Activation‐induced changes in platelet surface receptor expression and the contribution of the large‐platelet subpopulation to activation

Abstract

Objective Platelet surface receptors are also present subcellularly in organelle membranes and can be expressed on the surface upon platelet activation. However, some receptors were reported to be decreased after activation. We analyzed the mechanism of activation‐dependent expression for different receptors. Methods Flow cytometry using platelet‐rich plasma or washed platelets was used to analyze receptor‐expression changes after platelet activation by glycoprotein (GP) VI–specific agonists, crosslinked collagen‐related peptide (CRP‐XL) and convulxin (Cvx), and thrombin. Platelets prelabeled with fluorescent antibody specific for a receptor were allowed to adhere on immobilized collagen or fibrinogen and post‐stained with antibody against the same receptor labeled with another fluorophore, allowing us to differentiate preexisting receptors from newly expressed receptors. Results Surface expression of αIIbβ3 increased in CRP‐XL–, Cvx‐, or thrombin‐stimulated platelets, but GPIb decreased due to shedding and internalization. Both total and dimeric GPVI increased in thrombin‐induced platelets, but decreased in platelets stimulated by Cvx, as a result of internalization. The larger platelets showed a greater increase in surface receptor (α2β1, αIIbβ3, GPVI, GPIb) expression upon activation compared to the smaller ones. Pre‐ and postlabeling with antibody specific for the same receptor, but conjugated with different fluorophores, allowed us to differentiate the receptors expressed on the surface of resting platelets from receptors newly exposed to the surface upon platelet activation. Conclusions Increased receptor expressions after activation are mainly manifested in the larger platelets. On platelets adhered on fibrinogen, the newly expressed receptors, especially GPVI, are localized in the lamellipodia of the spread platelets.

Highly efficient platelet generation in lung vasculature reproduced by microfluidics

“…1g ). It has been reported that surface expression of CD61, CD42b, CD49b and GPVI were higher in larger platelets, commensurate with their larger surface area 26 . The subpopulation of generated platelets with the larger size has higher surface expression of CD61, CD42b, CD42d, CD49b and GPVI by comparison with the subpopulation with the smaller size (Supplementary Fig.…”

Immobilised collagen prevents shedding and induces sustained GPVI clustering and signalling in platelets

“…Indeed, GPVI spatial organisation and cluster formation at the plasma membrane play a central role in regulating signalling (17,26). Platelet GPVI levels at the plasma membrane can be regulated by endocytosis (27,28) or by receptor shedding, the latter being the subject of significant research (29). The extracellular region of GPVI can be cleaved by members of the ADAM family of sheddases (30,31).…”

Platelet functional abnormalities in pediatric patients with kaposiform hemangioendothelioma/Kasabach-Merritt phenomenon

“…Flow cytometry assessment of platelet CLEC-2 and GPVI expression for patients 9, 11, and 13 was performed as described previously. 34 , 35 Briefly, diluted to 150 × 10 6 platelet (plt)/mL platelet-rich plasma was incubated with anti–CLEC-2 or anti-GPVI antibodies for 10 minutes. After incubation with primary antibodies, Alexa-488–labeled secondary antibodies were added to the suspension and incubated for 10 minutes.…”