Mass spectrometry-based analysis on the impact of whole blood donation on the global plasma proteome.

BACKGROUND: Biomonitoring may provide important insights into the impact of a whole blood donation for individual blood donors. STUDY DESIGN AND METHODS: Here, we used unbiased mass spectrometry (MS)-based proteomics to assess longitudinal changes in the global plasma proteome, after a single blood donation for new and regular donors. Subsequently, we compared plasma proteomes of 76 male and female whole blood donors, that were grouped based on their ferritin and hemoglobin (Hb) levels. RESULTS: The longitudinal analysis showed limited changes in the plasma proteomes of new and regular donors after a whole blood donation during a 180-day follow-up period, apart from a significant short-term decrease in fibronectin. No differences were observed in the plasma proteomes of donors with high versus low Hb and/or ferritin levels. Plasma proteins with the highest variation between and within donors included pregnancy zone protein, which was associated with sex, Alfa 1-antitrypsin which was associated with the allelic variation, and Immunoglobulin D. Coexpression analysis revealed clustering of proteins that are associated with platelet, red cell, and neutrophil signatures as well as with the complement system and immune responses, including a prominent correlating cluster of immunoglobulin M (IgM), immunoglobulin J chain (JCHAIN), and CD5 antigen-like (CD5L). DISCUSSION: Overall, our proteomic approach shows that whole blood donation has a limited impact on the plasma proteins measured. Our findings suggest that plasma profiling can be successfully employed to consistently detect proteins and protein complexes that reflect the functionality and integrity of platelets, red blood cells, and immune cells in blood donors and thus highlights its potential use for donor health monitoring.

Factor VIII-driven changes in activated factor IX explored by hydrogen-deuterium exchange mass spectrometry.

The assembly of the enzyme-activated factor IX (FIXa) with its cofactor, activated factor VIII (FVIIIa) is a crucial event in the coagulation cascade. The absence or dysfunction of either enzyme or cofactor severely compromises hemostasis and causes hemophilia. FIXa is a notoriously inefficient enzyme that needs FVIIIa to drive its hemostatic potential, by a mechanism that has remained largely elusive to date. In this study, we employed hydrogen-deuterium exchange-mass spectrometry (HDX-MS) to investigate how FIXa responds to assembly with FVIIIa in the presence of phospholipids. This revealed a complex pattern of changes that partially overlaps with those changes that occur upon occupation of the substrate-binding site by an active site-directed inhibitor. Among the changes driven by both cofactor and substrate, HDX-MS highlighted several surface loops that have been implicated in allosteric networks in related coagulation enzymes. Inspection of FVIIIa-specific changes indicated that 3 helices are involved in FIXa-FVIIIa assembly. These are part of a basic interface that is also known as exosite II. Mutagenesis of basic residues herein, followed by functional studies, identified this interface as an extended FVIIIa-interactive patch. HDX-MS was also applied to recombinant FIXa variants that are associated with severe hemophilia B. This revealed that single amino acid substitutions can silence the extended network of FVIIIa-driven allosteric changes. We conclude that HDX-MS has the potential to visualize the functional impact of disease-associated mutations on enzyme-cofactor complexes in the hemostatic system.

Factor VIII/V C-domain swaps reveal discrete C-domain roles in factor VIII function and intracellular trafficking.

Factor VIII C-domains are believed to have specific functions in cofactor activity and in interactions with von Willebrand factor. We have previously shown that factor VIII is co-targeted with von Willebrand factor to the Weibel-Palade bodies in blood outgrowth endothelial cells, even when factor VIII carries mutations in the light chain that are associated with defective von Willebrand factor binding. In this study, we addressed the contribution of individual factor VIII C-domains in intracellular targeting, von Willebrand factor binding and cofactor activity by factor VIII/V C-domain swapping. Blood outgrowth endothelial cells were transduced with lentivirus encoding factor V, factor VIII or YFP-tagged C-domain chimeras, and examined by confocal microscopy. The same chimeras were produced in HEK293-cells for in vitro characterization and chemical foot-printing by mass spectrometry. In contrast to factor VIII, factor V did not target to Weibel-Palade bodies. The chimeras showed reduced Weibel-Palade body targeting, suggesting that this requires the factor VIII C1-C2 region. The factor VIII/V-C1 chimera did not bind von Willebrand factor and had reduced affinity for activated factor IX, whereas the factor VIII/V-C2 chimera showed a minor reduction in von Willebrand factor binding and normal interaction with activated factor IX. This suggests that mainly the C1-domain carries factor VIII-specific features in assembly with von Willebrand factor and activated factor IX. Foot-printing analysis of the chimeras revealed increased exposure of lysine residues in the A1/C2- and C1/C2-domain interface, suggesting increased C2-domain mobility and disruption of the natural C-domain tandem pair orientation. Apparently, this affects intracellular trafficking, but not extracellular function.

