,et al.  Cross-Linking GPVI-Fc by Anti-Fc Antibodies Potentiates Its Inhibition of Atherosclerotic Plaque- and Collagen-Induced Platelet Activation , 2016, JACC. Basic to translational science.

[33]  Christopher M. Williams,et al.  Coordinated Membrane Ballooning and Procoagulant Spreading in Human Platelets , 2015, Circulation.

[34]  J. Heemskerk,et al.  Multi-parameter assessment of thrombus formation on microspotted arrays of thrombogenic surfaces , 2014 .

[35]  Rachel Cavill,et al.  Identification of platelet function defects by multi-parameter assessment of thrombus formation , 2014, Nature Communications.

[36]  R. Andrews,et al.  Targeting GPVI as a novel antithrombotic strategy , 2014, Journal of blood medicine.

[37]  K. Hamulýak,et al.  Key role of integrin αIIbβ3 signaling to Syk kinase in tissue factor-induced thrombin generation , 2012, Cellular and Molecular Life Sciences.

[38]  B. Nieswandt,et al.  Platelet adhesion and activation mechanisms in arterial thrombosis and ischaemic stroke , 2011, Journal of thrombosis and haemostasis : JTH.

[39]  M. Gawaz,et al.  Novel Antiplatelet Drug Revacept (Dimeric Glycoprotein VI-Fc) Specifically and Efficiently Inhibited Collagen-Induced Platelet Aggregation Without Affecting General Hemostasis in Humans , 2011, Circulation.

[40]  A. Tolcher,et al.  Phase I Study of E7820, an Oral Inhibitor of Integrin α-2 Expression with Antiangiogenic Properties, in Patients with Advanced Malignancies , 2011, Clinical Cancer Research.

[41]  P. D. de Groot,et al.  Synergism between platelet collagen receptors defined using receptor-specific collagen-mimetic peptide substrata in flowing blood. , 2010, Blood.

[42]  J. Molkentin,et al.  Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen* , 2010, The Journal of Biological Chemistry.

[43]  P. Siljander,et al.  Collagen‐mimetic peptides mediate flow‐dependent thrombus formation by high‐ or low‐affinity binding of integrin α2β1 and glycoprotein VI , 2008, Journal of thrombosis and haemostasis : JTH.

[44]  E. Kremmer,et al.  Platelet GPVI binds to collagenous structures in the core region of human atheromatous plaque and is critical for atheroprogression in vivo , 2008, Basic Research in Cardiology.

[45]  Toshihiko Hayashi,et al.  Collagen-type specificity of glycoprotein VI as a determinant of platelet adhesion , 2008, Platelets.

[46]  D. Hammer,et al.  GPVI and α2β1 play independent critical roles during platelet adhesion and aggregate formation to collagen under flow , 2005 .

[47]  W. Ouwehand,et al.  The FASEB Journal • Research Communication Human atheromatous plaques stimulate thrombus formation by activating platelet glycoprotein VI , 2022 .

[48]  S. Watson,et al.  Adhesion of human and mouse platelets to collagen under shear: a unifying model , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[49]  M. V. van Zandvoort,et al.  Principal Role of Glycoprotein VI in &agr;2&bgr;1 and &agr;IIb&bgr;3 Activation During Collagen-Induced Thrombus Formation , 2004 .

[50]  J. Sixma,et al.  The role of collagen in thrombosis and hemostasis , 2004, Journal of thrombosis and haemostasis : JTH.

[51]  U. Heinzmann,et al.  Soluble glycoprotein VI dimer inhibits platelet adhesion and aggregation to the injured vessel wall in vivo , 2004, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[52]  Richard W Farndale,et al.  Identification of the primary collagen-binding surface on human glycoprotein VI by site-directed mutagenesis and by a blocking phage antibody. , 2004, Blood.

[53]  S. Santoro,et al.  The contributions of the α2β1 integrin to vascular thrombosis in vivo , 2003 .

[54]  S. Watson,et al.  Platelet-collagen interaction: is GPVI the central receptor? , 2003, Blood.

[55]  B. Nieswandt,et al.  Integrin α2-Deficient Mice Develop Normally, Are Fertile, but Display Partially Defective Platelet Interaction with Collagen* , 2002, The Journal of Biological Chemistry.

[56]  B. Nieswandt,et al.  Glycoprotein VI but not α2β1 integrin is essential for platelet interaction with collagen , 2001 .

[57]  Brian Savage,et al.  Specific Synergy of Multiple Substrate–Receptor Interactions in Platelet Thrombus Formation under Flow , 1998, Cell.

[58]  R. Farndale,et al.  The platelet reactivity of synthetic peptides based on the collagen III fragment alpha1(III)CB4. Evidence for an integrin alpha2beta1 recognition site involving residues 522-528 of the alpha1(III) collagen chain. , 1997, The Journal of biological chemistry.

[59]  R. Farndale,et al.  The Platelet Reactivity of Synthetic Peptides Based on the Collagen III Fragment a1(III)CB4 EVIDENCE FOR AN INTEGRIN a2b1 RECOGNITION SITE INVOLVING RESIDUES 522–528 OF THE a1(III) COLLAGEN CHAIN* , 1997 .

[60]  J. Sixma,et al.  Platelet adhesion to collagen type IV under flow conditions. , 1996, Blood.

[61]  J. Sixma,et al.  Human blood platelets showing no response to collagen fail to express surface glycoprotein Ia , 1985, Nature.