Coagulation factor XIIIa substrates in human plasma: identification and incorporation into the clot

Abstract

Coagulation factor XIII (FXIII) is a transglutaminase with a well defined role in the final stages of blood coagulation. Active FXIII (FXIIIa) catalyzes the formation of ε-(γ-glutamyl)lysine isopeptide bonds between specific Gln and Lys residues. The primary physiological outcome of this catalytic activity is stabilization of the fibrin clot during coagulation. The stabilization is achieved through the introduction of cross-links between fibrin monomers and through cross-linking of proteins with anti-fibrinolytic activity to fibrin. FXIIIa additionally cross-links several proteins with other functionalities to the clot. Cross-linking of proteins to the clot is generally believed to modify clot characteristics such as proteolytic susceptibility and hereby affect the outcome of tissue damage. In the present study, we use a proteomic approach in combination with transglutaminase-specific labeling to identify FXIIIa plasma protein substrates and their reactive residues. The results revealed a total of 147 FXIIIa substrates, of which 132 have not previously been described. We confirm that 48 of the FXIIIa substrates were indeed incorporated into the insoluble fibrin clot during the coagulation of plasma. The identified substrates are involved in, among other activities, complement activation, coagulation, inflammatory and immune responses, and extracellular matrix organization.

Keywords: Blood Coagulation Factors; Fibrinogenesis; Plasma; Proteomics; Transglutaminases.

FIGURE 1.

Gene Ontology summary of the identified FXIIIa substrates. The Gene Ontology annotations of the identified FXIIIa substrates are summarized by six categories with the number of proteins in each category in a pie chart. Seven unannotated proteins are not represented. The Others category include 15 proteins annotated to transport processes. These proteins are, among others, apolipoproteins, hemoglobin, ceruloplasmin, and vitamin D-binding protein. A large group of substrates are annotated to the Response to wounding and Immune system process categories. The Response to wounding category is mainly comprised of proteins annotated to coagulation and to the inflammatory response. In the Immune system processes category, proteins annotated to complement activation are heavily represented. The data suggest that FXIIIa-mediated cross-linking plays a role beyond clot stabilization during coagulation.

FIGURE 2.

Secondary structure localization of reactive Gln residues. Secondary structural information was extracted from the UniProt database for 389 of the 549 reactive Gln residues. The reactive residues could therefore be assigned to helix, strand, turn, or loop. The data suggest that reactive Gln residues are overrepresented in loops.