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P.D187H Mutation Impairs ADAMTS13 Activity and Secretion and Contributes to Thrombotic Thrombocytopenic Purpura in Mice

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P.D187H Mutation Impairs ADAMTS13 Activity and Secretion and Contributes to Thrombotic Thrombocytopenic Purpura in Mice View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Lirias Journal of Thrombosis and Haemostasis, 13: 283–292 DOI: 10.1111/jth.12804 ORIGINAL ARTICLE The novel ADAMTS13-p.D187H mutation impairs ADAMTS13 activity and secretion and contributes to thrombotic thrombocytopenic purpura in mice E. DE COCK,* C. HERMANS,† J. DE RAEYMAECKER,‡ K. DE CEUNYNCK,* B. DE MAEYER,* N. VANDEPUTTE,* A. VANDENBULCKE,* H. DECKMYN,* H. ROTTENSTEINER,§ M. DE MAEYER,‡ S. F. DE MEYER* andK. VANHOORELBEKE* *Laboratory for Thrombosis Research, KU Leuven Kulak, Kortrijk; †Division of Haematology, Haemostasis and Thrombosis Unit, Saint-Luc University Hospital, Brussels; ‡Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, Laboratory of Biomolecular Modeling, KU Leuven, Leuven, Belgium; and §Baxter Innovations GmbH, Vienna, Austria To cite this article: De Cock E, Hermans C, De Raeymaecker J, De Ceunynck K, De Maeyer B, Vandeputte N, Vandenbulcke A, Deckmyn H, Rottensteiner H, De Maeyer M, De Meyer SF, Vanhoorelbeke K. The novel ADAMTS13-p.D187H mutation impairs ADAMTS13 activity and secretion and contributes to thrombotic thrombocytopenic purpura in mice. J Thromb Haemost 2015; 13: 283–92. of ADAMTS13. The homozygous p.D187H mutation Summary. Background: Congenital thrombotic thrombo- down-regulated ADAMTS13 activity in vitro. Impaired cytopenic purpura (TTP) is characterized by mutations in proteolytic activity was linked to unstable Ca2+ binding the ADAMTS13 gene, which either impair protein secre- as visualized using a molecular dynamics simulation. In tion or influence ADAMTS13 (A Disintegrin-like And addition, the p.D187H mutation affects protein secretion Metalloprotease domain with ThromboSpondin type-1 in vitro.InAdamts13–/– mice, the homozygous p.D187H motif, member 13) activity. Phenotypic consequences of mutation reduced ADAMTS13 secretion and activity and these mutations have not yet been evaluated in animal contributed to TTP when these mice were triggered with models for TTP. Objectives: To identify the in vitro effect recombinant human von Willebrand factor. Conclusions: of a novel ADAMTS13 mutation and to investigate Our data indicate that the p.D187H mutation impairs whether this mutation induces TTP in vivo. Methods: All ADAMTS13 activity and secretion and is responsible for 29 ADAMTS13 exons with exon–intron boundaries of a TTP onset in mice. patient with pregnancy-onset TTP were sequenced. Wild- type and mutant ADAMTS13 proteins were both tran- Keywords: ADAMTS13 protein, human; ADAMTS13 siently and stably expressed in human embryonic kidney protein, mouse; animal model; mutation; thrombotic cells, and their activity was evaluated in vitro using fluor- thrombocytopenic purpura. escence resonance energy transfer and flow assays. Molec- ular dynamics simulations were performed to study Ca2+ stability. Adamts13–/– mice were hydrodynamically injected with wild-type and mutant expression plasmids Introduction and triggered with recombinant human von Willebrand When patients are deficient in the metalloprotease factor. Results: We identified a novel heterozygous ADAMTS13 (A Disintegrin-like And Metalloprotease c.559G>C mutation in exon 6 of the proposita’s ADAM- domain with ThromboSpondin type-1 motif, member 13), TS13 gene. This mutation resulted in a p.Asp187His sub- spontaneous microthrombi are formed leading to the rare stitution (p.D187H), which was located in the high but severe disorder thrombotic thrombocytopenic purpura affinity Ca2+-binding site in the metalloprotease domain (TTP) [1]. ADAMTS13 deficiency prevents trimming of ultra-large (UL) von Willebrand factor (VWF) multimers. Correspondence: Karen Vanhoorelbeke, Laboratory for Thrombosis These UL multimers spontaneously interact with platelets Research, IRF Life Sciences, KU Leuven Kulak, Etienne Sabbelaan and form aggregates that obstruct blood vessels in multi- 53, B-8500 Kortrijk, Belgium. ple organs, resulting in microvascular thrombosis, severe Tel.: +32 56 24 60 61; fax: +32 56 24 69 97. thrombocytopenia, and hemolytic anemia [1,2]. In the E-mail: [email protected] majority of TTP patients, ADAMTS13 deficiency is Received 9 May 2014 acquired due to anti-ADAMTS13 antibodies either neu- Manuscript handled by: C. Gachet tralizing the enzyme’s activity or accelerating its clearance Final decision: P. H. Reitsma, 16 November 2014 [3,4]. Fewer patients have congenital TTP, or Upshaw– © 2014 International Society on Thrombosis and Haemostasis 284 E. De Cock et al Schulman syndrome, defined by genetic mutations and 28 with a platelet count of 48 000 lLÀ1. Pathological polymorphisms within the ADAMTS13 gene [5,6]. AD- examination of the placenta revealed signs of chronic AMTS13 deficiency alone might not provoke TTP, as anoxia. Postpartum, the platelet count decreased to some patients remain asymptomatic until early adulthood. 