Publications by authors named "Jan Voorberg"

101 Publications

PAD4 takes charge during neutrophil activation: impact of PAD4 mediated NET formation on immune-mediated disease.

J Thromb Haemost 2021 Mar 27. Epub 2021 Mar 27.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6200 MD, Maastricht, The Netherlands.

Peptidyl arginine deiminase 4 (PAD4) is an enzyme expressed in neutrophils that, when activated, can drive the formation of neutrophil extracellular traps (NETs). Uncontrolled activation of PAD4 and subsequent citrullination of proteins is increasingly recognized as a driver of (auto) immune diseases. Currently, our understanding of PAD4 structure-function relationships and activity control in vivo is incomplete. In this review, employed molecular modelling to generate a three-dimensional structure of the complete PAD4 molecule. Using our model we discuss the catalytic conversion of the arginine substrate to citrulline. Besides mechanistic insight into PAD4 function, we give an overview of biological functions of PAD4 and mechanisms that influence its activation. In addition we discuss the crucial role of PAD4 mediated citrullination of histones in the formation of neutrophil extracellular traps(NET). Subsequently we focus on the role of PAD4 mediated NET formation and its role in pathogenesis of rheumatoid arthritis, sepsis and (immune-)thrombosis. Finally, we summarize current efforts to design different classes of PAD4 inhibitors that are being developed for improved treatment of autoimmune disorders as well as thrombo-inflammatory disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jth.15313DOI Listing
March 2021

N-glycan mediated shielding of ADAMTS13 prevents binding of pathogenic autoantibodies in immune-mediated TTP.

Blood 2021 Feb 5. Epub 2021 Feb 5.

Sanquin Research Amsterdam UMC Landsteiner Laboratory, Amsterdam, Netherlands.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an autoimmune disorder caused by the development of autoantibodies targeting different domains of ADAMTS13. Profiling studies have shown that residues R568, F592, R660, Y661 and Y665 within exosite-3 of the spacer domain provide an immunodominant region of ADAMTS13 for pathogenic autoantibodies that develop in patients with iTTP. Modification of these 5 core residues with the goal of reducing auto-antibody binding revealed a significant trade-off between autoantibody resistance and proteolytic activity. Here, we employed structural bioinformatics to identify a larger epitope landscape on the ADAMTS13 spacer domain. Models of spacer-antibody complexes predicted that residues R568, L591, F592, K608, M609, R636, L637, R639, R660, Y661, Y665 and L668 contribute to an expanded epitope within the spacer domain. Based on bioinformatics-guided predictions we designed a panel of N-glycan insertions in this expanded epitope to reduce the binding of spacer domain autoantibodies. One N-glycan variant (NGLY3-ADAMTS13, containing a K608N substitution) showed strongly reduced reactivity with TTP patient sera (28%) as compared to WT-ADAMTS13 (100%). Insertion of an N-glycan at amino acid position 608 did not interfere with processing of VWF positioning the resulting NGLY3-ADAMTS13 variant as a potential novel therapeutic option for treatment of iTTP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2020007972DOI Listing
February 2021

New kid on the BLOC.

Blood 2020 12;136(24):2729-2730

Sanquin Research.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2020007913DOI Listing
December 2020

Immunogenic hotspots in the spacer domain of ADAMTS13 in immune-mediated thrombotic thrombocytopenic purpura.

J Thromb Haemost 2021 02 31;19(2):478-488. Epub 2020 Dec 31.

Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium.

Background: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by anti-ADAMTS13 autoantibodies inducing a severe deficiency of ADAMTS13. Epitope mapping studies on samples obtained during acute iTTP episodes have shown that the iTTP immune response is polyclonal, with almost all patients having autoantibodies targeting the spacer domain of ADAMTS13.

Objectives: To identify the immunogenic hotspots in the spacer domain of ADAMTS13.

Patients/methods: A library of 11 full-length ADAMTS13 spacer hybrids was created in which amino acid regions of the spacer domain of ADAMTS13 were exchanged by the corresponding region of the spacer domain of ADAMTS1. Next, the full-length ADAMTS13 spacer hybrids were used in enzyme-linked immunosorbent assay to epitope map anti-spacer autoantibodies in 138 samples from acute and remission iTTP patients.

Results: Sixteen different anti-spacer autoantibody profiles were identified with a similar distribution in acute and remission patients. There was no association between the anti-spacer autoantibody profiles and disease severity. Almost all iTTP samples contained anti-spacer autoantibodies against the following three regions: amino acid residues 588-592, 602-610, and 657-666 (hybrids E, G, and M). Between 31% and 57% of the samples had anti-spacer autoantibodies against amino acid regions 572-579, 629-638, 667-676 (hybrids C, J, and N). In contrast, none of the samples had anti-spacer autoantibodies against amino acid regions 556-563, 564-571, 649-656, and 677-685 (hybrids A, B, L, and O).

Conclusion: We identified three hotspot regions (amino acid regions 588-592, 602-610, and 657-666) in the spacer domain of ADAMTS13 that are targeted by anti-spacer autoantibodies found in a large cohort of iTTP patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jth.15170DOI Listing
February 2021

Modifying ADAMTS13 to modulate binding of pathogenic autoantibodies of patients with acquired thrombotic thrombocytopenic purpura.

Haematologica 2020 11 1;105(11):2619-2630. Epub 2020 Nov 1.

Department of Molecular and Cellular Hemostasis, Sanquin-Academic Medical Center, The Netherlands.

