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DOI: 10.1160/TH06-08-0439
Sequential adhesion of platelets and leukocytes from flowing whole blood onto a collagen-coated surface: Requirement for a GpVI-binding site in collagen
Financial support: GER was supported by a British Heart Foundation Non-Clinical Senior Lectureship.Publication History
Received
10 August 2006
Accepted after resubmission
08 February 2007
Publication Date:
24 November 2017 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/200705/lookinside/thumbnails/10-1160-th06-08-0439-1.jpg)
Summary
The adhesion of leukocytes to immobilised platelets may contribute to inflammatory and thrombotic responses in damaged tissue. To investigate the conditions under which platelets and leukocytes might be deposited together in vessels, we perfused fluorescently-labelled whole blood through glass capillaries coated with various collagen preparations. Video-microscopic observations of the surface showed that platelets formed numerous, individual, rolling and stationary attachments to surfaces coated with acid-soluble, monomeric collagen. However, leukocyte interactions with the deposited platelets were rare. If the blood was washed out, the adherent platelets became more activated, and many rolling adherent leukocytes were observed if a second bolus of blood was perfused over them. This suggested that platelet activation had initially been inadequate to support leukocyte capture. Next, fibrillar collagen was adsorbed to the capillaries to present an ordered array of peptide motifs to platelet receptor glycoprotein (Gp)VI and transduce an activating signal. In this case, platelets were deposited in discrete, stable aggregates and the bound platelets captured many flowing leukocytes.Alternatively, acid-soluble collagen was seeded with collagen-related peptide (CRP) known to contain a GpVI-binding motif.Again, platelet adhesion became stable, and numerous flowing leukocytes were captured.Addition of antibody against GpVI or against P-selectin greatly reduced leukocyte adhesion to the platelets. Thus, in whole blood, platelets binding to exposed collagen need to be activated through GpVI in order to expose sufficient P-selectin to allow efficient capture of flowing leukocytes to take place.
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References
- 1 Massberg S, Enders G, Matos FC. et al. Fibrinogen deposition at the postischemic vessel wall promotes platelet adhesion during ischemia-reperfusion in vivo. Blood 1999; 94: 3829-3838.
- 2 Massberg S, Brand K, Gruner S. et al. A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation. J Exp Med 2002; 196: 887-896.
- 3 Kuligowski MP, Kitching AR, Hickey MJ. Leukocyte recruitment to the inflamed glomerulus: a critical role for platelet-derived P-selectin in the absence of rolling. J Immunol 2006; 176: 6991-6999.
- 4 Andrews RK, Shen Y, Gardiner EE. et al. Platelet adhesion receptors and (patho)physiological thrombus formation. Histol Histopathol 2001; 16: 969-980.
- 5 Nieswandt B, Watson SP. Platelet-collagen interaction: is GPVI the central receptor?. Blood 2003; 102: 449-461.
- 6 Siljander PR, Munnix IC, Smethurst PA. et al. Platelet receptor interplay regulates collagen-induced thrombus formation in flowing human blood. Blood 2004; 103: 1333-1341.
- 7 Buttrum SM, Hatton R, Nash GB. Selectin-mediated rolling of neutrophils on immobilized platelets. Blood 1993; 82: 1165-1174.
- 8 Lalor P, Nash GB. Adhesion of flowing leucocytes to immobilized platelets. Br J Haematol 1995; 89: 725-732.
- 9 Stone PC, Nash GB. Conditions under which immobilized platelets activate as well as capture flowing neutrophils. Br J Haematol 1999; 105: 514-522.
- 10 Yeo EL, Sheppard JA, Feuerstein IA. Role of P-selectin and leukocyte activation in polymorphonuclear cell adhesion to surface adherent activated platelets under physiologic shear conditions (an injury vessel wall model). Blood 1994; 83: 2498-2507.
- 11 Ostrovsky L, King AJ, Bond S. et al. A juxtacrine mechanism for neutrophil adhesion on platelets involves platelet-activating factor and a selectin-dependent activation process. Blood 1998; 91: 3028-3036.
- 12 Rebello SS, Huang J, Lucchesi BR. Role of extracellular ionized calcium in the in vitro assessment of GpIIb/IIIa receptor antagonists. J Thromb Thrombolysis 2000; 09: 23-28.
- 13 Abbitt KB, Nash GB. Characteristics of leucocyte adhesion directly observed in flowing whole blood in vitro. Br J Haematol 2001; 112: 55-63.
- 14 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: 903-911.
- 15 Cooke BM, Usami S, Perry I. et al. A simplified method for culture of endothelial cells and analysis of adhesion of blood cells under conditions of flow. Microvasc Res 1993; 45: 33-45.
- 16 Knight CG, Morton LF, Onley DJ. et al. Collagenplatelet interaction: Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen. Cardiovasc Res 1999; 41: 450-457.
- 17 Bahra P, Nash GB. Sparsely adherent platelets support capture and immobilization of flowing neutrophils. J Lab Clin Med 1998; 132: 223-228.
- 18 Abbitt KB, Nash GB. Rheological properties of the blood influencing selectin-mediated adhesion of flowing leukocytes. Am J Physiol 2003; 285: H229-H240.
- 19 Abbitt KB, Rainger GE, Nash GB. Effects of fluorescent dyes on selectin and integrin-mediated stages of adhesion and migration of flowing leukocytes. J Immunol Methods 2000; 239: 109-119.
- 20 Smith E, Lally F, Cocker L. et al. Phototoxicity and fluorotoxicity combine to alter the behaviour of neutrophils in fluorescence microscopy based flow adhesion assays. Microsc Res Tech 2006; 69: 875-884.
- 21 Sheikh S, Nash GB. Continuous activation and deactivation of integrin CD11b/CD18 during de novo expression enables rolling neutrophils to immobilize on platelets. Blood 1996; 87: 5040-5050.
- 22 Pasquet JM, Gross B, Quek L. et al. LAT is required for tyrosine phosphorylation of phospholipase cgamma2 and platelet activation by the collagen receptor GPVI. Mol Cell Biol 1999; 19: 8326-8334.
- 23 Asselin J, Knight CG, Farndale RW. et al. Monomeric (glycine-proline-hydroxyproline)10 repeat sequence is a partial agonist of the platelet collagen receptor glycoprotein VI. Biochem J 1999; 339: 413-418.
- 24 Gibbins JM, Okuma M, Farndale R. et al. Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor gamma-chain. FEBS Lett 1997; 413: 255-259.
- 25 King MR, Heinrich V, Evans E. et al. Nano-to- Micro Scale Dynamics of P-Selectin Detachment from Leukocyte Interfaces. III. Numerical Simulation of Tethering under Flow. Biophys J 2005; 88: 1676-1683.
- 26 von Hundelshausen P, Weber KS, Huo Y. et al. RANTES deposition by platelets triggers monocyte arrest on inflamed and atherosclerotic endothelium. Circulation 2001; 103: 1772-1777.
- 27 Nash GB, Morland C, Sheikh S. et al. Adhesion between leucocytes and platelets:rheology, mechanisms and consequences. In: Progress in Applied Microcirculation, S Karger. 1996: 98-113.
- 28 Lawrence MB, Springer TA. Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell 1991; 65: 859-873.
- 29 Skilbeck CA, Walker PG, David T. et al. Disturbed flow promotes deposition of leucocytes from flowing whole blood in a model of a damaged vessel wall. Br J Haematol 2004; 126: 418-427.