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Non-redundant Roles of Platelet Glycoprotein VI and IIb/IIIa via Syk Kinase in Fibrin-dependent Thrombus Shielding under Flow

1University of Maastricht, Maastricht, the Netherlands, 2University of Birmingham, Birmingham, United Kingdom, 3ISAS Institute, Dortmund, Germany, 4Dept. of Haematology-Internal Medicine, MUMC+, Maastricht, the Netherlands, 5University of Cambridge, Cambridge, United Kingdom, 6Synapse Research Institute, Maastricht, the Netherlands, 7Institute of Cardiovascular and Metabolic Medicine, Leeds, United Kingdom, 8Laboratory for Vascular Translational Science , INSERM, Paris, France, 9COMPARE, The Universities of Birmingham and Nottingham, Birmingham, United Kingdom

Abstract Number: PB1736

Meeting: ISTH 2020 Congress

Theme: Platelets and Megakaryocytes » Platelet Receptors

Background: Fibrin in the thrombus core strengthens platelet aggregation by binding to integrin aIIbb3 (glycoprotein (GP) IIb/IIIa). Lately GPVI has been shown to bind also to fibrin(ogen).

Aims: To determine how fibrin-GPVI interaction contribute to and consolidate thrombus formation.

Methods: Fibrin-coated surfaces were generated from fibrinogen and thrombin, containing or not transglutaminase factor XIIIa and von Willebrand factor-binding peptide (VWF-BP). The thrombus-forming potential of all surfaces was assessed in the presence or absence of GPVI, GPIIb/IIIa or GPIba activity. Whole-blood flow was performed using the Maastricht microfluidics device under coagulant conditions at arterial wall-shear rate.

Results: Fibrin fibres with(out) FXIIIa moderately supported single platelet adhesion and activation in flowed whole blood. Thrombus build-up on fibrin surfaces was more delayed than that on collagen-III, and aggregated platelets were positive for secretion markers, but not for phosphatidylserine expression. Coated fibrinogen only supported platelet adhesion. On fibrin surfaces, VWF-BP enhanced adhesion but not thrombus build-up. Blocking GPVI with 9O12 antibody (50 mg/ml) substantially but incompletely reduced aggregation and secretion, with limited effect on adhesion. Inhibition of Syk (PRT-060318, 10 mM) nearly abolished thrombus parameters, with residual adhesion remaining. Absence of GPIIb/IIIa (Glanzmann blood) dramatically suppressed platelet adhesion regardless of arterial/venous shear. Both 9O12 and PRT-060318 suppressed fibrin-induced platelet aggregation in suspension and reduced Ca2+ signals under flow. Inhibition of GPIba with RAG35 antibody affected platelet adhesion with minor effect on platelet secretion.

Conclusions: With or without FXIIIa, fibrin surfaces moderately support thrombus formation, when compared to collagens. Formation of the shielding thrombus relies on GPIIb/IIIa and Syk activation, and on essential but low GPVI activity. These results indicate that fibrin triggers a low-level activation of GPVI, which is dependent on GPIIb/IIIa for inducing Syk-mediated platelet activation. This work provides new insights into the potential of targeting GPVI-fibrin for treatment of thrombosis.

To cite this abstract in AMA style:

Perrella G, Huang J, Provenzale I, Swieringa F, Heubel-Moenen FCJI, Farndale RW, Roest M, Thomas M, Ariëns RAS, Jandrot-Perrus M, Watson SP, van der Meijden PEJ, Heemskerk JWM. Non-redundant Roles of Platelet Glycoprotein VI and IIb/IIIa via Syk Kinase in Fibrin-dependent Thrombus Shielding under Flow [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/non-redundant-roles-of-platelet-glycoprotein-vi-and-iib-iiia-via-syk-kinase-in-fibrin-dependent-thrombus-shielding-under-flow/. Accessed December 5, 2021.

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