Antibodies that develop in patients with immune thrombotic thrombocytopenic purpura (iTTP) commonly target the spacer epitope R568/F592/R660/Y661/Y665 (RFRYY). In this study we present a detailed contribution of each residue in this epitope for autoantibody binding. Different panels of mutations were introduced here to create a large collection of full-length ADAMTS13 variants comprising conservative (Y←→F), semi-conservative (Y/F→L), non-conservative (Y/F→N) or alanine (Y/F/R→A) substitutions. Previously reported Gain-of-Function (GoF, KYKFF) and truncated 'MDTCS' variants were also included. Sera of 18 patients were screened against all variants. Conservative mutations of the aromatic residues did not reduce the binding of autoantibodies. Moderate resistance was achieved by replacing R568 and R660 by lysines or alanines. Semi-conservative mutations of aromatic residues show a moderate effectiveness in autoantibody resistance. Non-conservative asparagine or alanine mutations of aromatic residues are the most effective. In the mixtures of autoantibodies from the majority (89%) of patients screened, autoantibodies targeting the spacer RFRYY epitope have preponderance compared to other epitopes. Reductions in ADAMTS13 proteolytic activity were observed for all full-length mutant variants, in varying degrees. The greatest activity reductions were observed in the most autoantibody-resistant variants (15-35% residual activity in FRETS-VWF73). Among these, a triple-alanine mutant RARAA showed activity in a VWF multimer assay. This study shows that non-conservative and alanine modifications of residues within the exosite-3 spacer RFRYY epitope in full-length ADAMTS13 resist the binding of autoantibodies from iTTP patients, while retaining residual proteolytic activity. Our study provides a framework for the design of autoantibody-resistant ADAMTS13 variants for further therapeutic development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2019.226068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604655PMC
November 2020

Generation and validation of small ADAMTS13 fragments for epitope mapping of anti-ADAMTS13 autoantibodies in immune-mediated thrombotic thrombocytopenic purpura.

Res Pract Thromb Haemost 2020 Jul 25;4(5):918-930. Epub 2020 Jun 25.

Laboratory for Thrombosis Research IRF Life Sciences KU Leuven Campus Kulak Kortrijk Kortrijk Belgium.

Background: In immune-mediated thrombotic thrombocytopenic purpura (iTTP), patients develop an immune response against the multidomain enzyme ADAMTS13. ADAMTS13 consists of a metalloprotease (M) and disintegrin-like (D) domain, 8 thrombospondin type 1 repeats (T1-T8), a cysteine-rich (C), a spacer (S), and 2 CUB domains (CUB1-2). Previous epitope mapping studies have used relatively large overlapping ADAMTS13 fragments.

Objectives: We aimed at developing small nonoverlapping ADAMTS13 fragments to fine map anti-ADAMTS13 autoantibodies in iTTP patients.

Methods: A library of 16 ADAMTS13 fragments, comprising several small (M, DT, C, S, T2-T5, T6-T8, CUB1, CUB2), and some larger fragments with overlapping domains (MDT, MDTC, DTC, CS, T2-T8, CUB1-2, MDTCS, T2-C2), were generated. All fragments, and ADAMTS13, were expressed as a fusion protein with albumin domain 1, and purified. The folding of the fragments was tested using 17 anti-ADAMTS13 monoclonal antibodies with known epitopes. An epitope mapping assay using small ADAMTS13 fragments was set up, and validated by analyzing 18 iTTP patient samples.

Results: Validation with the monoclonal antibodies demonstrated that single S and CUB1 were not correctly folded, and therefore CS and CUB1-2 fragments were selected instead of single C, S, CUB1, and CUB2 fragments. Epitope mapping of antibodies of patients with iTTP confirmed that 6 nonoverlapping ADAMTS13 fragments M, DT, CS, T2-T5, T6-T8, and CUB1-2 were sufficient to accurately determine the antibody-binding sites.

Conclusion: We have developed a tool to profile patients with iTTP according to their anti-ADAMTS13 antibodies for a better insight in their immune response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/rth2.12379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354404PMC
July 2020

Orchestration of Primary Hemostasis by Platelet and Endothelial Lysosome-Related Organelles.

Arterioscler Thromb Vasc Biol 2020 06 7;40(6):1441-1453. Epub 2020 May 7.

From the Department of Molecular and Cellular Hemostasis, Sanquin Research and Landsteiner Laboratory (E.K., R.B., J.V.), Amsterdam University Medical Center, University of Amsterdam, the Netherlands.

Megakaryocyte-derived platelets and endothelial cells store their hemostatic cargo in α- and δ-granules and Weibel-Palade bodies, respectively. These storage granules belong to the lysosome-related organelles (LROs), a heterogeneous group of organelles that are rapidly released following agonist-induced triggering of intracellular signaling pathways. Following vascular injury, endothelial Weibel-Palade bodies release their content into the vascular lumen and promote the formation of long VWF (von Willebrand factor) strings that form an adhesive platform for platelets. Binding to VWF strings as well as exposed subendothelial collagen activates platelets resulting in the release of α- and δ-granules, which are crucial events in formation of a primary hemostatic plug. Biogenesis and secretion of these LROs are pivotal for the maintenance of proper hemostasis. Several bleeding disorders have been linked to abnormal generation of LROs in megakaryocytes and endothelial cells. Recent reviews have emphasized common pathways in the biogenesis and biological properties of LROs, focusing mainly on melanosomes. Despite many similarities, LROs in platelet and endothelial cells clearly possess distinct properties that allow them to provide a highly coordinated and synergistic contribution to primary hemostasis by sequentially releasing hemostatic cargo. In this brief review, we discuss in depth the known regulators of α- and δ-granules in megakaryocytes/platelets and Weibel-Palade bodies in endothelial cells, starting from transcription factors that have been associated with granule formation to protein complexes that promote granule maturation. In addition, we provide a detailed view on the interplay between platelet and endothelial LROs in controlling hemostasis as well as their dysfunction in LRO related bleeding disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/ATVBAHA.120.314245DOI Listing
June 2020

Open ADAMTS13, induced by antibodies, is a biomarker for subclinical immune-mediated thrombotic thrombocytopenic purpura.

Blood 2020 07;136(3):353-361

Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium.

