Over‐expression of Dyrk1A affects bleeding by modulating plasma fibronectin and fibrinogen level in mice

Abstract Down syndrome is the most common chromosomal abnormality in humans. Patients with Down syndrome have hematologic disorders, including mild to moderate thrombocytopenia. In case of Down syndrome, thrombocytopenia is not associated with bleeding, and it remains poorly characterized regarding molecular mechanisms. We investigated the effects of overexpression of Dyrk1A, an important factor contributing to some major Down syndrome phenotypes, on platelet number and bleeding in mice. Mice overexpressing Dyrk1A have a decrease in platelet number by 20%. However, bleeding time was found to be reduced by 50%. The thrombocytopenia and the decreased bleeding time observed were not associated to an abnormal platelet receptors expression, to a defect of platelet activation by ADP, thrombin or convulxin, to the presence of activated platelets in the circulation or to an abnormal half‐life of the platelets. To propose molecular mechanisms explaining this discrepancy, we performed a network analysis of Dyrk1A interactome and demonstrated that Dyrk1A, fibronectin and fibrinogen interact indirectly through two distinct clusters of proteins. Moreover, in mice overexpressing Dyrk1A, increased plasma fibronectin and fibrinogen levels were found, linked to an increase of the hepatic fibrinogen production. Our results indicate that overexpression of Dyrk1A in mice induces decreased bleeding consistent with increased plasma fibronectin and fibrinogen levels, revealing a new role of Dyrk1A depending on its indirect interaction with these two proteins.


| INTRODUC TI ON
Down syndrome (DS) or trisomy 21 is the most common chromosomal abnormality in humans. DS is a complex genetic condition due to an extra copy of human chromosome 21 (HSA21) resulting in many genes deregulation. 1 DS is associated with multiple disease spectrum. 1 Among them, all individuals with DS exhibit mild to severe intellectual disability. 2 Patients with DS also have hematologic disorders, including thrombocytopenia, which is seen in 66% of DS babies. 3,4 Even if thrombocytopenia is mild to moderate and not associated with bleeding, it remains poorly characterized regarding molecular mechanisms.
Dual specificity Tyrosine (Y) phosphorylation-Regulated Kinase 1A (Dyrk1A) encodes a proline-directed serine/threonine kinase, whose gene is located on HSA21 in the DS critical region. Dyrk1A is an important factor contributing to intellectual disability, memory deficit and Alzheimer's disease (AD)-type dementia, the main features of the DS phenotypes. 5,6 The key functions of Dyrk1A have been well documented in neuronal defects observed in DS patients. 7 With its location within the DS critical region, Dyrk1A has been suggested to play a crucial role in brain alterations both in trisomy and monosomy 21 patients. 8 Minimal deletions overlapping 21q22 microdeletion containing among others the Dyrk1A and RUNX1 gene and covers the DS critical region have been described in partial monosomy 21 patients. 9,10 Interestingly, these patients present not only intellectual disability but also idiopathic thrombocytopenia. Shinawi et al 11  Surprisingly, patients with partial 21 monosomy were diagnosed with low platelet counts, although RUNX1 is not deleted. 10 Therefore, we hypothesized that Dyrk1A may also affect platelet counts and bleeding. More recently to explore differences in the circulating proteomes of DS individuals, Sullivan and colleagues compared a series of studies focusing on secreted proteins and those with extracellular domains. 12 299 proteins were differentially detected between the plasma of DS individual versus euploid controls. Among them, multiple proteins within the response to wounding/regulation of coagulation were identified in DS samples, including fibrinogen. 12 Fibrinogen is a plasma protein involved in primary and secondary haemostasis, thus playing a crucial role in clot formation. We therefore assessed Dyrk1A interaction with proteins extracted from Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) of healthy individuals and unrelated individuals with DS and investigated the effects of Dyrk1A overexpression on plasma fibronectin and fibrinogen, platelet number and half-life, platelet receptors expression, platelet activation and bleeding in mice.

| Experimental mice
All procedures were carried out in accordance with the ethical standards of French and European regulations (European Communities Council Directive, 86/609/EEC). Official authorization from the French Ministry of Agriculture was granted to perform research and experiments on animals (authorization number 75-369) and the experimental protocol was approved by the institutional animal care and use committee of the Paris Diderot University (CEEA40). Mice were housed in a controlled environment with unlimited access to food and water on a 12-h light/dark cycle. The number of mice used, and suffering were minimized as much as possible. The murine bacterial artificial chromosome 189 N3 (mBACtgDyrk1A) strain was previously constructed by electroporating HM-1 embryonic stem cells with the retrofitted BAC-189 N3. 13 The mBACtgDyrk1A (TgDyrk1A) and Dp (16)1Yey mice were maintained on a C57Bl/6J background and genotyped as described. 13,14 Male and female mice from the same litter, 3 months of age, were used. Number and suffering of mice being reduced as much as possible.

