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Thrombospondin and fibrinogen bind serotonin-derivatized proteins on COAT-platelets
1W. K. Warren Medical Research Institute, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Insights
Activated platelets, known as COAT-platelets, bind serotonin to thrombospondin and fibrinogen. This interaction stabilizes procoagulant proteins on the platelet surface, enhancing clot formation and stability.
Area of Science:
- Biochemistry
- Hematology
- Cell Biology
Background:
- Platelet activation by agonists like collagen and thrombin generates COAT-platelets (collagen and thrombin activated).
- COAT-platelets are characterized by an enrichment of membrane-bound, procoagulant proteins.
- Serotonin derivatization of alpha-granule proteins enhances their retention on COAT-platelets via interactions with specific binding sites.
Purpose of the Study:
- To identify the specific platelet proteins that provide serotonin binding sites on COAT-platelets.
- To elucidate the mechanism by which serotonin conjugation stabilizes procoagulant proteins on activated platelets.
Main Methods:
- Utilized photoreactive cross-linking with an albumin-(serotonin)6 conjugate during COAT-platelet production to identify binding proteins.
- Employed in vitro binding assays using biotin-albumin-(serotonin)6 to verify interactions with purified proteins and protein fragments.
Main Results:
- Thrombospondin and fibrinogen were identified as the primary proteins providing serotonin binding sites on COAT-platelets.
- Demonstrated saturable binding of serotonin conjugate to thrombospondin, fibrinogen, and fibrinogen fragment D in vitro.
- Confirmed that serotonin-derivatized proteins bind to both their known receptors and these newly identified serotonin binding sites.
Conclusions:
- Thrombospondin and fibrinogen act as serotonin receptors on COAT-platelets, contributing to protein retention.
- This dual-binding mechanism (to protein receptors and serotonin sites) creates stable, multivalent complexes on the COAT-platelet surface.
- The findings provide a deeper understanding of COAT-platelet function in hemostasis and thrombosis.
Abstract:
Activation of platelets with 2 agonists, collagen and thrombin, reveals a subpopulation of cells referred to as COAT-platelets (collagen and thrombin activated). These cells are enriched in several membrane-bound, procoagulant proteins, including fibrinogen, thrombospondin, factor V, von Willebrand factor, and fibronectin. alpha-Granule proteins bound to COAT-platelets are derivatized with serotonin by a transglutaminase-mediated process, and the interaction of conjugated serotonins with unidentified serotonin binding sites on the platelet surface enhances retention of these proteins. We now demonstrate that both thrombospondin and fibrinogen provide the requisite serotonin binding sites. Thrombospondin and fibrinogen were identified using photoreactive cross-linking to an albumin-(serotonin)(6) conjugate during COAT-platelet production. We subsequently verified that biotin-albumin-(serotonin)(6) binds in vitro to thrombospondin, fibrinogen, and fibrinogen fragment D in a saturable manner. These data support a model for COAT-platelets where serotonin-derivatized procoagulant proteins interact with their respective receptors (eg, fibrinogen with glycoprotein IIb/IIIa or factor V with phosphatidylserine) as well as serotonin binding sites on fibrinogen and thrombospondin, resulting in a stable, multivalent complex on the cell surface.