Immobilized fibrinogen activates human platelets through glycoprotein VI

Abstract

Glycoprotein VI, a major platelet activation receptor for collagen and fibrin, is considered a particularly promising, safe antithrombotic target. In this study, we show that human glycoprotein VI signals upon platelet adhesion to fibrinogen. Full spreading of human platelets on fibrinogen was abolished in platelets from glycoprotein VI- deficient patients suggesting that fibrinogen activates platelets through glycoprotein VI. While mouse platelets failed to spread on fibrinogen, human-glycoprotein VI-transgenic mouse platelets showed full spreading and increased Ca2+ signaling through the tyrosine kinase Syk. Direct binding of fibrinogen to human glycoprotein VI was shown by surface plasmon resonance and by increased adhesion to fibrinogen of human glycoprotein VI-transfected RBL-2H3 cells relative to mock-transfected cells. Blockade of human glycoprotein VI with the Fab of the monoclonal antibody 9O12 impaired platelet aggregation on preformed platelet aggregates in flowing blood independent of collagen and fibrin exposure. These results demonstrate that human glycoprotein VI binds to immobilized fibrinogen and show that this contributes to platelet spreading and platelet aggregation under flow.

Platelet preparation for function testing in the laboratory and clinic: Historical and practical aspects

“…In sharp contrast, it has been shown that this complex plays a key role in thrombus growth, for example, in platelet‐platelet interactions, by mediating the recruitment of circulating platelets to von Willebrand factor (VWF) exposed at the surface of activated platelets within the thrombus . Moreover, we recently reported that platelet aggregation under flow also involves GPVI, whereas this receptor does not participate in platelet‐platelet interactions under the low shear conditions found in LTA . In addition, the part played by soluble agonists such as thrombin, ADP, or TxA 2 might be overestimated in suspension assays like LTA where they are not washed away by the flow.…”

“…Washed platelet suspensions are thus ideal for investigations using LTA, flow cytometry, electron microscopy, western blotting of platelet lysates, or radioligand binding, to mention only a few applications …”

Proteolytic processing of platelet receptors

“…In one study, the GPVI‐fibrin interaction occurred only with GPVI in dimeric form and could be abrogated by pretreatment with collagen or CRP, implying at least partial overlap of the binding site for these ligands, however, fibrin‐GPVI monomer interactions and separate CRP and fibrin binding sites were proposed in another study . Under certain experimental conditions and in collaboration with αIIbβ3, the fibrin monomer component fibrinogen also can engage GPVI . Understanding how these two GPVI ligands intersect and contribute to GPVI function is important, as selective disruption of one type of GPVI‐ligand interaction, either through competitive inhibition at the ligand‐binding site, or at the level of GPVI dimerization represents an enticing new approach to develop antiplatelet agents with minimal effects on hemostasis.…”

“…108 Under certain experimental conditions and in collaboration with αIIbβ3, the fibrin monomer component fibrinogen also can engage GPVI. 33,34 Understanding how these two GPVI ligands intersect and contribute to GPVI function is important, as selective disruption of one type of GPVI-ligand interaction, either through competitive inhibition at the ligand-binding site, or at the level of GPVI dimerization represents an enticing new approach to develop antiplatelet agents with minimal effects on hemostasis.…”

“…The cytoplasmic domain of FcRγ contains an immunoreceptor tyrosine activation motif (ITAM) and together the GPVI/FcRγ complex transmits ligand‐induced signalling events into the platelet by triggering phosphorylation of two tyrosine residues with the ITAM and subsequent activation of p72‐spleen tyrosine kinase (Syk) . Along with collagen, GPVI can bind laminin, fibrin, fibrinogen, histones, adiponectin, and the extracellular matrix metalloproteinase inducer (EMMPRIN) expressed on monocytes and leukocytes. Intact GPVI is also essential for efficient generation of thrombin at the platelet surface .…”