Abstract
GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization. Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth. Graphic Abstract: A graphic abstract is available for this article.
Highlights
We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization
Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity
The importance of GPVI in thrombus formation is emphasized by observations that absence of GPVI impairs experimental thrombosis in mouse models, including no occlusive thrombus formation observed in knockout mice following
Summary
The data that support the findings of this study are available from the corresponding author upon reasonable request. ELISAs were performed based on previously described methods,[23] with slight modifications. 96-well Maxisorp Nuncimmuno plates were coated with 10 nmol/L BSA, purified fibrinogen, type 1 collagen fibrils or CRP-XL, or 100 nmol/L GPVI-Fc/GPVI monomer, in duplicates overnight at 4 °C. Immobilized fibrinogen was converted to fibrin by addition of 0.1 U/mL thrombin for 20 minutes at room temperature. Plates were washed to remove excess protein and blocked with 3% BSA-PBS for 1 hour at room temperature. Plates were incubated with purified GPVI constructs at 100 nmol/L, or BSA control, for 1 hour. For concentration-dependent experiments, 0 to 400 nmol/L GPVI constructs were used, and 100
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