Glycoprotein VI is not a Functional Platelet Receptor for Fibrin Formed in Plasma or Blood

Abstract

Glycoprotein VI (GPVI), a platelet collagen receptor, is crucial in mediating atherothrombosis. Besides collagen, injured plaques expose tissue factor (TF) that triggers fibrin formation. Previous studies reported that GPVI also is a platelet receptor for fibrinogen and fibrin. We studied the effect of anti-GPVI antibodies and inhibitors of GPVI signaling kinases (Syk and Btk) on platelet adhesion and aggregate formation onto immobilized fibrinogen and different types of fibrin under arterial flow conditions. Fibrin was prepared from isolated fibrinogen (“pure fibrin”), recombinant fibrinogen (“recombinant fibrin”), or generated more physiologically from endogenous fibrinogen in plasma (“plasma fibrin”) or by exposing TF-coated surfaces to flowing blood (“blood fibrin”). Inhibition of GPVI and Syk did not inhibit platelet adhesion and aggregate formation onto fibrinogen. In contrast anti-GPVI antibodies, inhibitors of Syk and Btk and the anti-GPIb antibody 6B4 inhibited platelet aggregate formation onto pure and recombinant fibrin. However, inhibition of GPVI and GPVI signaling did not significantly reduce platelet coverage of plasma fibrin and blood fibrin. Plasma fibrin contained many proteins incorporated during clot formation. Advanced optical imaging revealed plasma fibrin as a spongiform cushion with thicker, knotty, and long fibers and little activation of adhering platelets. Albumin intercalated in plasma fibrin fibers left only little space for platelet attachment. Pure fibrin was different showing a dense mesh of thin fibers with strongly activated platelets. We conclude that fibrin formed in plasma and blood contains plasma proteins shielding GPVI-activating epitopes. Our findings do not support a role of GPVI for platelet activation by physiologic fibrin.

Keywords

Glycoprotein VI - platelet - fibrinogen/fibrin - atherothrombosis - glycoprotein Ib

^∗ The review process for this paper was fully handled by Gregory Y. H. Lip, Editor-in-Chief.

