Single-chain factor XII exhibits activity when complexed to polyphosphate

BackgroundThe mechanism underpinning factorXII autoactivation was originally characterized with non-physiological surfaces, such as dextran sulfate (DS), ellagic acid, and kaolin. Several natural' anionic activating surfaces, such as platelet polyphosphate (polyP), have now been identified. ObjectiveTo analyze the autoactivation of FXII by polyP of a similar length to that found in platelets (polyP(70)). Methods and resultsPolyP(70) showed similar efficacy to DS in stimulating autoactivation of FXII, as detected with amidolytic substrate. Western blotting revealed different forms of FXII with the two activating surfaces: two-chain FXIIa was formed with DS, whereas single-chain FXII (scFXII; 80kDa) was formed with polyP(70). Dissociation of scFXII from polyP(70) abrogated amidolytic activity, suggesting reversible exposure of the active site. Activity of scFXII-polyP(70) was enhanced by Zn2+ and was sensitive to NaCl concentration. A bell-shaped concentration response to polyP(70) was evident, as is typical of surface-mediated reactions. Reaction of scFXII-polyP(70) with various concentrations of S2302 generated a sigmoidal curve, in contrast to a hyperbolic curve for FXIIa, from which a Hill coefficient of 3.67 was derived, indicative of positive cooperative binding. scFXII-polyP(70) was more sensitive to inhibition by H-d-Pro-Phe-Arg-chloromethylketone and corn trypsin inhibitor than FXIIa, but inhibition profiles for C1-inhibitor were similar. Active scFXII-polyP(70) was also able to cleave its physiological targets FXI and prekallikrein to their active forms. ConclusionsAutoactivation of FXII by polyP, of the size found in platelets, proceeds via an active single-chain intermediate. scFXII-polyP(70) shows activity towards physiological substrates, and may represent the primary event in initiating contact activation invivo.