| Summary: | Haemostasis is a highly regulated, fundamental, physiological process featuring numerous peripheral membrane proteins. Of these, the membrane and calcium binding properties of the vitamin K-dependent proteins are dependent on a common N-terminal γ-carboxyglutamic acid (Gla)-containing domain. Previous work on Gla proteins has provided a wealth of affinity and kinetic membrane binding information. These studies have employed a number of biophysical techniques using artificial phosphatidylserine-containing model membranes. However, many aspects of the membrane binding interaction, in terms of mechanism and modulation by protein cofactor remain obscure. This thesis examines two methods for studying the membrane binding properties of human plasma derived Gla proteins with emphasis on prothrombin.
In Chapter 3 differential centrifugation combined with immunoaffinity detection was used to quantify the effect the cofactor Factor Va had on the membrane binding affinity of prothrombin for membrane. Factor Va bound to anionic phospholipid membrane undoubtedly enhanced the membrane binding affinity of prothrombin relative to prothrombin binding in the absence of the cofactor. Thus, these results indicate that Factor Va can recruit prothrombin or prethrombin 1, a Gla-domain less fragment of prothrombin, to the membrane surface, plausibly contributing to its cofactor function.
In Chapters 4 and 5, surface plasmon resonance (SPR) was used to evaluate the Ca²⁺-specific binding properties of a number of Gla proteins to immobilized membranes. Membrane affinity, molar binding preference and kinetics controlling complex formation and complex breakdown
varied widely between Gla proteins. The comparative results obtained by SPR indicate that the majority of homologous Gla proteins bind membranes with a complex mechanism which may involve membrane induced protein dimers. Unlike prothrombin, the binding profiles for fragment 1 and fragment 1.2 fitted closely to a one-site binding model. Apparent biphasic association and biphasic dissociation phases were observed for prothrombin and commonly amongst the other Gla proteins at a wide range of protein concentrations including physiological concentrations. For prothrombin, dimerization appears to be specific to the protease domain as neither fragment 1 nor fragment 1.2 displays such binding complexities. It is possible that dimerization increases the half-life of membrane-bound Gla proteins thereby promoting their participation in complex assembly and function. === Medicine, Faculty of === Biochemistry and Molecular Biology, Department of === Graduate
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