Membrane binding properties of prothrombin and other gamma-carboxyglutamic acid-containing coagulation proteins

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 ofBiochemistry and Molecular Biology, Department ofGraduat

Effects of mellitic acid (MA) and sodium fluoride (NaF) on the histological appearance of murine fetal tibiae cultured in vitro

The aim of this studv was to develop a standardized image analysis method for localization and quantitative measurement of calcified structures of murine fetal tibiae cultured in vitro as a completion and verification of previous biochemical studies. The calcified structures of bone stained by von Kossa silver technique and the epiphyseal cartilages showing intensive metachromasia with toluidine-blue staining were converted with grey-value window programs and afterwards the areas of the selected structures were measured. The histomorphological investigations showed that the murine tibiae, incubated for a period of 6 days in a medium with addition of 5 mmol mellitic acid, showed both a significant reduction of calcium deposits and an increase of epiphyseal intercellular cartilage matrix. The tibiae incubated in a medium with addition of 0.5 mmol sodium fluoride significantly showed an increase of calcium deposits in the thickened lammellae of the cornpacta. These histomorphological results confirm previous biochemical studies

Modulation of Complement Activation and Amplification on Nanoparticle Surfaces by Glycopolymer Conformation and Chemistry

The complement system plays an integral part of a host’s innate immunity, and its activation is highly dependent on the chemistry and structure of a “foreign” target surface. We determined that the conformational state of glycopolymer chains, defined by the grafting density (chains/nm²), on the nanoparticle (NP) surface acts as a “molecular switch” for complement activation and amplification, and the protein corona on the NP surface dictates this process. A grafting density threshold was determined, below which minimal complement activation was observed and above which substantial complement activation was detected. The glycopolymer-grafted NPs activated complement <i>via</i> the alternative pathway. The chemical structure of pendent sugar units on the grafted polymer was also an important determinant for complement activation. NPs grafted with glucose-containing polymer activated complement at a lower grafting density compared to NPs grafted with galactose-containing polymer. Analysis of complement activation products C3a and SC5b-9 followed a similar pattern. Complement activation on the NP surface was independent of particle size or concentration for a given conformational state of grafted polymer. To gain insight into a putative surface-dependent mechanism of complement activation, we determined the nature of adsorbed protein corona on various NPs through quantitative mass spectrometry. Elevated levels of two pro-complement proteins, factors B and C3, present on the NP surface grafted with glycopolymer chains at high grafting density compared to low grafting density surface, may be responsible for its complement activity. Galactose polymer modified NPs adsorbed more of the negative regulator of complement, factor H, than the glucose surface, providing an explanation for its lower level of complement activation