Single‐chain factor XII exhibits activity when complexed to polyphosphate

• Published in: Journal of thrombosis and haemostasis Vol. 12; no. 9; pp. 1513 - 1522
• Main Authors: Engel, R., Brain, C. M., Paget, J., Lionikiene, A. S., Mutch, N. J.
• Format: Journal Article
• Language: English
• Published: England Elsevier Limited 01-09-2014
• Subjects: Amino Acid Chloromethyl Ketones - chemistry; Anions - chemistry; Arginine - chemistry; blood coagulation; Blood Platelets - metabolism; Catalytic Domain; Dextran Sulfate - chemistry; Disulfides - chemistry; Ellagic Acid - chemistry; Enzyme Precursors - chemistry; Factor XII - chemistry; factor XII; Hemostasis; Humans; Kaolin - chemistry; Plant Proteins - chemistry; polyphosphates; Polyphosphates - chemistry; Prekallikrein - chemistry; Protein Binding; RNA - chemistry; Surface Properties; zymogens; zymogens; hemostasis; blood coagulation; factor XII; polyphosphates
• ISSN: 1538-7933; 1538-7836; 1538-7836
• DOI: 10.1111/jth.12663

Summary
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 Zn2+ 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.