Recently, we showed that ADAMTS13 circulates in an open conformation during the acute phase of immune-mediated thrombotic thrombocytopenic purpura (iTTP). Although the cause of this conformational change remains elusive, ADAMTS13 is primarily closed in iTTP patients in remission with ADAMTS13 activity >50% and undetectable anti-ADAMTS13 autoantibodies, as well as after rituximab treatment, suggesting a role for anti-ADAMTS13 autoantibodies. Therefore, immunoglobulin G from 18 acute iTTP patients was purified and added to closed ADAMTS13 in healthy donor plasma. This resulted in open ADAMTS13 in 14 of 18 (78%) samples, proving that anti-ADAMTS13 autoantibodies can induce an open ADAMTS13 conformation. To further elucidate the conformation of ADAMTS13 in iTTP patients, we studied a novel iTTP patient cohort (n = 197) that also included plasma samples from iTTP patients in remission in whom ADAMTS13 activity was <50%. The open ADAMTS13 conformation was found during acute iTTP, as well as in patients in remission with ADAMTS13 activity <50% and in half of the patients with ADAMTS13 activity >50%, although free anti-ADAMTS13 autoantibodies were not always detected. Thus, open ADAMTS13 is a hallmark of acute iTTP, as well as a novel biomarker that can be used to detect subclinical iTTP in patients in remission. Finally, a long-term follow-up study in 1 iTTP patient showed that the open conformation precedes a substantial drop in ADAMTS13 activity. In conclusion, we have shown that anti-ADAMTS13 autoantibodies from iTTP patients induce an open ADAMTS13 conformation. Most importantly, an open ADAMTS13 conformation is a biomarker for subclinical iTTP and could become an important tool in TTP management.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2019004221DOI Listing
July 2020

Sec22b determines Weibel-Palade body length by controlling anterograde ER-Golgi transport.

Haematologica 2021 Apr 1;106(4):1138-1147. Epub 2021 Apr 1.

Dept. of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands.

Von Willebrand factor (VWF) is a multimeric hemostatic protein that is synthesized in endothelial cells, where it is stored for secretion in elongated secretory organelles, so-called Weibel-Palade bodies (WPBs). Hemostatic activity of VWF is strongly tied to WPB length, but how endothelial cells control the dimensions of their WPBs is unclear. In this study we used a targeted shRNA screen to identify the longin-SNARE Sec22b as a novel determinant of WPB size and VWF trafficking. We found that Sec22b depletion resulted in loss of the typically elongated WPB morphology along with disintegration of the Golgi and dilation of rough ER (rER) cisternae. This was accompanied by reduced proteolytic processing of VWF, accumulation of VWF in the dilated rER and reduced basal and stimulated VWF secretion. Our data demonstrate that the elongation of WPBs, and thus adhesive activity of its cargo VWF, is determined by the rate of anterograde transport between ER and Golgi, which depends on Sec22b-containing SNARE complexes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2019.242727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018124PMC
April 2021

Tolerating Factor VIII: Recent Progress.

Front Immunol 2019 10;10:2991. Epub 2020 Jan 10.

Uniformed Services University of the Health Sciences, Bethesda, MD, United States.

Development of neutralizing antibodies against biotherapeutic agents administered to prevent or treat various clinical conditions is a longstanding and growing problem faced by patients, medical providers and pharmaceutical companies. The hemophilia A community has deep experience with attempting to manage such deleterious immune responses, as the lifesaving protein drug factor VIII (FVIII) has been in use for decades. Hemophilia A is a bleeding disorder caused by genetic mutations that result in absent or dysfunctional FVIII. Prophylactic treatment consists of regular intravenous FVIII infusions. Unfortunately, 1/4 to 1/3 of patients develop neutralizing anti-FVIII antibodies, referred to clinically as "inhibitors," which result in a serious bleeding diathesis. Until recently, the only therapeutic option for these patients was "Immune Tolerance Induction," consisting of intensive FVIII administration, which is extraordinarily expensive and fails in ~30% of cases. There has been tremendous recent progress in developing novel potential clinical alternatives for the treatment of hemophilia A, ranging from encouraging results of gene therapy trials, to use of other hemostatic agents (either promoting coagulation or slowing down anti-coagulant or fibrinolytic pathways) to "bypass" the need for FVIII or supplement FVIII replacement therapy. Although these approaches are promising, there is widespread agreement that preventing or reversing inhibitors remains a high priority. Risk profiles of novel therapies are still unknown or incomplete, and FVIII will likely continue to be considered the optimal hemostatic agent to support surgery and manage trauma, or to combine with other therapies. We describe here recent exciting studies, most still pre-clinical, that address FVIII immunogenicity and suggest novel interventions to prevent or reverse inhibitor development. Studies of FVIII uptake, processing and presentation on antigen-presenting cells, epitope mapping, and the roles of complement, heme, von Willebrand factor, glycans, and the microbiome in FVIII immunogenicity are elucidating mechanisms of primary and secondary immune responses and suggesting additional novel targets. Promising tolerogenic therapies include development of FVIII-Fc fusion proteins, nanoparticle-based therapies, oral tolerance, and engineering of regulatory or cytotoxic T cells to render them FVIII-specific. Importantly, these studies are highly applicable to other scenarios where establishing immune tolerance to a defined antigen is a clinical priority.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.02991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965068PMC
November 2020

Update - PROFILE: Early-Stage Researchers Advancing Insights on TTP through a Unique PhD Track.

Hemasphere 2019 08 7;3(4):e281. Epub 2019 Aug 7.

Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/HS9.0000000000000281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745918PMC
August 2019

Alternative trafficking of Weibel-Palade body proteins in CRISPR/Cas9-engineered von Willebrand factor-deficient blood outgrowth endothelial cells.

Res Pract Thromb Haemost 2019 Oct 1;3(4):718-732. Epub 2019 Aug 1.

Molecular and Cellular Hemostasis Sanquin Research and Landsteiner Laboratory Amsterdam UMC University of Amsterdam Amsterdam The Netherlands.

Background: Synthesis of the hemostatic protein von Willebrand factor (VWF) drives formation of endothelial storage organelles called Weibel-Palade bodies (WPBs). In the absence of VWF, angiogenic and inflammatory mediators that are costored in WPBs are subject to alternative trafficking routes. In patients with von Willebrand disease (VWD), partial or complete absence of VWF/WPBs may lead to additional bleeding complications, such as angiodysplasia. Studies addressing the role of VWF using VWD patient-derived blood outgrowth endothelial cells (BOECs) have reported conflicting results due to the intrinsic heterogeneity of patient-derived BOECs.

Objective: To generate a VWF-deficient endothelial cell model using clustered regularly interspaced short palindromic repeats (CRISPR) genome engineering of blood outgrowth endothelial cells.

Methods: We used CRISPR/CRISPR-associated protein 9 editing in single-donor cord blood-derived BOECs (cbBOECs) to generate clonal cbBOECs. Clones were selected using high-throughput screening, mutations were validated by sequencing, and cells were phenotypically characterized.

