A site on factor XII required for productive interactions with polyphosphate.

BACKGROUND

During plasma contact activation, factor XII (FXII) binds to surfaces through its heavy chain and undergoes conversion to the protease FXIIa. FXIIa activates prekallikrein and factor XI (FXI). Recently, we showed that the FXII first epidermal growth factor-1 (EGF1) domain is required for normal activity when polyphosphate is used as a surface.

OBJECTIVES

The aim of this study was to identify amino acids in the FXII EGF1 domain required for polyphosphate-dependent FXII functions.

METHODS

FXII with alanine substitutions for basic residues in the EGF1 domain were expressed in HEK293 fibroblasts. Wild-type FXII (FXII-WT) and FXII containing the EGF1 domain from the related protein Pro-HGFA (FXII-EGF1) were positive and negative controls. Proteins were tested for their capacity to be activated, and to activate prekallikrein and FXI, with or without polyphosphate, and to replace FXII-WT in plasma clotting assays and a mouse thrombosis model.

RESULTS

FXII and all FXII variants were activated similarly by kallikrein in the absence of polyphosphate. However, FXII with alanine replacing Lys, Lys, and Lys (FXII-Ala) or Lys, His, and Lys (FXII-Ala) were activated poorly in the presence of polyphosphate. Both have <5% of normal FXII activity in silica-triggered plasma clotting assays and have reduced binding affinity for polyphosphate. Activated FXIIa-Ala displayed profound defects in surface-dependent FXI activation in purified and plasma systems. FXIIa-Ala reconstituted FXII-deficient mice poorly in an arterial thrombosis model.

CONCLUSION

FXII Lys, Lys, Lys, and Lys form a binding site for polyanionic substances such as polyphosphate that is required for surface-dependent FXII function.