14 000 lLÀ1 with clear signs of hemolysis and microangi- In these patients, a second hit or trigger such as acute opathy (schistocytes 4%, elevated lactate dehydrogenase, infections or pregnancy causes the onset of TTP [7]. low haptoglobin). Platelets then normalized within ADAMTS13 is located on chromosome 9q34 and con- 10 days with steroids alone. sists of 29 exons [8,9]. More than 150 different mutations When she was tested in 2006, her platelet count was (missense, nonsense, and splice site mutations; small dele- normal. Complete thrombophilia workup was performed tions and insertions) randomly distributed throughout the but did not reveal any acquired or congenital abnormality ADAMTS13 gene have been reported so far [10]. To date, with the exception of a slight increase in factor VIII approximately two-thirds of the congenital TTP patients (FVIII; 166%) and VWF (160% for the antigen and are compound heterozygous, while the remaining patients 168% for the activity measured by collagen binding bear homozygous ADAMTS13 mutations. In vitro char- assay) levels. Because of her history of microangiopathy, acterization of many of these ADAMTS13 mutations ADAMTS13 activity was measured (she was under remis- revealed that the majority impaired protein secretion, sion at that time) and found to be < 8% (TECHNO- although mutations that also influence ADAMTS13 activ- ZYMEâ ADAMTS13-activity ELISA; Technoclone, ity (e.g., p.Ser119Phe [11], p.Arg193Trp [12], p.Pro457Leu Vienna, Austria), and no anti-ADAMTS13 antibodies [13], and p.Arg1060Trp [14]) have been reported [5]. Phe- were detected in her plasma (mixing studies with normal notypic consequences of these mutations have never been plasma using the TECHNOZYMEâ ADAMTS13-activity tested in animal models. Such studies might nevertheless ELISA). There were no plasma samples available from provide valuable information on the contribution of the period of her pregnancy to test for ADAMTS13 anti- specific ADAMTS13 mutations to the pathophysiology gen and activity levels. From the family history it became of TTP. clear that her mother had three pregnancies, two of which In this study, the genetic defect in a pregnancy-onset were complicated by preeclampsia. There was, however, TTP patient was identified to correctly diagnose her with no history suspicion of microangiopathy in any other hereditary TTP. In addition, the expression and activity member of her family. Family screening for ADAMTS13 of the recombinant mutant protein were evaluated in deficiency has not yet been feasible. Because of the clini- vitro. Finally, we used a TTP mouse model in which cal expression and ADAMTS13 phenotype, the patient Adamts13–/– mice were triggered with recombinant was diagnosed with TTP. Mutation analysis of ADAM- human VWF (rVWF) [15] [16] to study the effect of the TS13 was performed in this study to confirm the diagno- ADAMTS13 mutant on the pathophysiology of TTP. sis of hereditary TTP. Informed consent was obtained in accordance with the Declaration of Helsinki. Patient, materials, and methods Identification of the mutation Case history Genomic DNA was extracted from the peripheral leuko- A now 41-year-old woman was initially referred to the cytes from the patient using the salting out procedure as hospital in 2006 for hematological assessment following described [17]. All 29 ADAMTS13 exons with exon– two complicated pregnancies. Her first pregnancy at the intron boundaries were amplified by polymerase chain age of 31 was complicated by mild thrombocytopenia reaction with primers as described elsewhere [18]. Prod- detected at week 12 with severe intrauterine growth retar- ucts were sequenced in both directions, and sequencing dation. HELPP (hemolysis, elevated liver enzymes, and a reactions were outsourced (GATC Biotech AG, Kon- low platelet count) syndrome was suspected, and medical stanz, Germany). pregnancy termination was performed at week 17. During her second pregnancy at the age of 34, abnormal uterine Expression of recombinant wild-type and p.D187H artery Doppler waveforms were found at week 26. She ADAMTS13 was admitted at week 28 for fetal monitoring. Fetal growth stagnation and mild thrombocytopenia (platelet The identified missense mutation p.D187H was intro- count of 108 000 lLÀ1) with biological signs of hemolysis duced in the pcDNA6.1 ADAMTS13 expression vector and microangiopathy (elevated lactate dehydrogenase lev- using the QuickChangeTM XL Site Directed Mutagenesis els, reduced haptoglobin levels, but absence of schisto- Kit (Stratagene, La Jolla, CA, USA). Constructs were cytes) were confirmed. There was no disturbance of liver sequenced
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