Abstract
The roles of factor XIIIa-specific cross-links in thrombus formation, regression, or probability for embolization are largely unknown. A molecular understanding of fibrin architecture at the level of these cross-links could inform the development of therapeutic strategies to prevent the sequelae of thromboembolism. Here, we present an MS-based method to map native factor XIIIa cross-links in the insoluble matrix component of whole-blood or plasma-fibrin clots and in in vivo thrombi. Using a chaotrope-insoluble digestion method and quantitative cross-linking MS, we identified the previously mapped fibrinogen peptides that are responsible for covalent D-dimer association, as well as dozens of novel cross-links in the αC region of fibrinogen α. Our findings expand the known native cross-linked species from one to over 100 and suggest distinct antiparallel registries for interprotofibril association and covalent attachment of serpins that regulate clot dissolution.
Highlights
The roles of factor XIIIa–specific cross-links in thrombus formation, regression, or probability for embolization are largely unknown
A key step in clot formation is the generation of fibrin cross-links, a process that is catalyzed by the transglutaminase activity of factor XIIIa (FXIIIa)
Based on proteomic methods to characterize insoluble extracellular matrices [15,16,17] and chemical cross-links [18, 19], we developed an analytical method to characterize FXIIIa crosslinks in ex vivo generated fibrin clots (Fig. 1A; detailed analytical methods are provided in the supporting methods)
Summary
We present an MS-based method to map native factor XIIIa cross-links in the insoluble matrix component of wholeblood or plasma-fibrin clots and in in vivo thrombi. FXIIIa activity is a critical determinant of venous thrombus composition, size, and subsequent tissue repair [4], highlighting the importance of fibrin cross-linking to regulate both the clot architecture and regulation by sequestration of plasmin and inhibitors that will influence matrix remodeling kinetics. Cross-linking MS (CL-MS) has quickly become a widely used method to map protein–protein interactions and provide distance constraints to elucidate topology in multiprotein complexes using chemical cross-linking reagents [12, 13] This approach has been primarily used to map cross-links in isolated proteins and recently in cell culture and tissues to obtain more global information [14]. We develop an approach for the characterization of fibrin clots to obtain molecular detail regarding the specificity of FXIIIa cross-link sites and substrates
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