| Cell lines, culture conditions and protein analysis
LCLs were derived from B lymphocytes of three healthy individuals and three unrelated individuals with DS as described. 15 LCLs are easy to grow and are widely used to study genotype-phenotype correlation. 16 Parents of patients from the Institut Jérôme Lejeune gave their informed consent, and the French biomedical ethics committee gave its approval for this study (Comité de Protection des Personnes dans la Recherche Biomédicale number 03025). LCLs were cultured in Opti-MEM with GlutaMax (Invitrogen, Cergy, France) supplemented with 5% fetal bovine serum from a unique batch and 1% penicillin and streptomycin mix (10,000 U/mL). Cell lines were grown at 37°C in humidified incubators under 5% CO2.
Cells were harvested by centrifugation, were washed in PBS, followed by another centrifugation and were stored at −80°C. Cell lysates were obtained from 5 × 10 6 cell pellets treated with 300 μL of lysis buffer [Tris, 50 mM, pH 8; NaCl, 150 mM; Igepal, 1% (Sigma-Aldrich, France); SDS, 0.1% containing protease inhibitors (1 mM Pefabloc SC, 5 μg/mL E64 and 2.5 μg/mL Leupeptin). After centrifugation for 10 min at 15,000 × g at 4°C, the cell lysate was stored at −80°C. To assess Dyrk1A interaction with proteins, co-IP experiments were performed with 200 μg of protein incubated 2 h with 2 μg rAb anti-Dyrk1A (Abnova) at 4°C. The immunocomplexes were incubated with protein A/G Magnetic beads (Thermo Scientific Pierce; Fisher Scientific SAS, Illkirch, France) overnight at 4°C, and beads were washed. Dyrk1A co-IP was analysed by mass spectrometry (MS). All tryptic digests of protein extracts were analysed with a LTQ Velos Orbitrap equipped with an EASY-Spray nanoelectrospray ion source and coupled to an Easy nano-LC Proxeon 1000 system (all devices were obtained from Thermo Fisher Scientific, San Jose, CA) and chromatographic separation of the peptides. All the experimental conditions for LC-MS/MSMS acquisitions were as previously described. 17 MS/MS data were processed with an in-house Mascot search server (Matrix Science, Boston, MA; version 2.4.1).
The mass tolerance was set to 7 ppm for precursor ions and 0.5 Da for fragments. The maximum number of missed trypsin cleavages was limited to two. The SwissProt database with the Homo sapiens taxonomy was used for the MS/MS identification step.

| Network analysis of Dyrk1A interactome
The affinity purification-mass spectrometry experiment produced a list of proteins that are expected to interact with Dyrk1A, either directly or indirectly. Therefore, to restore the connectivity in the Dyrk1A interactome, we submitted the list of candidate partners to the Proteo3Dnet web server. 18 Briefly, the method finds proteinprotein interactions (PPIs) by integrating data from three sources: BioGRID. In the results presented below, the proteins are designated by their UniProtID from which "_HUMAN" has been truncated.

| Preparation of serum samples, protein extraction and ELISA essays
Mice were anaesthetised with ketamine/xylazine (100 mg/kg and 10 mg/kg, respectively) and blood samples were obtained by retroorbital sinus sampling with heparinized capillaries, collected into tubes containing an 1/10 volume of 3.8% sodium citrate. The plasma was isolated by centrifugation (1500 g) for 15 min at R.T. and kept at −80°c until use. Mice were sacrificed, and the liver dissected and stored at −80°C until analysis. The plasma Dyrk1A levels were assessed by a solid phase immobilized epitope immunoassay, as described. 23 Plasma and liver fibrinogen (Abcam ELISA kit (# ab213478, Paris, France)), plasma fibronectin (Abcam ELISA kit ((# ab108849, Paris, France))), plasma C-reactive protein (CRP) (Abcam ELISA kit ((# ab222511, Paris, France))), plasma Interleukin-6 (IL-6) (Abcam ELISA kit ((# ab222503, Paris, France))) were assessed using sandwich ELISA. After removal of unbound conjugates, bound enzyme activity was assessed by use of a chromogenic substrate for measurement at 450 nm by a microplate reader (Flex Station 3, Molecular Devices, Ltd., Wokhingham, UK).

