Cryptic D-Dimer Antigen: A Sensitive Marker of Factor XIIIa Activity during Intravascular Coagulation.
Authors: Suwanawiboon Bundarika , Haroon Zishan A. , Lai Thung S. , Wu Jogin R. , Greenberg Charles S. 
Published: October 2019

Abstract
FactorXIIIa (FXIIIa) plays an important role in regulating hemostatic and thrombotic events. FXIIIa modifies the structure of fibrin making the insoluble fibrin gel resistant to plasmin degradation. Methods to detect FXIIIa crosslinking of soluble fibrin(ogen) complexes (SFCs) are not readily available. The purpose of this study was to 1) develop a sensitive assay to detect FXIIIa crosslinked (XL)-SFCs designated: cryptic D-dimer assay, 2) determine whether soluble XL-SFCs are formed prior to formation of an insoluble clot, 3) determine whether soluble XL-SFCs circulated in human plasma and could be incorporated into a fibrin clot, 4) determine whether levels of soluble XL-SFCs are elevated in patients suspected of having intravascular coagulation. We postulated that the cryptic D-dimer antigen would be created by FXIIIa crosslinking of SFCs and could be detected after plasmin digestion. We added purified recombinant FXIIIa (119 nM) to afibrinogenemic plasma with varying concentrations of purified fibrinogen (10–200 μg/mL) and assayed for D-dimer antigen levels pre- and post- plasmin treatment. D-dimer levels were determined by a quantitative latex agglutination assay (MiniQuant® D-dimer, Ireland). The assay utilizes a monoclonal antibody specific for an epitope that is present on plasmin modified crosslinked D-dimer domain on either fibrinogen or fibrin. We found that generation of cryptic D-dimer antigen was a) dependent on FXIIIa, b) uncovered only after plasmin digestion and c) increased with escalating fibrinogen concentration. The level of cryptic D-dimer antigen increased from 0.45 μg/ml to 5.4 μg/ml in the presence of fibrinogen (10–200 μg/ml), FXIIIa and 5 mM calcium chloride. We had earlier confirmed that the cryptic D-dimer antigen could be detected in both EDTA and citrated plasmas and the exposure of this antigen was not modified by calcium present during plasmin digestion. We also determined that the cryptic D-dimer antigen was generated 150 sec before a visible clot appeared in recalcifed plasma in plastic tubes. We then investigated whether cryptic D-dimer antigen circulated in healthy individuals and if these levels changed in patients. Cryptic D-dimer antigen was detected (mean level 1 μg/ml, <1% of plasma fibrinogen level) in ten normal plasma samples. In comparison, patients with elevated D-dimer by ELISA assay showed cryptic D-dimer mean level in excess of 5 μg/ml. Cryptic D-dimer levels increased with rising native D-dimer (no plasmin treatment) levels in patients. To assess the thrombogenic potential of SFCs with cryptic D-dimer antigens, we added reptilase (5 BU/ml) to plasma and examined whether the cryptic D-dimer antigen or native D-dimer could bind to a fibrin clot. We found that more than 70% of the cryptic D-dimer antigen was clottable (n=20) as compared to only 48 % of the native D-dimer (n=20). This suggests that the majority of cryptic D-dimer antigen is present on clottable SFCs. In conclusion, we developed an assay that can detect the ability of FXIIIa to modify SFCs before the appearance of a fibrin clot and may serve as a sensitive marker of intravascular fibrin formation. The cryptic D-dimer antigen is a novel analyte that is present on clottable SFCs and its levels increased in individuals experiencing intravascular blood coagulation. In addition, a basal level of FXIIIa activity exists in human plasma and that regulating this process could have an important impact on the fate of intravascular fibrin and thrombotic events.