Structure-function relationship and flow-induced conformational changes in coagulation proteins and cellular receptors

The dynamics of proteins at interactive surfaces contributes to molecular recognition events that drive assembly into biologically active conformations. We are addressing the issue of dynamics by recent innovative functional proteomics approaches. To this end, we have implemented chemical labelling techniques using isobaric tandem mass tags (TMTs) to probe conformational changes in enzyme‐cofactor and enzyme‐receptor complexes. We further have been implementing hydrogen‐deuterium exchange (HDX‐MS) to monitor the conformational details of intra‐ and inter‐domain interactions in protein complexes. This “footprinting” approach allows the identification of exposed surface elements, and provides a key to understand their molecular interaction. We now use these technologies to dissect the structure-function of factor VIII and it’s interaction partners, including the clearance receptor LRP1, FVIII-specific antibodies, and the molecular structure of the tenase complex, the Factor IXa-Factor VIII-Factor X enzyme‐cofactor complex that provides the heart of the coagulation cascade.

Blood flow is an integral part of hemostasis, yet is completely overlooked in most studies on hemostatic protein interactions with the vasculature. The current project aims to identify the effect of blood flow on crosstalk between hemostatic proteins and the vasculature. More specifically, this project aims to 1) dissect flow-dependent receptor-ligand interactions 2) identify flow-induced conformational changes in membrane receptors and the vascular membrane surface and 3) uncover the role of key players of primary hemostasis, including platelets and VWF, and secondary hemostasis, including FVIII, in coagulation-induced signaling in the presence and absence of flow. For these studies, advanced mass spectrometry-based approaches are used including chemical footprinting using tandem-mass tags, hydrogen-deuterium exchange, cell surface proteomics and phosphoproteomics. Mass spectrometry-based studies will be complemented using biochemical and cell biological approaches including surface plesmon resonance and confocal microscopy. These studies will help to understand mechanosensory protein-receptor interactions and coagulation-induced vascular signaling.  Understanding these interactions and signaling events is essential to develop rational approaches for the design of coagulation factors with an improved function and to identify potential novel targets for therapeutic intervention of hemostatic disorders.   

 

Phosphoproteomics of endothelial cell and blood platelets activation

Bleeding and clotting, inflammatory and vascular diseases

Activation of endothelial cells and blood platelets is essential for blood clot formation and hemostasis. Endothelial cells and blood platelets express a variety of receptors that can be activated by multiple ligands resulting in endothelial and platelet activation, shape change and aggregation. The signaling pathways downstream of these receptors have remained largely unexplored. The current project aims to unravel which signaling pathways are triggered upon endothelial and platelet activation by various agonists using mass spectrometry-based phosphoproteomics. This approach has recently been used successfully to dissect thrombin-mediated signaling events in endothelial cells. The same technology will also be used to better define the aberrant functional and signaling properties of a cohort of patients with congenital platelet disorders. The findings will be combined with next generation sequencing approaches to define signaling networks crucial for platelet function. Altogether, this will provide us with a roadmap of early signaling events that contribute to platelet (dys)function.

Proteomics of hematopoiesis

Hematopoiesis is the process of self-renewal or differentiation of hematopoietic stem cells into all blood cell types through multi-lineage diversification. Current efforts to obtain detailed knowledge on the driving forces behind hematopoiesis are directed at dissecting changes in gene expression at the level of RNA transcription. However, there is a large gap in our knowledge on cell-specific processes that regulate translation of RNA into protein, and therefore studies based on RNA expression alone can be misleading and fail to identify key proteins and cellular processes. It is therefore of prime importance to study the differentiation of hematopoietic stem cells into functional blood cells as well as circulating blood cells not only at the RNA level, but more importantly also at the protein level. This project aims to obtain protein expression profiles for all circulating blood cells, to uncover changes in protein expression profiles during hematopoiesis and to gain more insight into the cell-specific processes that regulate translation of RNA into protein. 

Key publications

• Stokhuijzen E, Koornneef JM, Nota B, van den Eshof BL, van Alphen FPJ, van den Biggelaar M, van der Zwaan C, Kuijk C, Mertens K, Fijnvandraat K, Meijer AB. Differences between Platelets Derived from Neonatal Cord Blood and Adult Peripheral Blood Assessed by Mass Spectrometry. J Proteome Res. 2017 Oct 6;16(10):3567-3575
• Stavenuiter F, Ebberink EHTM, Mertens K, Meijer AB. Role of glycine 221 in catalytic activity of hyaluronan-binding protein 2. J Biol Chem. 2017 Apr 14;292(15):6381-6388.
• Kuijpers TW, Tool ATJ, van der Bijl I, de Boer M, van Houdt M, de Cuyper IM, Roos D, van Alphen F, van Leeuwen K, Cambridge EL, Arends MJ, Dougan G, Clare S, Ramirez-Solis R, Pals ST, Adams DJ, Meijer AB, van den Berg TK. Combined immunodeficiency with severe inflammation and allergy caused by ARPC1B deficiency. J Allergy Clin Immunol. 2017 Jul;140(1):273-277.e10
• Van den Biggelaar M, Madsen JJ, Faber JH, Zuurveld MG, van der Zwaan C, Olsen OH, Stennicke HR, Mertens K, Meijer AB. Factor VIII Interacts with the Endocytic Receptor Low-Density Lipoprotein Receptor-Related Protein 1 via an Extended Surface Comprising 'Hot-Spot' Lysine Residues. J Biol Chem 2015 Jul 3;290(27):16463-76.