| Platelet counts and volume
Blood sampling was performed under anaesthesia (ketamine/xylazine (100 mg/kg and 10 mg/kg, respectively)) and blood counts were determined with an automatic cell counter (Scil Vet ABC Plus, Horiba Medical, France)

| Harmine treatment
Mice were injected intraperitoneally (without anaesthesia) in the evening overnight, with 10 mg/kg of harmine hydrochloride hydrate (Fisher Scientific, Illkirch, France) dissolved in 0.9% NaCl. The next morning, mice were injected once more for 1 h. Control mice were injected with 0.9% NaCl (Vehicle).

| Measure of tail bleeding time
Tail bleeding time was measured by 3 mm tail-tip cut in anaesthetised mice (ketamine/xylazine (100 mg/kg and 10 mg/kg, respectively)). After cutting, the tail was immediately immersed in 50 mL of 0.9% sodium chloride at 37°C. A stopwatch was started immediately to determine the time required for the bleeding to stop. To ensure that bleeding did not start again, we monitored for at least 60 sec the tail bleeding. Tail bleeding assays were stopped at 600 sec if the bleeding did not stop

| Isolation of mouse platelets, flow cytometry and western blotting
Mice were anaesthetised by intraperitoneal injection of xylazin (10 mg/kg) and ketamine (100 mg/kg), then whole blood was collected by cardiac puncture. Quantification of αIIbβ3 activation in conditions of unstirred platelets in whole blood was assessed by flow cytometry using the JON/A monoclonal antibody specific for the activated form of αIIbβ3. Isolated platelets were obtained as previously described. 24 Platelet surface β-galactose exposure was determined using fluorescein isothiocyanate (FITC)-conjugated RCA or ECL (RCA #FL-1081; ECL #FL-1141; Vector Laboratories) as described. 25 Washed platelets (3 × 10 8 /mL) were stimulated with a range of activators (convulxin, ADP, or thrombin). After incubation for 10 min without stirring, platelets were diluted at 10 8

| Platelet depletion
Mice were injected intraperitoneally (without anaesthesia) with the R300 antibody (Emfret, Eibelstadt, Germany) directed against GPIB (1 mg/g) in the evening and platelets counts were determined the next morning. C301 antibody (Emfret, Eibelstadt, Germany) was used as control.

| Human platelet isolation and protein analysis
Human blood was collected on acid citrate dextrose solution and washed platelets were obtained by standard procedures within 2-3 h. 26

| Data analysis
Statistical analysis was done with the Student's t-test using Statview software (Statview 3, Abacus Corporation). For multiple pairwise comparisons between genotypes and treatments, statistical analysis was done with two-way anova followed by the Bonferroni/Dunnet post hoc test using Statview software. The results are expressed as mean ± SEM (standard error of the mean). Data were considered significant when p < 0.05.

| Dyrk1A, fibronectin and fibrinogen interact indirectly
To assess Dyrk1A interaction with proteins, coimmunoprecipitation (co-IP) experiments were performed with proteins extracted from LCLs of three healthy individuals and three unrelated individuals with DS. The resulting Dyrk1A interactome includes FINC (the fibronectin), plasma fibronectin being an important self-limiting regulator to prevent haemorrhage as well as excessive thrombus formation, 27 but not the fibrinogen (Table S1). This may be due to a particularly indirect and/or weak Dyrk1A-fibrinogen interaction that could not be captured in vitro. Therefore, to propose a molecular mechanism linking the Dyrk1A and fibrinogen levels, we performed a computational analysis that included (i) Dyrk1A, (ii) the 16 proteins that co-precipitated with Dyrk1A and were detected by MS in at least two of the six LCLs, and (iii) the 3 α, β and γ fibrinogen chains.
The list of 20 candidate partners was submitted to the Proteo3Dnet server for a preliminary analysis, which led to adding the undetected