Distinct roles of Ser-764 and Lys-773 at the N terminus of von Willebrand factor in complex assembly with coagulation factor VIII.

Complex formation between coagulation factor VIII (FVIII) and von Willebrand factor (VWF) is of critical importance to protect FVIII from rapid in vivo clearance and degradation. We have now employed a chemical footprinting approach to identify regions on VWF involved in FVIII binding. To this end, lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII, which does not bind VWF. Nano-LC-MS analysis showed that the lysine residues of almost all identified VWF peptides were not differentially modified upon incubation of VWF with FVIII or activated FVIII. However, Lys-773 of peptide Ser-766-Leu-774 was protected from chemical modification in the presence of FVIII. In addition, peptide Ser-764-Arg-782, which comprises the first 19 amino acid residues of mature VWF, showed a differential modification of both Lys-773 and the α-amino group of Ser-764. To verify the role of Lys-773 and the N-terminal Ser-764 in FVIII binding, we employed VWF variants in which either Lys-773 or Ser-764 was replaced with Ala. Surface plasmon resonance analysis and competition studies revealed that VWF(K773A) exhibited reduced binding to FVIII and the FVIII light chain, which harbors the VWF-binding site. In contrast, VWF(S764A) revealed more effective binding to FVIII and the FVIII light chain compared with WT VWF. The results of our study show that the N terminus of VWF is critical for the interaction with FVIII and that Ser-764 and Lys-773 have opposite roles in the binding mechanism.

Variant mapping using mass spectrometry-based proteotyping as a diagnostic tool in von Willebrand disease.

BACKGROUND: von Willebrand disease (VWD) is the most common inherited bleeding disorder, characterized by either partial or complete von Willebrand factor (VWF) deficiency or by the occurrence of VWF proteoforms of altered functionality. The gene encoding VWF is highly polymorphic, giving rise to a variety of proteoforms with varying plasma concentrations and clinical significance. OBJECTIVES: To address this complexity, we translated genomic variation in VWF to corresponding VWF proteoforms circulating in blood. METHODS: VWF was characterized in VWD patients (n = 64) participating in the Willebrand in the Netherlands study by conventional laboratory testing, DNA sequencing and complementary discovery, and targeted mass spectrometry-based plasma proteomic strategies. RESULTS: Unbiased plasma profiling combined with immune enrichment of VWF verified VWF and its binding partner factor VIII as key determinants of VWD and revealed a remarkable heterogeneity in VWF amino acid sequence coverage among patients. Subsequent VWF proteotyping enabled identification of both polymorphisms (eg, p.Thr789Ala, p.Gln852Arg, and p.Thr1381Ala), as well as pathogenic variants (n = 16) along with their corresponding canonical sequences. Targeted proteomics using stable isotope-labeled peptides confirmed unbiased proteotyping for 5 selected variants and suggested differential proteoform quantities in plasma. The variant-to-wild-type peptide ratio was determined in 6 type 2B patients heterozygous for p.Arg1306Trp, confirming the relatively low proteoform concentration of the pathogenic variant. The elevated VWF propeptide/VWF ratio indicated increased clearance of specific VWF proteoforms. CONCLUSION: This study highlights how VWF proteotyping from plasma could be the first step to bridge the gap between genotyping and functional testing in VWD.

Probing activation-driven changes in coagulation factor IX by mass spectrometry.

BACKGROUND: Activated factor IX (FIXa) is an inefficient enzyme that needs activated factor VIII (FVIII) for full activity. Recently, we identified a network of FVIII-driven changes in FIXa employing hydrogen-deuterium eXchange mass spectrometry (HDX-MS). Some changes also occurred in active-site inhibited FIXa, but others were not cofactor-driven, in particular those within the 220-loop (in chymotrypsin numbering). OBJECTIVE: The aim of this work is to better understand the zymogen-to-enzyme transition in FIX, with specific focus on substrate-driven changes at the catalytic site. METHODS: Footprinting mass spectrometry by HDX and Tandem-Mass Tags (TMT) labelling were used to explore changes occurring upon the conversion from FIX into FIXa. Mutagenesis and kinetic studies served to assess the role of the 220-loop. RESULTS: HDX-MS displayed remarkably few differences between FIX and FIXa. In comparison with FIX, FIXa did exhibit decreased deuterium uptake at the N-terminus region. This was more prominent when the FIXa active site was occupied by an irreversible inhibitor. TMT-labelling showed that the N-terminus is largely protected from labelling, and that inhibitor binding increases protection to a minor extent. Occupation of the active site also reduced deuterium uptake within the 220-loop backbone. Mutagenesis within the 220-loop revealed that a putative H-bond network contributes to FIXa activity. TMT labeling of the N-terminus suggested that these 220-loop variants are more zymogen-like than wild-type FIXa. CONCLUSION: In the absence of cofactor and substrate, FIXa is predominantly zymogen-like. Stabilization in its enzyme-like form involves, apart from FVIII-binding, also interplay between the 220-loop, N-terminus, and the substrate binding site.