Results: Two BOEC clones were obtained and were entirely devoid of WPBs, while their overall cell morphology was unaltered. Several WPB proteins, including CD63, syntaxin-3 and the cargo proteins angiopoietin (Ang)-2, interleukin (IL)-6, and IL-8 showed alternative trafficking and secretion in the absence of VWF. Interestingly, Ang-2 was relocated to the cell periphery and colocalized with Tie-2.

Conclusions: CRISPR editing of provides a robust method to create VWF- deficient BOECs that can be directly compared to their wild-type counterparts. Results obtained with our model system confirmed alternative trafficking of several WPB proteins in the absence of VWF and support the theory that increased Ang-2/Tie-2 interaction contributes to angiogenic abnormalities in VWD patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/rth2.12242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6782018PMC
October 2019

Interaction networks of Weibel-Palade body regulators syntaxin-3 and syntaxin binding protein 5 in endothelial cells.

J Proteomics 2019 08 13;205:103417. Epub 2019 Jun 13.

Molecular and Cellular Hemostasis, Amsterdam UMC, University of Amsterdam, The Netherlands; Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands. Electronic address:

The endothelium stores the hemostatic protein Von Willebrand factor (VWF) in endothelial storage organelles called Weibel-Palade bodies (WPBs). During maturation, WPBs recruit a complex of Rab GTPases and effectors that associate with components of the SNARE machinery that control WPB exocytosis. Recent genome wide association studies have found links between genetic variations in the SNAREs syntaxin-2 (STX2) and syntaxin binding protein 5 (STXBP5) and VWF plasma levels, suggesting a role for SNARE proteins in regulating VWF release. Moreover, we have previously identified the SNARE proteins syntaxin-3 and STXBP1 as regulators of WPB release. In this study we used an unbiased iterative interactomic approach to identify new components of the WPB exocytotic machinery. An interactome screen of syntaxin-3 identifies a number of SNAREs and SNARE associated proteins (STXBP2, STXBP5, SNAP23, NAPA and NSF). We show that the VAMP-like domain (VLD) of STXBP5 is indispensable for the interaction with SNARE proteins and this capacity of the VLD could be exploited to identify an extended set of novel endothelial SNARE interactors of STXBP5. In addition, an STXBP5 variant with an N436S substitution, which is linked to lower VWF plasma levels, does not show a difference in interactome when compared with WT STXBP5. SIGNIFICANCE: The hemostatic protein Von Willebrand factor plays a pivotal role in vascular health: quantitative or qualitative deficiencies of VWF can lead to bleeding, while elevated levels of VWF are associated with increased risk of thrombosis. Tight regulation of VWF secretion from WPBs is therefore essential to maintain vascular homeostasis. We used an unbiased proteomic screen to identify new components of the regulatory machinery that controls WPB exocytosis. Our data expand the endothelial SNARE protein network and provide a set of novel candidate WPB regulators that may contribute to regulation of VWF plasma levels and vascular health.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jprot.2019.103417DOI Listing
August 2019

Defective AP-3-dependent VAMP8 trafficking impairs Weibel-Palade body exocytosis in Hermansky-Pudlak Syndrome type 2 blood outgrowth endothelial cells.

Haematologica 2019 10 10;104(10):2091-2099. Epub 2019 Jan 10.

Molecular and Cellular Hemostasis, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam

Weibel-Palade bodies are endothelial secretory organelles that contain von Willebrand factor, P-selectin and CD63. Release of von Willebrand factor from Weibel-Palade bodies is crucial for platelet adhesion during primary hemostasis. Endosomal trafficking of proteins like CD63 to Weibel-Palade bodies during maturation is dependent on the adaptor protein complex 3 complex. Mutations in the gene, which encodes the adaptor protein complex 3 β1 subunit, result in Hermansky-Pudlak syndrome 2, a rare genetic disorder that leads to neutropenia and a mild bleeding diathesis. This is caused by abnormal granule formation in neutrophils and platelets due to defects in trafficking of cargo to secretory organelles. The impact of these defects on the secretory pathway of the endothelium is largely unknown. In this study, we investigated the role of adaptor protein complex 3-dependent mechanisms in trafficking of proteins during Weibel-Palade body maturation in endothelial cells. An patient-derived endothelial model of Hermansky-Pudlak syndrome type 2 was established using blood outgrowth endothelial cells that were isolated from a patient with compound heterozygous mutations in Hermansky-Pudlak syndrome type 2 endothelial cells and CRISPR-Cas9-engineered endothelial cells contain Weibel-Palade bodies that are entirely devoid of CD63, indicative of disrupted endosomal trafficking. Hermansky-Pudlak syndrome type 2 endothelial cells have impaired Ca2mediated and cAMP-mediated exocytosis. Whole proteome analysis revealed that, apart from adaptor protein complex 3 β1, also the μ1 subunit and the v-SNARE VAMP8 were depleted. Stimulus-induced von Willebrand factor secretion was impaired in CRISPR-Cas9-engineered VAMP8endothelial cells. Our data show that defects in adaptor protein complex 3-dependent maturation of Weibel-Palade bodies impairs exocytosis by affecting the recruitment of VAMP8.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2018.207787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886443PMC
October 2019

Prevention of the anti-factor VIII memory B-cell response by inhibition of Bruton tyrosine kinase in experimental hemophilia A.

Haematologica 2019 05 13;104(5):1046-1054. Epub 2018 Dec 13.

