Avantika Golas

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).

Anchorage-dependent mammalian cells are typically grown in vitro on hydrophilic glass and plastic substrata in a medium supplemented with 5–20% v/v blood-serum proteins. Inoculated single cells gravitate from suspension to within close proximity of substrata surfaces whereupon initial contact and attachment occurs followed by progressive cell adhesion, spreading, and ultimately proliferation. A critical examination of the role of proteins and water in the initial attachment phase concludes that the cell attachment phase is not mediated by biological recognition of surface-adsorbed ligands by cell membrane receptors as frequently depicted in various textbook explanations of cell adhesion. This conclusion is based on extensive experimental evidence showing that blood proteins do not adsorb on hydrophilic surfaces that are most conducive to cell growth but do adsorb on hydrophobic surfaces that are not conducive to cell growth. As a consequence, the conventional idea that initial cell attachment is mediated by various adhesin factors adsorbed from serum-protein solutions is viewed as untenable. Rather, it is concluded that the initial contact-and-attachment of cells to hydrophilic surfaces is controlled by physicochemical interactions unrelated to biological recognition. The general physics of these interactions is known but an adequate descriptive theory that can be tested against experimentally measured cell adhesion kinetics has yet to be developed. The role of these physicochemical interactions in stimulating biological machinery within cells to fully adhere and proliferate on surfaces of biotechnical interest is unknown but is of great significance to the science underlying various biomedical and biotechnical applications of materials.