• References

• 1 Badimon L, Vilahur G. Thrombosis formation on atherosclerotic lesions and plaque rupture. J Intern Med 2014; 276 (06) 618-632
• 2 Fuster V, Moreno PR, Fayad ZA, Corti R, Badimon JJ. Atherothrombosis and high-risk plaque: part I: evolving concepts. J Am Coll Cardiol 2005; 46 (06) 937-954
• 3 Reininger AJ, Bernlochner I, Penz SM. , et al. A 2-step mechanism of arterial thrombus formation induced by human atherosclerotic plaques. J Am Coll Cardiol 2010; 55 (11) 1147-1158
• 4 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
• 5 van Zanten GH, de Graaf S, Slootweg PJ. , et al. Increased platelet deposition on atherosclerotic coronary arteries. J Clin Invest 1994; 93 (02) 615-632
• 6 Badimon JJ, Lettino M, Toschi V. , et al. Local inhibition of tissue factor reduces the thrombogenicity of disrupted human atherosclerotic plaques: effects of tissue factor pathway inhibitor on plaque thrombogenicity under flow conditions. Circulation 1999; 99 (14) 1780-1787
• 7 Hantgan RR, Hindriks G, Taylor RG, Sixma JJ, de Groot PG. Glycoprotein Ib, von Willebrand factor, and glycoprotein IIb:IIIa are all involved in platelet adhesion to fibrin in flowing whole blood. Blood 1990; 76 (02) 345-353
• 8 Heemskerk JW, Mattheij NJ, Cosemans JM. Platelet-based coagulation: different populations, different functions. J Thromb Haemost 2013; 11 (01) 2-16
• 9 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
• 10 Jamasbi J, Megens RT, Bianchini M. , et al. Differential inhibition of human atherosclerotic plaque-induced platelet activation by dimeric GPVI-Fc and anti-GPVI antibodies: Functional and imaging studies. J Am Coll Cardiol 2015; 65 (22) 2404-2415
• 11 Nieswandt B, Watson SP. Platelet-collagen interaction: is GPVI the central receptor?. Blood 2003; 102 (02) 449-461
• 12 Kuijpers MJ, Schulte V, Bergmeier W. , et al. Complementary roles of glycoprotein VI and alpha2beta1 integrin in collagen-induced thrombus formation in flowing whole blood ex vivo. FASEB J 2003; 17 (06) 685-687
• 13 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
• 14 Grüner S, Prostredna M, Aktas B. , et al. Anti-glycoprotein VI treatment severely compromises hemostasis in mice with reduced alpha2beta1 levels or concomitant aspirin therapy. Circulation 2004; 110 (18) 2946-2951
• 15 Herr AB, Farndale RW. Structural insights into the interactions between platelet receptors and fibrillar collagen. J Biol Chem 2009; 284 (30) 19781-19785
• 16 ClinicalTrials.gov. Revacept in symptomatic carotid stenosis. 2012 [cited 2018 Dec 20]; Available at: https://clinicaltrials.gov/ct2/show/NCT01645306 . Accessed March 31, 2020
• 17 ClinicalTrials.gov. Intracoronary stenting and antithrombotic regimen: lesion platelet adhesion as selective target of endovenous Revacept (ISAR-Plaster). 2017 [cited 2018 Dec 5]; Available at: https://clinicaltrials.gov/ct2/show/NCT03312855 . Accessed March 31, 2020
• 18 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
• 19 ClinicalTrials.gov. Acute ischemic stroke interventional study (ACTIMIS). 2019 [cited 2019 June 26, 2019]; Available at: https://clinicaltrials.gov/ct2/show/NCT03803007 . Accessed March 31, 2020
• 20 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
• 21 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
• 22 Denzinger V, Busygina K, Jamasbi J. , et al. Optimizing platelet GPVI inhibition versus hemostatic impairment by ibrutinib and the novel Btk-inhibitors acalabrutinib, ONO/GS-4059, BGB-3111 and evobrutinib. Thromb Haemost 2019; 119: 397-406
• 23 Jung SM, Moroi M, Soejima K. , et al. Constitutive dimerization of glycoprotein VI (GPVI) in resting platelets is essential for binding to collagen and activation in flowing blood. J Biol Chem 2012; 287 (35) 30000-30013
• 24 Mammadova-Bach E, Ollivier V, Loyau S. , et al. Platelet glycoprotein VI binds to polymerized fibrin and promotes thrombin generation. Blood 2015; 126 (05) 683-691
• 25 Alshehri OM, Hughes CE, Montague S. , et al. Fibrin activates GPVI in human and mouse platelets. Blood 2015; 126 (13) 1601-1608
• 26 Mangin PH, Onselaer MB, Receveur N. , et al. Immobilized fibrinogen activates human platelets through glycoprotein VI. Haematologica 2018; 103 (05) 898-907
• 27 Induruwa I, Moroi M, Bonna A. , et al. Platelet collagen receptor Glycoprotein VI-dimer recognizes fibrinogen and fibrin through their D-domains, contributing to platelet adhesion and activation during thrombus formation. J Thromb Haemost 2018; 16 (02) 389-404
