BACKGROUND: The mechanism underpinning factor XII 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. OBJECTIVE: To analyze the autoactivation of FXII by polyP of a similar length to that found in platelets (polyP70 ). METHODS AND RESULTS: PolyP70 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 αFXIIa was formed with DS, whereas single-chain FXII (scFXII; 80 kDa) was formed with polyP70 . Dissociation of scFXII from polyP70 abrogated amidolytic activity, suggesting reversible exposure of the active site. Activity of scFXII-polyP70 was enhanced by Zn(2+) and was sensitive to NaCl concentration. A bell-shaped concentration response to polyP70 was evident, as is typical of surface-mediated reactions. Reaction of scFXII-polyP70 with various concentrations of S2302 generated a sigmoidal curve, in contrast to a hyperbolic curve for αFXIIa, from which a Hill coefficient of 3.67 was derived, indicative of positive cooperative binding. scFXII-polyP70 was more sensitive to inhibition by H-d-Pro-Phe-Arg-chloromethylketone and corn trypsin inhibitor than αFXIIa, but inhibition profiles for C1-inhibitor were similar. Active scFXII-polyP70 was also able to cleave its physiological targets FXI and prekallikrein to their active forms. CONCLUSIONS: Autoactivation of FXII by polyP, of the size found in platelets, proceeds via an active single-chain intermediate. scFXII-polyP70 shows activity towards physiological substrates, and may represent the primary event in initiating contact activation in vivo.
BACKGROUND: The mechanism underpinning factor XII 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. OBJECTIVE: To analyze the autoactivation of FXII by polyP of a similar length to that found in platelets (polyP70 ). METHODS AND RESULTS: PolyP70 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 αFXIIa was formed with DS, whereas single-chain FXII (scFXII; 80 kDa) was formed with polyP70 . Dissociation of scFXII from polyP70 abrogated amidolytic activity, suggesting reversible exposure of the active site. Activity of scFXII-polyP70 was enhanced by Zn(2+) and was sensitive to NaCl concentration. A bell-shaped concentration response to polyP70 was evident, as is typical of surface-mediated reactions. Reaction of scFXII-polyP70 with various concentrations of S2302 generated a sigmoidal curve, in contrast to a hyperbolic curve for αFXIIa, from which a Hill coefficient of 3.67 was derived, indicative of positive cooperative binding. scFXII-polyP70 was more sensitive to inhibition by H-d-Pro-Phe-Arg-chloromethylketone and corn trypsin inhibitor than αFXIIa, but inhibition profiles for C1-inhibitor were similar. Active scFXII-polyP70 was also able to cleave its physiological targets FXI and prekallikrein to their active forms. CONCLUSIONS: Autoactivation of FXII by polyP, of the size found in platelets, proceeds via an active single-chain intermediate. scFXII-polyP70 shows activity towards physiological substrates, and may represent the primary event in initiating contact activation in vivo.
Authors: Rami Abu-Fanne; Victoria Stepanova; Rustem I Litvinov; Suhair Abdeen; Khalil Bdeir; Mohamed Higazi; Emad Maraga; Chandrasekaran Nagaswami; Alexander R Mukhitov; John W Weisel; Douglas B Cines; Abd Al-Roof Higazi Journal: Blood Date: 2018-11-15 Impact factor: 22.113
Authors: Joanne L Mitchell; Ausra S Lionikiene; Georgi Georgiev; Anja Klemmer; Chelsea Brain; Paul Y Kim; Nicola J Mutch Journal: Blood Date: 2016-09-30 Impact factor: 22.113
Authors: Yasin Kokoye; Ivan Ivanov; Qiufang Cheng; Anton Matafonov; S Kent Dickeson; Shauna Mason; Daniel J Sexton; Thomas Renné; Keith McCrae; Edward P Feener; David Gailani Journal: Thromb Res Date: 2016-02-18 Impact factor: 3.944