Hydrogen-Deuterium Exchange Mass Spectrometry Identifies Activated Factor IX-Induced molecular Changes in Activated Factor VIII.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was employed to gain insight into the changes in factor VIII (FVIII) that occur upon its activation and assembly with activated factor IX (FIXa) on phospholipid membranes. HDX-MS analysis of thrombin-activated FVIII (FVIIIa) revealed a marked increase in deuterium incorporation of amino acid residues along the A1-A2 and A2-A3 interface. Rapid dissociation of the A2 domain from FVIIIa can explain this observation. In the presence of FIXa, enhanced deuterium incorporation at the interface of FVIIIa was similar to that of FVIII. This is compatible with the previous finding that FIXa contributes to A2 domain retention in FVIIIa. A2 domain region Leu631-Tyr637, which is not part of the interface between the A domains, also showed a marked increase in deuterium incorporation in FVIIIa compared with FVIII. Deuterium uptake of this region was decreased in the presence of FIXa beyond that observed in FVIII. This implies that FIXa alters the conformation or directly interacts with this region in FVIIIa. Replacement of Val634 in FVIII by alanine using site-directed mutagenesis almost completely impaired the ability of the activated cofactor to enhance the activity of FIXa. Surface plasmon resonance analysis revealed that the rates of A2 domain dissociation from FVIIIa and FVIIIa-Val634Ala were indistinguishable. HDX-MS analysis showed, however, that FIXa was unable to retain the A2 domain in FVIIIa-Val634Ala. The combined results of this study suggest that the local structure of Leu631-Tyr637 is altered by FIXa and that this region contributes to the cofactor function of FVIII.

Unique surface-exposed hydrophobic residues in the C1 domain of factor VIII contribute to cofactor function and von Willebrand factor binding.

BACKGROUND: The identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and von Willebrand factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 domain, this implies that the FVIII C1 domain comprises unique surface-exposed elements involved in VWF and FIXa interaction. OBJECTIVE: The aim of this study is to identify residues in the FVIII C1 domain that contribute to VWF and FIXa binding. METHODS: Structures and primary sequences of FVIII and FV were compared to identify surface-exposed residues unique to the FVIII C1 domain. The identified residues were replaced with alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays. RESULTS: Five surface-exposed hydrophobic residues unique to the FVIII C1 domain, ie, F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130, and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface-exposed V2125, which is spatially next to F2127, affected only VWF binding. CONCLUSION: The surface-exposed hydrophobic residues in C1 domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 domain in FVIII life cycle.

Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver.

Transduction with recombinant adeno-associated virus (AAV) vectors is limited by the need to convert its single-stranded (ss) genome to transcriptionally active double-stranded (ds) forms. For AAV-mediated hemophilia B (HB) gene therapy, we have overcome this obstacle by constructing a liver-restricted mini-human factor IX (hFIX) expression cassette that can be packaged as complementary dimers within individual AAV particles. Molecular analysis of murine liver transduced with these self-complementary (sc) vectors demonstrated rapid formation of active ds-linear genomes that persisted stably as concatamers or monomeric circles. This unique property resulted in a 20-fold improvement in hFIX expression in mice over comparable ssAAV vectors. Administration of only 1 x 10(10) scAAV particles led to expression of hFIX at supraphysiologic levels (8I U/mL) and correction of the bleeding diathesis in FIX knock-out mice. Of importance, therapeutic levels of hFIX (3%-30% of normal) were achieved in nonhuman primates using a significantly lower dose of scAAV than required with ssAAV. Furthermore, AAV5-pseudotyped scAAV vectors mediated successful transduction in macaques with pre-existing immunity to AAV8. Hence, this novel vector represents an important advance for hemophilia B gene therapy.