INSERM, UMR S 1138, Centre de Recherche des Cordeliers, Paris, France

Hemophilia A is a rare hemorrhagic disorder caused by the lack of functional pro-coagulant factor VIII. Factor VIII replacement therapy in patients with severe hemophilia A results in the development of inhibitory anti-factor VIII IgG in up to 30% of cases. To date, immune tolerance induction, with daily injection of large amounts of factor VIII, is the only strategy to eradicate factor VIII inhibitors. This strategy is, however, efficient in only 60-80% of patients. We investigated whether blocking B-cell receptor signaling upon inhibition of Bruton tyrosine kinase prevents anti-factor VIII immune responses in a mouse model of severe hemophilia A. Factor VIII-naïve and factor VIII-sensitized factor VIII-deficient mice were fed with the selective inhibitor of Bruton tyrosine kinase, (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxyl] phenyl)-1H pyrazole-4-carboxamide (PF-06250112), to inhibit B-cell receptor signaling prior to challenge with exogenous factor VIII. The consequences on the anti-factor VIII immune response were studied. Inhibition of Bruton tyrosine kinase during the primary anti-factor VIII immune response in factor VIII-naïve mice did not prevent the development of inhibitory anti-factor VIII IgG. In contrast, the anti-factor VIII memory B-cell response was consistently reduced upon treatment of factor VIII-sensitized mice with the Bruton tyrosine kinase inhibitor. The Bruton tyrosine kinase inhibitor reduced the differentiation of memory B cells and following adoptive transfer to factor VIII-naïve animals. Taken together, our data identify inhibition of Bruton tyrosine kinase using PF-06250112 as a strategy to limit the reactivation of factor VIII-specific memory B cells upon re-challenge with therapeutic factor VIII.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2018.200279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518880PMC
May 2019

Generation of anti-idiotypic antibodies to detect anti-spacer antibody idiotopes in acute thrombotic thrombocytopenic purpura patients.

Haematologica 2019 06 6;104(6):1268-1276. Epub 2018 Dec 6.

Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Belgium

In autoantibody-mediated autoimmune diseases, autoantibody profiling allows patients to be stratified and links autoantibodies with disease severity and outcome. However, in immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients, stratification according to antibody profiles and their clinical relevance has not been fully explored. We aimed to develop a new type of autoantibody profiling assay for iTTP based on the use of anti-idiotypic antibodies. Anti-idiotypic antibodies against 3 anti-spacer autoantibodies were generated in mice and were used to capture the respective anti-spacer idiotopes from 151 acute iTTP plasma samples. We next deciphered these anti-spacer idiotope profiles in iTTP patients and investigated whether these limited idiotope profiles could be linked with disease severity. We developed 3 anti-idiotypic antibodies that recognized particular idiotopes in the anti-spacer autoantibodies II-1, TTP73 or I-9, that are involved in ADAMTS13 binding; 35%, 24% and 42% of patients were positive for antibodies with the II-1, TTP73 and I-9 idiotopes, respectively. Stratifying patients according to the corresponding 8 anti-spacer idiotope profiles provided a new insight into the anti-spacer II-1, TTP73 and I-9 idiotope profiles in these patients. Finally, these limited idiotope profiles showed no association with disease severity. We successfully developed 3 anti-idiotypic antibodies that allowed us to determine the profiles of the anti-spacer II-1, TTP73 and I-9 idiotopes in iTTP patients. Increasing the number of patients and/or future development of additional anti-idiotypic antibodies against other anti-ADAMTS13 autoantibodies might allow idiotope profiles of clinical, prognostic value to be identified.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2018.205666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545854PMC
June 2019

Anti-HLA antibodies with complementary and synergistic interaction geometries promote classical complement activation on platelets.

Haematologica 2019 02 27;104(2):403-416. Epub 2018 Sep 27.

Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center

High titers of HLA antibodies are associated with platelet refractoriness, causing poor platelet increments after transfusions in a subset of patients with HLA antibodies. Currently, we do not know the biological mechanisms that explain the variability in clinical responses in HLA alloimmunized patients receiving platelet transfusions. Previously we showed that a subset of anti-HLA IgG-antibodies induces FcγRIIa-dependent platelet activation and enhanced phagocytosis. Here, we investigated whether anti-HLA IgG can induce complement activation on platelets. We found that a subset of anti-HLA IgG induced complement activation via the classical pathway, causing C4b and C3b deposition and formation of the membrane-attack complex. This resulted in permeabilization of platelet membranes and increased calcium influx. Complement activation also caused enhanced α-granule release, as measured by CD62P surface exposure. Blocking studies revealed that platelet activation was caused by FcγRIIa-dependent signaling as well as HLA antibody induced complement activation. Synergistic complement activation employing combinations of monoclonal IgGs suggested that assembly of oligomeric IgG complexes strongly promoted complement activation through binding of IgGs to different antigenic determinants on HLA. In agreement with this, we observed that preventing anti-HLA-IgG hexamer formation using an IgG-Fc:Fc blocking peptide, completely inhibited C3b and C4b deposition. Our results show that HLA antibodies can induce complement activation on platelets including membrane attack complex formation, pore formation and calcium influx. We propose that these events can contribute to fast platelet clearance in patients refractory to platelet transfusions with HLA alloantibodies, who may benefit from functional-platelet matching and treatment with complement inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2018.201665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355480PMC
February 2019

Anti-ADAMTS13 Autoantibodies against Cryptic Epitopes in Immune-Mediated Thrombotic Thrombocytopenic Purpura.

Thromb Haemost 2018 Oct 20;118(10):1729-1742. Epub 2018 Sep 20.

Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by severe ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13) deficiency, the presence of anti-ADAMTS13 autoantibodies and an open ADAMTS13 conformation with a cryptic epitope in the spacer domain exposed. A detailed knowledge of anti-ADAMTS13 autoantibodies will help identifying pathogenic antibodies and elucidating the cause of ADAMTS13 deficiency. We aimed at cloning anti-ADAMTS13 autoantibodies from iTTP patients to study their epitopes and inhibitory characteristics. We sorted anti-ADAMTS13 autoantibody expressing B cells from peripheral blood mononuclear cells of 13 iTTP patients to isolate anti-ADAMTS13 autoantibody sequences. Ninety-six B cell clones producing anti-ADAMTS13 autoantibodies were identified from which 30 immunoglobulin M (IgM) and 5 IgG sequences were obtained. For this study, we only cloned, expressed and purified the five IgG antibodies. In vitro characterization revealed that three of the five cloned IgG antibodies, TTP73-1, ELH2-1 and TR8C11, indeed recognize ADAMTS13. Epitope mapping showed that antibodies TTP73-1 and TR8C11 bind to the cysteine-spacer domains, while the antibody ELH2-1 recognizes the T2-T3 domains in ADAMTS13. None of the antibodies inhibited ADAMTS13 activity. Given the recent findings regarding the open ADAMTS13 conformation during acute iTTP, we studied if the cloned antibodies could recognize cryptic epitopes in ADAMTS13. Interestingly, all three antibodies recognize cryptic epitopes. In conclusion, we cloned three anti-ADAMTS13 autoantibodies from iTTP patients that recognize cryptic epitopes. Hence, these data nicely fit our recent finding that the conformation of ADAMTS13 is open during acute iTTP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1055/s-0038-1669459DOI Listing
October 2018

Weibel-Palade Body Localized Syntaxin-3 Modulates Von Willebrand Factor Secretion From Endothelial Cells.

