Introduction: Surface contact activation of the blood zymogen factor XII (FXII, Hageman factor) has been implicated as an important cause for poor hemocompatibility of biomaterials. Recent studies on contact activation of FXII in neat-buffer (FXII autoactivation) allowed for close examination of FXII/surface interactions [1][2]. Divergent observations of FXII activation in buffer and in plasma lead us to ponder the influences of plasma proteins not directly involved in coagulation cascade on activation of FXII. This study investigated FXII contact activation by different types of activators in a system of incrementally increased complexity by adding selected plasma proteins spanning three decades of molecular weight.
Materials and Methods: Glass particles (425-600 nm, Sigma Aldrich) were cleaned and silanized by octadecyltrichlorosilane (OTS) or 3-aminopropyltrichlorosilane (APTES) following standard procedures[2]. Bovine albumin (1 mg/mL), bovine ubiquitin (0.1 mg/mL) and bovine IgG (10 mg/mL) were prepared in PBS separately or as a mixture. 500 µL of 30 µg/ml FXII in PBS with or without these supplemental proteins was brought into contact with 100 mg of different activators for 1 hr on a rotating hematology mixer. The products present in the supernatant were then assessed by a plasma coagulation time assay for procoagulant yields, and a chromogenic assay with Pefa-5963 for amidolytic yield.
Results and Discussion: Plasma proteins promote FXII autoactivation by hydrophilic Glass (Figure 1). Procoagulant yield of FXII contact activation by hydrophilic Glass was significantly increased in the order of BSA>cocktail>IgG>Ubiquitin by 3-20 times. This observation partially explains why procoagulant yields of the same surface area of glass induced in plasma are greater than in neat-buffer. Generation of amidolytic yield was enhanced in similar ways to that of procoagulant yield but at lower levels of 1.3 to 2.9 times. The ratio of procoagulant to amidolytic yield was elevated in the order of cocktail > BSA > IgG >Ubiquitin. It has suggested that plasma proteins favor the amplification of procoagulant over amidolytic yields. Plasma proteins like BSA having similar MW as FXII seem to have the most effect on FXII autoactivation. It is possible that influences of plasma proteins on FXII autoactivation are dependent on the MW of proteins.
Effects of plasms proteins on OTS and APTES induced FXII contact activation (Figure 2). Plasma proteins promoted the procoagulant yield of OTS by 2-11 times in the order of BSA>cocktail> Ubiquitin> IgG, a similar trend as that of hydrophilic Glass. Procoagulant yields of APTES were amplified by 2-5 times in the order of cocktail>IgG>Ubiquitin> BSA, opposite to the order of Glass and OTS, suggesting that plasma proteins affect FXII autoactivation by different modes depending on properties of the activators.
Conclusion: Plasma proteins appear to enhance FXII autoactivation in terms of procoagulant and amidolytic yields in different manners depending on both properties of proteins and activators.
References:
[1] Zhou et al., Biomaterials, 2006, 27, 4325
[2] Golas et al. , Biomaterials, 2010, 31, 1068