Proteomics and biomolecular mass spectrometry of hemostatic processes

Research lines

Structure and function of coagulation factors

The coagulation cascade comprises several serine proteases that act in combination with a non-enzymatic co-factor on phospholipid-containing membranes. Over the past decade we have been focusing on the mechanism by which activated factor IX assembles with its co-factor, factor VIII. These proteins are indispensable for proper functioning of the coagulation cascade as a functional absence of factor VIII and factor IX is associated with the bleeding disorders hemophilia A and hemophilia B. Factor VIII is composed of a series of repeated domains which appear in the order A1-a1-A2-a2-B-a3-A3-C1-C2. The A-domains of factor VIII mediate the binding to activated factor IX and factor X, whereas the C domains have been implicated in binding to phospholipids. After activation, the A2 domain rapidly dissociates from activated factor VIII resulting in a dampening of the activity of the activated factor X-generating complex.

In this line of research, we employ chemical footprinting mass spectrometry (MS) as well as hydrogen-deuterium exchange MS to assess the molecular mechanism behind cofactor function and the enzymatic activity of serine proteases. These approaches are complemented by enzyme-kinetics and site-directed mutagenesis studies of the coagulations proteins. Previously, we identified residues that contribute to the stability of activated factor VIII. Our findings revealed that the lysine couple K1967-K1968 within region T1964-Y1971 has an opposite contribution to activated factor VIII stability. In addition, results showed that region 1803-1810 not only contributes to activated factor VIII stability, it also includes a binding region for activated factor IX.  

Cellular uptake mechanisms behind the clearance of coagulation factors

Coagulation factors are rapidly cleared from the circulation. As a consequence, hemophilia patients require frequent intravenous infusions during coagulation factor substitution therapy. This line of research focusses on the cellular mechanisms that contribute to the clearance of coagulation factor VIII. The mechanisms by which von Willebrand factor (VWF) modulates factor VIII half-life is addressed as well. Chemical footprinting mass spectrometry (MS) and hydrogen-deuterium exchange MS are employed to identify binding sites that contribute to the interaction of factor VIII with its clearance receptors and VWF. The cellular uptake mechanisms of (modified) coagulation factors are further addressed with confocal microscopy and flow cytometry studies. Previously, we have identified LDL receptor-related protein (LRP) as one cellular receptor that contributes to factor VIII clearance.

Ligand binding of LRP is mediated by clusters of small complement-type repeats (CR). It has been proposed that at least two CRs are required for high-affinity interaction by utilizing two spatially distinct lysine residues on the ligand surface. We have recently addressed the role of the critical lysines of factor VIII not only for LRP binding but also for the endocytosis of this cofactor. Competition experiments employing an antagonist of the LDL receptor family members revealed that there is a cell surface binding event for factor VIII, which is independent of LRP. Cell surface binding proved to be less effective for the fluorescent factor VIII-YFP variants K2092A, F2093A and K2092A/F2093A.

Surface plasmon resonance analysis showed that these substitutions affect LRP binding as well. Finally, flow cytometry analysis revealed a major reduction of endocytic uptake of these factor VIII-YFP variants. Our results demonstrate that C1 domain residues K2092-F2093 are of major importance for FVIII endocytosis by contributing to cell surface binding and receptor binding. 

Uptake and storage of coagulation factors in megakaryocytes and platelets and disorders therein.      

The α-granules of platelets contain a large variety of proteins with diverse and opposing functions. Since platelets themselves possess little biosynthetic capacity, most of the α-granule proteins are synthesized by megakaryocytes, the platelet precursor cells. Assembly of α-granules and cargo distribution also occurs in the megakaryocyte. In contrast to other α-granule proteins, fibrinogen and factor V are exceptional as they are derived completely from endocytosis from plasma. The mechanisms that drive the endocytosis and subsequent packaging of these proteins into α-granules are poorly understood.

It further seems remarkable that megakaryocytes comprise specialized mechanisms for the endocytosis and subsequent storage of factor V and fibrinogen only. In this line of research, we employ proteomics approaches on platelets and maturating megakaryocytes derived from hematopoietic progenitors to study α-granule biogenesis as well as platelet and megakaryocyte dysfunction in general. As a spin off from this from this research line, we recently found that rare platelet disorders reveal unique protein expression profiles that not only facilitates the diagnosis of these rare platelet disorders. In combination with next generation sequencing approaches, it also provides the basis for the identification of novel disorders.

Key publications

  • Differences between Platelets Derived from Neonatal Cord Blood and Adult Peripheral Blood Assessed by Mass Spectrometry. 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. J Proteome Res. 2017 Oct 6;16(10):3567-3575
  • Role of glycine 221 in catalytic activity of hyaluronan-binding protein 2. Stavenuiter F, Ebberink EHTM, Mertens K, Meijer AB. J Biol Chem. 2017 Apr 14;292(15):6381-6388.
  • Combined immunodeficiency with severe inflammation and allergy caused by ARPC1B deficiency. 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. 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.