Arterioscler Thromb Vasc Biol 2018 07 7;38(7):1549-1561. Epub 2018 Jun 7.

From the Plasma Proteins, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, The Netherlands (M.S., E.K., B.L.v.d.E., A.G., M.H., D.v.B., H.M., M.v.d.B., J.V., R.B.)

Objective: Endothelial cells store VWF (von Willebrand factor) in rod-shaped secretory organelles, called Weibel-Palade bodies (WPBs). WPB exocytosis is coordinated by a complex network of Rab GTPases, Rab effectors, and SNARE (soluble NSF attachment protein receptor) proteins. We have previously identified STXBP1 as the link between the Rab27A-Slp4-a complex on WPBs and the SNARE proteins syntaxin-2 and -3. In this study, we investigate the function of syntaxin-3 in VWF secretion.

Approach And Results: In human umbilical vein endothelial cells and in blood outgrowth endothelial cells (BOECs) from healthy controls, endogenous syntaxin-3 immunolocalized to WPBs. A detailed analysis of BOECs isolated from a patient with variant microvillus inclusion disease, carrying a homozygous mutation in (STX3), showed a loss of syntaxin-3 protein and absence of WPB-associated syntaxin-3 immunoreactivity. Ultrastructural analysis revealed no detectable differences in morphology or prevalence of immature or mature WPBs in control versus STX3 BOECs. VWF multimer analysis showed normal patterns in plasma of the microvillus inclusion disease patient, and media from STX3 BOECs, together indicating WPB formation and maturation are unaffected by absence of syntaxin-3. However, a defect in basal as well as Ca- and cAMP-mediated VWF secretion was found in the STX3 BOECs. We also show that syntaxin-3 interacts with the WPB-associated SNARE protein VAMP8 (vesicle-associated membrane protein-8).

Conclusions: Our data reveal syntaxin-3 as a novel WPB-associated SNARE protein that controls WPB exocytosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/ATVBAHA.117.310701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039413PMC
July 2018

A subset of anti-HLA antibodies induces FcγRIIa-dependent platelet activation.

Haematologica 2018 10 1;103(10):1741-1752. Epub 2018 Jun 1.

Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, the Netherlands.

HLA antibodies are associated with refractoriness to platelet transfusion, leading to rapid platelet clearance, sometimes coinciding with clinical side effects such as fever and chills. The presence of HLA antibodies is not always manifested by clinical symptoms. It is currently unclear why refractoriness to platelet transfusion is only observed in a subset of patients. Here, we utilized the availability of a unique panel of human monoclonal antibodies to study whether these were capable of activating platelets. Three out of eight human HLA-specific monoclonal antibodies induced activation of HLA-matched platelets from healthy donors as evidenced by enhanced α-granule release, aggregation, and αb activation. The propensity of HLA monoclonal antibodies to activate platelets was independent of the HLA subtype to which they were directed, but was dependent on the recognized epitope. Activation was fully inhibited either by blocking FcγRIIa, or by blocking FcγRIIa-dependent signaling with Syk inhibitor IV. Furthermore, activation required the presence of the IgG-Fc part, as F(ab') fragments of HLA monoclonal antibodies were unable to induce platelet activation. Mixing experiments revealed that activation of platelets occurred in an intra-platelet dependent manner. Accordingly, a proportion of sera from refractory patients with HLA antibodies induced FcγRIIa-dependent platelet activation. Our data show that a subset of HLA antibodies is capable of crosslinking HLA and FcγRIIa thereby promoting platelet activation and enhancing these cells' phagocytosis by macrophages. Based on these findings we suggest that FcγRIIa-dependent platelet activation may contribute to the decreased platelet survival in platelet-transfusion-dependent patients with HLA antibodies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2018.189365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6165798PMC
October 2018

Emerging Concepts in Immune Thrombocytopenia.

Front Immunol 2018 30;9:880. Epub 2018 Apr 30.

Department of Hematology, Erasmus University Medical Centre, Rotterdam, Netherlands.

Immune thrombocytopenia (ITP) is an autoimmune disease defined by low platelet counts which presents with an increased bleeding risk. Several genetic risk factors (e.g., polymorphisms in immunity-related genes) predispose to ITP. Autoantibodies and cytotoxic CD8 T cells (Tc) mediate the anti-platelet response leading to thrombocytopenia. Both effector arms enhance platelet clearance through phagocytosis by splenic macrophages or dendritic cells and by induction of apoptosis. Meanwhile, platelet production is inhibited by CD8 Tc targeting megakaryocytes in the bone marrow. CD4 T helper cells are important for B cell differentiation into autoantibody secreting plasma cells. Regulatory Tc are essential to secure immune tolerance, and reduced levels have been implicated in the development of ITP. Both Fcγ-receptor-dependent and -independent pathways are involved in the etiology of ITP. In this review, we present a simplified model for the pathogenesis of ITP, in which exposure of platelet surface antigens and a loss of tolerance are required for development of chronic anti-platelet responses. We also suggest that infections may comprise an important trigger for the development of auto-immunity against platelets in ITP. Post-translational modification of autoantigens has been firmly implicated in the development of autoimmune disorders like rheumatoid arthritis and type 1 diabetes. Based on these findings, we propose that post-translational modifications of platelet antigens may also contribute to the pathogenesis of ITP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2018.00880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5937051PMC
June 2019

Dissecting the pathophysiology of immune thrombotic thrombocytopenic purpura: interplay between genes and environmental triggers.

Haematologica 2018 07 19;103(7):1099-1109. Epub 2018 Apr 19.

