Development and Use of IgM/J‐Chain Fusion Proteins for Characterization of Immunoglobulin Superfamily Ligand‐Receptor Interactions

Ammann, Johannes U.; Trowsdale, John

doi:10.1002/0471140864.ps1924s75

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“…14,15 Proteins were expressed by transient transfection of HEK293T cells. Cell-culture medium containing GPVI CBD or GPVI-Fcg was purified by Ni-NTA or protein-A affinity chromatography, respectively.…”

Section: Gpvi Constructionsmentioning

confidence: 99%

Fibrin and D-dimer bind to monomeric GPVI

et al. 2017

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Key Points• GPVI is the major signaling receptor for fibrin in human platelets; the GPVI binding site is located in the fibrin D-dimer region.• D-dimer blocks platelet aggregation by fibrin and collagen but not by a collagen-related peptide, suggesting a distinct binding epitope.Fibrin has recently been shown to activate platelets through the immunoglobulin receptor glycoprotein VI (GPVI). In the present study, we show that spreading of human platelets on fibrin is abolished in patients deficient in GPVI, confirming that fibrin activates human platelets through the immunoglobulin receptor. Using a series of proteolytic fragments, weshow that D-dimer, but not the E fragment of fibrin, binds to GPVI and that immobilized D-dimer induces platelet spreading through activation of Src and Syk tyrosine kinases. In contrast, when platelets are activated in suspension, soluble D-dimer inhibits platelet aggregation induced by fibrin and collagen, but not by a collagen-related peptide composed of a repeat GPO sequence or by thrombin. Using surface plasmon resonance, we demonstrate that fibrin binds selectively to monomeric GPVI with a K D of 302 nM, in contrast to collagen, which binds primarily to dimeric GPVI. These results establish GPVI as the major signaling receptor for fibrin in human platelets and provide evidence that fibrin binds to a distinct configuration of GPVI. This indicates that it may be possible to develop agents that selectively block the interaction of fibrin but not collagen with the immunoglobulin receptor. Such agents are required to establish whether selective targeting of either interaction has the potential to lead to development of an antithrombotic agent with a reduced effect on bleeding relative to current antiplatelet drugs.

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Section: Gpvi Constructionsmentioning

confidence: 99%