Comparison of FXII contact activation with or without proteolytic cleavage

Abstract

Event Abstract Back to Event Comparison of FXII contact activation with or without proteolytic cleavage Yuan Yan1, Lichong Xu1 and Christopher A. Siedlecki1 1 The Pennsylvania State University Hershey Medical Center, Department of Surgery and Bioengineering, United States Introduction: Surface-induced plasma coagulation is potentiated by contact activation of the blood zymogen factor XII (FXII, Hageman factor). Recent studies revealed that FXII surface activation exhibited different characteristics in neat-buffer and in plasma[1]. aFXIIa and βFXIIa are well known products of FXII contact activation via enzymatic cleavage in the presence of plasma proteins such as prekallikrein, plasmin, and trypsin[2]. Activation products of FXII in buffer (FXII autoactivation) are yet to be fully understood. This study compared the products of FXII activation with or without adding prekallikrein (PK) with regard to activated FXII composition, procoagulant and amidolytic activities. Materials and Methods: Activators used were glass particles (425-600 µm, Sigma Aldrich) in either cleaned or silanized form. 500 µL of 30 µg/ml FXII in PBS was brought into contact with 100 mg of different activators with or without added PK for 1 hr on a rotating hematology mixer. The products present in the supernatant were then assessed by high-resolution electrophoresis (Agilent Protein 230 kit) for molecular weight analysis, a plasma coagulation time assay for procoagulant yield, and a chromogenic assay with Pefa-5963 for amidolytic yield. Results and Discussion: Electrophoretic analyses of FXII activation products in neat-buffer with or without PK. In the absence of PK, the products of FXII activation showed only one band in the size of 95-98 kDa regardless of surface activator, indicating that no proteolytic cleavage takes place in FXII autoactivation. Instead, these FXII-derived products appear to be of the same molecular weight as initial FXII. Note that they present as~ 20% higher molecular weight as observed by automated electrophoresis due to the glycosylation of FXII[3]. In the presence of PK, FXII-derived fragments with smaller molecular weight of about 33 kDa were observed. This matches the size of shared catalytic domain by both aFXIIa and βFXIIa. Yields of FXII-derived products by different activators are shown in Table 1 according to the concentrations calculated by electrophoresis software. Functional analyses of FXII activation products in buffer with or without PK (Figure 1). FXII autoactivation takes place at all surface types, not only at anionic, as demonstrated by measurable procoagulant and amidolytic activities. However, procoagulant yields from OTS and APTES were much lower than that of Glass, suggesting activators of varying surface energy induce different FXII conformers displaying remarkably different procoagulant activity. The presence of PK (or proteolytic cleavage) amplified both procoagulant and amidolytic yields from all surface types in a manner roughly proportional to the amount of βFXIIa and total FXIIa, respectively. Conclusion: FXII contact activation in buffer does not produce proteolytic fragments. It is likely that FXII undergoes non-enzymatic conformational changes in response to surface contact, thus showing different procoagulant and amidolytic activities depending on surface energy. The presence of prekallikrein amplifies both activities, likely as the result of subsequent enzymatic cleavage of FXII into aFXIIa and βFXIIa.