• 28 Slater A, Perrella G, Onselaer MB. , et al. Does fibrin(ogen) bind to monomeric or dimeric GPVI, or not at all?. Platelets 2019; 30 (03) 281-289
• 29 Ebrahim M, Jamasbi J, Adler K. , et al. Dimeric glycoprotein VI binds to collagen but not to fibrin. Thromb Haemost 2018; 118 (02) 351-361
• 30 Mosesson MW. Fibrinogen and fibrin structure and functions. J Thromb Haemost 2005; 3 (08) 1894-1904
• 31 Talens S, Leebeek FW, Demmers JA, Rijken DC. Identification of fibrin clot-bound plasma proteins. PLoS One 2012; 7 (08) e41966
• 32 Talens S, Leebeek FWG, Veerhuis R, Rijken DC. Decoration of fibrin with extracellular chaperones. Thromb Haemost 2019; 19 (10) 1624-1631
• 33 Nikolajsen CL, Dyrlund TF, Poulsen ET, Enghild JJ, Scavenius C. Coagulation factor XIIIa substrates in human plasma: identification and incorporation into the clot. J Biol Chem 2014; 289 (10) 6526-6534
• 34 Hirashima M, Imamura T, Yano K. , et al. High-level expression and preparation of recombinant human fibrinogen as biopharmaceuticals. J Biochem 2016; 159 (02) 261-270
• 35 Cabeza N, Li Z, Schulz C. , et al. Surface expression of collagen receptor Fc receptor-gamma/glycoprotein VI is enhanced on platelets in type 2 diabetes and mediates release of CD40 ligand and activation of endothelial cells. Diabetes 2004; 53 (08) 2117-2121
• 36 Savage B, Saldívar E, Ruggeri ZM. Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell 1996; 84 (02) 289-297
• 37 Elia N, Ott C, Lippincott-Schwartz J. Incisive imaging and computation for cellular mysteries: lessons from abscission. Cell 2013; 155 (06) 1220-1231
• 38 Cauwenberghs N, Vanhoorelbeke K, Vauterin S. , et al. Epitope mapping of inhibitory antibodies against platelet glycoprotein Ibalpha reveals interaction between the leucine-rich repeat N-terminal and C-terminal flanking domains of glycoprotein Ibalpha. Blood 2001; 98 (03) 652-660
• 39 Bültmann A, Li Z, Wagner S. , et al. Impact of glycoprotein VI and platelet adhesion on atherosclerosis--a possible role of fibronectin. J Mol Cell Cardiol 2010; 49 (03) 532-542
• 40 Schönberger T, Ziegler M, Borst O. , et al. The dimeric platelet collagen receptor GPVI-Fc reduces platelet adhesion to activated endothelium and preserves myocardial function after transient ischemia in mice. Am J Physiol Cell Physiol 2012; 303 (07) C757-C766
• 41 Walker JB, Nesheim ME. The molecular weights, mass distribution, chain composition, and structure of soluble fibrin degradation products released from a fibrin clot perfused with plasmin. J Biol Chem 1999; 274 (08) 5201-5212
• 42 Lorand L. Factor XIII: structure, activation, and interactions with fibrinogen and fibrin. Ann N Y Acad Sci 2001; 936: 291-311
• 43 Loscalzo J, Inbal A, Handin RI. von Willebrand protein facilitates platelet incorporation in polymerizing fibrin. J Clin Invest 1986; 78 (04) 1112-1119
• 44 Sane DC, Moser TL, Greenberg CS. Limited proteolysis of vitronectin by plasmin destroys heparin binding activity. Thromb Haemost 1991; 66 (03) 310-314
• 45 Wolberg AS. Plasma and cellular contributions to fibrin network formation, structure and stability. Haemophilia 2010; 16 (03) (Suppl. 03) 7-12
• 46 Nair CH, Dhall DP. Studies on fibrin network structure: the effect of some plasma proteins. Thromb Res 1991; 61 (03) 315-325
• 47 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
• 48 Endenburg SC, Hantgan RR, Lindeboom-Blokzijl L. , et al. On the role of von Willebrand factor in promoting platelet adhesion to fibrin in flowing blood. Blood 1995; 86 (11) 4158-4165
• 49 Arthur JF, Gardiner EE, Matzaris M. , et al. Glycoprotein VI is associated with GPIb-IX-V on the membrane of resting and activated platelets. Thromb Haemost 2005; 93 (04) 716-723
• 50 Gardiner EE, Arthur JF, Shen Y. , et al. GPIbalpha-selective activation of platelets induces platelet signaling events comparable to GPVI activation events. Platelets 2010; 21 (04) 244-252
• 51 Siess W. Molecular mechanisms of platelet activation. Physiol Rev 1989; 69 (01) 58-178
• 52 Briedé JJ, Wielders SJ, Heemskerk JW, Baruch D, Hemker HC, Lindhout T. von Willebrand factor stimulates thrombin-induced exposure of procoagulant phospholipids on the surface of fibrin-adherent platelets. J Thromb Haemost 2003; 1 (03) 559-565