title: Enzymes produced by autoactivation of blood factor XII in buffer: A contribution from the Hematology at Biomaterial Interfaces Research Group.
abstract: High-resolution electrophoresis of FXII-derived proteins produced by contact activation of FXII in buffer solutions (i.e. in absence of plasma proteins) with hydrophilic and silanized-glass activators spanning the observable range of water wettability (hydrophilic to hydrophobic), shows no evidence of proteolytic cleavage of FXII into αFXIIa or βFXIIa. The autoactivation mixture contains only a single-chain protein with a molecular weight of ∼80 kDa, confirming Oscar Ratnoff's previous finding of a single-chain activated form of FXII that he called 'HFea'. Functional assays have shown that these autoactivation products exhibit procoagulant potential (protease activity inducing clotting of blood) or amidolytic potential (cleaves amino bonds in s-2302 chromogen but do not cause coagulation of plasma) or both amidolytic potential and procoagulant potential. Some of these proteins also have the remarkable potential to 'suppress autoactivation' (i.e. suppress creation of enzymes with procoagulant potential). It is thus hypothesized that autoactivation of FXII in the absence of plasma proteins generates not just a single type of activated conformer, as suggested by previous researchers, but rather an ensemble of conformer products with collective activity that varies with activator surface energy used in contact activation of FXII. Furthermore, reaction of αFXIIa with FXII in buffer solution does not produce additional αFXIIa by the putative autoamplification reaction FXIIa + FXII → 2FXIIa as has been proposed in past literature to account for the discrepancy between chromogenic and plasma-coagulation assays for αFXIIa in buffer solution. Instead, net procoagulant activity measured directly by plasma-coagulation assays, decreases systematically with increasing FXII solution concentration. Under the same reaction conditions, chromogenic assay reveals that net amidolytic activity increases with increasing FXII solution concentration. Thus, although autoamplification does not occur it appears that there is some form of "FXII self reaction" that influences products of αFXIIa reaction with FXII. Electrophoretic measurements indicate that no proteolytic cleavage takes in this reaction leading us to conclude that change in activity is most likely due to change(s) in FXII conformation (with related change in enzyme activity).
authors.affiliation: Department of Materials Science and Engineering, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Avantika
authors.initials: A
authors.lastname: Golas
authors.affiliation: Department of Materials Science and Engineering, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Harit
authors.initials: H
authors.lastname: Pitakjakpipop
authors.affiliation: Department of Materials Science and Engineering, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Rahn
authors.affiliation: Department of Bioengineering, The Pennsylvania State University, University Park, PA 16802, USA; Department of Surgery, Pennsylvania State University College of Medicine, Biomedical Engineering Institute, Hershey, PA 17033, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Siedlecki
authors.affiliation: Department of Materials Science and Engineering, The Pennsylvania State University, University Park, PA 16802, USA; Department of Bioengineering, The Pennsylvania State University, University Park, PA 16802, USA. Electronic address: EAV3@PSU.EDU.
authors.forename: Erwin A
authors.initials: EA
authors.lastname: Vogler
mesh_terms: D001672:Biocompatible Materials / Q000494:pharmacology*; D001777:Blood Coagulation / Q000187:drug effects; D002021:Buffers; D004586:Electrophoresis; D004789:Enzyme Activation / Q000187:drug effects; D005174:Factor XII / Q000737:chemistry* / Q000378:metabolism*; D015956:Factor XIIa / Q000378:metabolism; D006405:Hematology; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D019679:Kininogen, High-Molecular-Weight / Q000494:pharmacology; D011288:Prekallikrein / Q000494:pharmacology; D012106:Research; D012996:Solutions; D013997:Time Factors
chemical_list: D001672:Biocompatible Materials; D002021:Buffers; D019679:Kininogen, High-Molecular-Weight; D012996:Solutions; D005174:Factor XII; D011288:Prekallikrein; D015956:Factor XIIa
keywords: Autoactivation; Autoamplification; Autohydrolysis; Conformers; FXII; Plasma coagulation