Abstract
Blood coagulation (hemostasis) is a process that is driven by plasma proteins which form a complex network of molecular interactions. An essential part of the coagulation system is formed by a cascade of proenzymes (i.e. coagulation factors) that are sequentially converted into active enzymes. Activation of these coagulation enzymes is regulated by cofactors present in the blood plasma. However, the way regulation is achieved on a molecular level remains an open question for most of the coagulation factors.
In the present thesis we have addressed these issues by use of footprinting techniques in combination with mass spectrometry. Footprinting makes use of chemical modification of amino acids. The extent of chemical modification can be seen as a measure of accessibility of the modified amino acids, either within a single protein or in a complex of proteins. The footprinting approach can, therefore, be used to probe changes within a single protein or to explore binding sites within a protein complex.
As proof-of-principle, footprinting with lysine-reactive tags was applied in a previously well-characterized protein complex between the plasma proteins Receptor-Associated Protein and Low-density-lipoprotein Receptor-related Protein. The same footprinting technique was used to characterize chimeras of the cofactor, factor VIII which contained protein domains of the homologous coagulation factor V.
Subsequent studies were aimed to elucidate the activation process of plasma proenzymes like Hyaluronan-binding-protein 2 and coagulation factors IX, X and prothrombin. The active protease domains of these serine proteases share essentially the same protein fold. By use of footprinting, subtle changes could be probed in the interplay between different parts of these protease domains. This way, activation of factor IX appeared different compared to those of factor X and prothrombin. The activity of activated IX was furthermore studied by alanine substitutions made in the factor IX protease domain.
In the present thesis we have addressed these issues by use of footprinting techniques in combination with mass spectrometry. Footprinting makes use of chemical modification of amino acids. The extent of chemical modification can be seen as a measure of accessibility of the modified amino acids, either within a single protein or in a complex of proteins. The footprinting approach can, therefore, be used to probe changes within a single protein or to explore binding sites within a protein complex.
As proof-of-principle, footprinting with lysine-reactive tags was applied in a previously well-characterized protein complex between the plasma proteins Receptor-Associated Protein and Low-density-lipoprotein Receptor-related Protein. The same footprinting technique was used to characterize chimeras of the cofactor, factor VIII which contained protein domains of the homologous coagulation factor V.
Subsequent studies were aimed to elucidate the activation process of plasma proenzymes like Hyaluronan-binding-protein 2 and coagulation factors IX, X and prothrombin. The active protease domains of these serine proteases share essentially the same protein fold. By use of footprinting, subtle changes could be probed in the interplay between different parts of these protease domains. This way, activation of factor IX appeared different compared to those of factor X and prothrombin. The activity of activated IX was furthermore studied by alanine substitutions made in the factor IX protease domain.
| Original language | English |
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| Award date | 11 Dec 2017 |
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| Print ISBNs | 978-94-6233-810-4 |
| Publication status | Published - 11 Dec 2017 |
Keywords
- footprinting
- mass spectrometry
- coagulation
- hemostasis