Centre de Référence des Microangiopathies Thrombotiques, Hôpital Saint-Antoine, AP-HP, Paris, France

Although outstanding progress has been made in understanding the pathophysiology of thrombotic thrombocytopenic purpura (TTP), knowledge of the immunopathogenesis of the disease is only at an early stage. Anti-ADAMTS13 auto-antibodies were shown to block proteolysis of von Willebrand factor and/or induce ADAMTS13 clearance from the circulation. However, it still remains to identify which immune cells are involved in the production of anti-ADAMTS13 autoantibodies, and therefore account for the remarkable efficacy of the B-cell depleting agents in this disease. The mechanisms leading to the loss of tolerance of the immune system towards ADAMTS13 involve the predisposing genetic factors of the human leukocyte antigen class II locus DRB1*11 and DQB1*03 alleles as well as the protective allele DRB1*04, and modifying factors such as ethnicity, sex and obesity. Future studies have to identify why these identified genetic risk factors are also frequently to be found in the healthy population although the incidence of immune-mediated thrombotic thrombocytopenic purpura (iTTP) is extremely low. Moreover, the development of recombinant ADAMTS13 opens a new therapeutic era in the field. Interactions of recombinant ADAMTS13 with the immune system of iTTP patients will require intensive investigation, especially for its potential immunogenicity. Better understanding of iTTP immunopathogenesis should, therefore, provide a basis for the development of novel therapeutic approaches to restore immune tolerance towards ADAMTS13 and thereby better prevent refractoriness and relapses in patients with iTTP. In this review, we address these issues and the related challenges in this field.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2016.151407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6029525PMC
July 2018

Mass spectrometry-assisted identification of ADAMTS13-derived peptides presented on HLA-DR and HLA-DQ.

Haematologica 2018 06 22;103(6):1083-1092. Epub 2018 Mar 22.

Department of Plasma Proteins, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, the Netherlands.

Formation of microthrombi is a hallmark of acquired thrombotic thrombocytopenic purpura. These microthrombi originate from insufficient processing of ultra large von Willebrand factor multimers by ADAMTS13 due to the development of anti-ADAMTS13 autoantibodies. Several studies have identified the major histocompatibility complex class II alleles HLA-DRB1*11, HLA-DQB1*03 and HLA-DQB1*02:02 as risk factors for acquired thrombotic thrombocytopenic purpura development. Previous research in our department indicated that ADAMTS13 CUB2 domain-derived peptides FINVAPHAR and LIRDTHSLR are presented on HLA-DRB1*11 and HLA-DRB1*03, respectively. Here, we describe the repertoire of ADAMTS13 peptides presented on HLA-DQ. In parallel, the repertoire of ADAMTS13-derived peptides presented on HLA-DR was monitored. Using HLA-DR- and HLA-DQ-specific antibodies, we purified HLA/peptide complexes from ADAMTS13-pulsed monocyte-derived dendritic cells. Using this approach, we identified ADAMTS13-derived peptides presented on HLA-DR for all 9 samples analyzed; ADAMTS13-derived peptides presented on HLA-DQ were identified in 4 out of 9 samples. We were able to confirm the presentation of the CUB2 domain-derived peptides FINVAPHAR and LIRDTHSLR on HLA-DR. In total, 12 different core-peptide sequences were identified on HLA-DR and 8 on HLA-DQ. For HLA-DR11, several potential new core-peptides were found; 4 novel core-peptides were exclusively identified on HLA-DQ. Furthermore, an analysis was performed using the EpiMatrix and JanusMatrix tools to evaluate the eluted peptides, in the context of HLA-DR, for putative effector or regulatory T-cell responses at the population level. The results from this study provide a basis for the identification of immuno-dominant epitopes on ADAMTS13 involved in the onset of acquired thrombotic thrombocytopenic purpura.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2017.179119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058777PMC
June 2018

Inhibitors in Nonsevere Hemophilia A: What Is Known and Searching for the Unknown.

Semin Thromb Hemost 2018 Sep 13;44(6):568-577. Epub 2018 Feb 13.

Department of Oncology, Center for Bleeding Disorders and Coagulation, Careggi University Hospital, Firenze, Italy.

Nonsevere hemophilia A (NSHA) is an inherited X-linked bleeding disorder, caused by mutations of the gene, leading to decreases of clotting factor VIII (FVIII) levels to 1 to 40 IU/dL. Desmopressin is the first therapeutic option for NSHA, but 40 to 50% of patients fail to attain adequate postinfusion FVIII levels. Thus, in these cases, FVIII concentrates remain the mainstay of treatment. The development of neutralizing FVIII antibodies (inhibitors) is a major challenge with replacement therapy. In contrast to severe disease, NSHA patients have a lifelong risk of inhibitor development. Recent data indicate that inhibitors are associated with a deterioration of clinical outcome, illustrated by an increase in bleeding and mortality rate. genotype is an important risk factor for inhibitor occurrence together with surgical interventions and a high dose of FVIII concentrate. Adequate prevention and treatment of inhibitors in NSHA patients is limited by a lack of understanding of the underlying immunological mechanisms. Elucidation of the immunology driving inhibitor development is required to identify high-risk patients, to understand the association between clinical risk factors and inhibitor occurrence, and to provide the opportunity to develop new preventive and therapeutic strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1055/s-0037-1621717DOI Listing
September 2018

Anti-ADAMTS13 Antibodies and a Novel Heterozygous p.R1177Q Mutation in a Case of Pregnancy-Onset Immune-Mediated Thrombotic Thrombocytopenic Purpura.

TH Open 2018 Jan 8;2(1):e8-e15. Epub 2018 Jan 8.

Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium.

In this study, we investigated a case of pregnancy-onset thrombotic thrombocytopenic purpura (TTP). The patient had severely decreased ADAMTS13 ( isintegrin nd etalloprotease with hrombo pondin type 1 motif, member 13) activity levels during acute phase and the presence of inhibitory anti-ADAMTS13 autoantibodies was demonstrated, which led to the diagnosis of immune-mediated TTP. However, ADAMTS13 activity was only mildly restored during remission, although inhibitory anti-ADAMTS13 antibodies were no longer detected. We hypothesized that genetic abnormalities could account for this discrepancy between ADAMTS13 activity and antigen. Genetic analysis revealed the presence of two heterozygous substitutions on the same allele: a single nucleotide polymorphism (SNP) c.2699C > T (p.A900V), located in the beginning of the T5 domain, and a mutation c.3530G > A (p.R1177Q) located in the third linker region of ADAMTS13. In vitro testing of those substitutions by expression of recombinant proteins revealed a normal secretion but a reduced ADAMTS13 activity by the novel p.R1177Q mutation, which could partially explain the subnormal activity levels found during remission. Although changes in the linker region might induce conformational changes in ADAMTS13, the p.R1177Q mutation in the third linker region of ADAMTS13 did not expose a cryptic epitope in the metalloprotease domain. In conclusion, we report on an immune-mediated pregnancy-onset TTP patient who had inhibitory anti-ADAMTS13 autoantibodies during acute phase, but not during remission. Genetic analysis confirmed the diagnosis of immune-mediated TTP and revealed the novel p.R1177Q mutation which mildly impaired ADAMTS13 activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1055/s-0037-1615252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524854PMC
January 2018

Desmopressin in moderate hemophilia A patients: a treatment worth considering.

Haematologica 2018 03 5;103(3):550-557. Epub 2018 Jan 5.

Department of Pediatric Hematology, Immunology and Infectious diseases, Emma Children's Hospital, Amsterdam, the Netherlands

Desmopressin increases endogenous factor VIII levels in hemophilia A. Large inter-individual variation in the response to desmopressin is observed. Patients with a lower baseline factor VIII activity tend to show a reduced response, therefore, desmopressin is less frequently used in moderate hemophilia A patients (baseline factor VIII activity 1-5 international units/deciliter), even though factor VIII levels may rise substantially in some of them. We aim to describe the response to desmopressin in moderate hemophilia A patients and to identify predictors. We selected data on 169 patients with moderate hemophilia from the multicenter Response to DDAVP In non-severe hemophilia A patients: in Search for dEterminants (RISE) cohort study. Adequate response to desmopressin was defined as a peak factor VIII level ≥ 30, and excellent response as ≥ 50 international units/deciliter after desmopressin administration. We used univariate and multiple linear regression techniques to analyze predictors of the peak factor VIII level. Response was considered adequate in 68 patients (40%), of whom 25 showed excellent response (15%). Intravenous administration, age, pre-desmopressin factor VIII activity and von Willebrand factor antigen, peak von Willebrand factor activity and desmopressin-induced rise in von Willebrand factor antigen were significant predictors of peak factor VIII level and explained 65% of the inter-individual variation. In 40% of moderate hemophilia A patients, desmopressin response was adequate, thus it is important not to with-hold this group of patients from desmopressin responsiveness. Among the six predictors that we identified for desmopressin-induced factor VIII rise, factor VIII activity and desmopressin-induced rise in von Willebrand factor antigen had the strongest effect.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2017.180059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830393PMC
March 2018

Insights into 3D Structure of ADAMTS13: A Stepping Stone towards Novel Therapeutic Treatment of Thrombotic Thrombocytopenic Purpura.

Thromb Haemost 2018 01 5;118(1):28-41. Epub 2018 Jan 5.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.

ADAMTS13 (A D: isintegrin A: nd M: etalloprotease with a T: hromboS: pondin type-1 motif, member 13: ) and von Willebrand factor (VWF) can be considered as scale weights which control platelet adhesion during primary haemostasis. In a very uncommon condition designated thrombotic thrombocytopenic purpura (TTP), functional absence of ADAMTS13 tips the balance toward VWF-mediated platelet adhesion in the microcirculation. TTP is associated with a high mortality and arises from either a congenital or acquired autoimmune deficiency of the plasma enzyme ADAMTS13. In case of acquired ADAMTS13 deficiency, autoantibodies bind to and inhibit the function of ADAMTS13. Currently available treatments of TTP aim to supply ADAMTS13 through plasma exchange or are aimed at B-cell depletion with rituximab. None of the available therapeutics, however, aims at protection of ADAMTS13 from circulating autoantibodies. In this review, our aim is to describe the structure-function relationship of ADAMTS13 employing homology models and previously published crystal structures. Structural bioinformatics investigation of ADAMTS13 reveals many insights and explains how mutations and autoantibodies may lead to the pathophysiology of TTP. The results of these studies provide a roadmap for the further development of rationally designed therapeutics for the treatment of patients with acquired TTP. In addition, we share our opinion on the state of the art of the open-closed conformations of ADAMTS13 which regulate the activity of this highly specific VWF cleaving protease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1160/TH17-06-0404DOI Listing
January 2018

The class I scavenger receptor CD163 promotes internalization of ADAMTS13 by macrophages.

Blood Adv 2017 Jan 16;1(5):293-305. Epub 2017 Jan 16.

Department of Plasma Proteins, Sanquin Research and Landsteiner Laboratory, Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands.

Internalization of ADAMTS13 by macrophages may contribute to its clearance from the circulation. Here we investigated endocytic mechanisms that contribute to the uptake of ADAMTS13 by macrophages. Human monocyte-derived macrophages were used to monitor the uptake of fluorescently labeled recombinant ADAMTS13 by flow cytometry. Internalization of ADAMTS13 was blocked upon addition of the cell-permeable dynamin inhibitor dynasore. Partial blocking of ADAMTS13 uptake was observed by using mannan; however, uptake was not affected by an antibody that blocked binding to the macrophage mannose receptor CD206, which suggests that other endocytic receptors contribute to the internalization of ADAMTS13 by macrophages. A pull-down with ADAMTS13 and subsequent mass spectrometric analysis identified the class I scavenger receptor CD163 as a candidate receptor for ADAMTS13. Blocking experiments with monoclonal anti-CD163 antibody EDHu-1 resulted in decreased ADAMTS13 internalization by macrophages. Pronounced inhibition of ADAMTS13 uptake by EDHu-1 was observed in CD163 high-expressing macrophages. In agreement with these findings, CD163-expressing Chinese hamster ovary cells were capable of rapidly internalizing ADAMTS13. Surface plasmon resonance revealed binding of ADAMTS13 to scavenger receptor cysteine-rich domains 1-9 and 1-5 of CD163. Taken together, our data identify CD163 as a major endocytic receptor for ADAMTS13 on macrophages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2016001321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744037PMC
January 2017