| Plasma fibronectin and fibrinogen levels are increased in mice over-expressing Dyrk1A
We first quantified fibronectin and fibrinogen levels in plasma of mice overexpressing Dyrk1A. There are three independent mouse regions homologous to HSA21, the largest region is found on mouse chromosome 16 (MMU16) which contains the gene encoding Dyrk1A. We used the Dp (16)1Yey mouse model which carried the duplication of the entire MMU16 region syntenic to HSA21, 14 and   found an increased plasma fibronectin fibrinogen level compared to control (WT) littermate mice (Table 1). To show if the increased fibronectin and fibrinogen level is due to Dyrk1A overexpression, we used a transgenic line overexpressing Dyrk1A, the mBACtgDyrk1A mouse model. 13 Fibronectin and Fibrinogen levels were increased in plasma of mice over-expressing Dyrk1A (TgBACDyrk1A) compared to control (WT) mice (Table 1). Plasma fibronectin and fibrinogen are synthesized by hepatocytes and secreted into the blood plasma. We analysed the liver levels in mice over-expressing Dyrk1A and found an increased fibrinogen content (9.4 ± 1.4 μg/mL versus 5.1 ± 1 μg/ mL (n = 6 for each) (p < 0.04)) without difference in fibronectin content (data not shown). These results suggest that increased plasma fibrinogen levels would be due to increased hepatic production, the increased plasma fibronectin level being rather due to its increased half time in mice overexpressing Dyrk1A.
Even if the liver is the source of at least 98% of the circulating fibrinogen, expression, synthesis, and secretion have been in vitro described in a variety of non-hepatic cells in response to inflammatory mediators. 28 We therefore quantified plasma CRP and IL-6 levels, CRP in plasma increasing during acute phase response to tissue injury or inflammatory stimuli especially IL-6. No difference was found for plasma CRP (4.2 ± 0.2 ng/mL versus 4.4 ± 0.2 ng/mL (n = 6 for each)) and plasma IL-6 (35.6 ± 1 pg/mL versus 40 ± 2.5 pg/ mL (n = 6 for each)) levels in mice over-expressing Dyrk1A compared to control mice.

| Platelet fibrinogen levels are not modified in mice over-expressing Dyrk1A
Platelet alpha-granule fibrinogen is endocytosed from plasma.
We therefore analysed the platelet levels in mice over-expressing Dyrk1A and found no difference compared to control (WT) mice ( Figure 2). There is therefore no increase in platelet fibrinogen endocytosis.

| Platelet activation is normal in mice overexpressing Dyrk1A
The next step was to investigate the impact of the overexpression of Dyrk1A on platelet function in vitro. In conditions of unstirred platelets after thrombin, ADP or Convulxin activation, no difference between TgDyrk1A platelets and WT platelets was observed for αIIbβ3 integrin activation and α granule secretion, measured by flow cytometry with a rat mAb JON/A(PE) specific for the activated conformation of the mouse integrin and a FITC-labelled rat anti-mouse CD62P mAb for P-selectin expression ( Figure S1 and S2) suggesting that the thrombocytopenia observed and the decrease bleeding time are not due to a thrombotic tendency in these mice. Moreover, no difference was observed in the expression level of the major receptors (αIIbβ3, GPVI, GPIbα and GPIbβ) analysed by flow cytometry ( Figure S3).
Interestingly, cytoskeletal proteins were analysed. No difference was found for actin ( Figure 2), but Filamin A level was increased in platelets of mice over-expressing Dyrk1A. (Figure 2).

| Platelet number is decreased in mice overexpressing Dyrk1A
To analyse the putative link between thrombocytopenia and overexpression of Dyrk1A, blood was collected and analysed for platelet count (Figure 3). Mice overexpressing Dyrk1A (TgDyrk1A mice) have a significantly lower platelet number compared to control (WT) mice ( Figure 3). However, no difference was found for the volume F I G U R E 2 Comparison of platelet fibrinogen and control protein levels obtained from control mice (WT) and mice over-expressing Dyrk1A (TgDyrk1A). Platelet lysates were prepared and Filamin A (FlnA), α-Fibrinogen (Fg), β-Actin, or 14-3-3 z was determined by western blotting. Immunoreactive bands were visualized by chemiluminescence and quantified using a luminescent image analyser system. Data represent mean ± SD (n = 3) and results are expressed as fold increase relative to controls (β-Actin, or 14-3-3 z). Statistical analysis was performed using t-test, *p < 0.05. of platelets ( Figure 3). No difference was also found for white blood cells, red cells, lymphocytes and haemoglobin levels. Furthermore, the platelet β-galactose exposure was found like WT mice and no splenomegaly was detected ( Figure S4). Taken as a whole, our data as well as the absence of abnormal levels of activated platelets detected in the circulation of TgDyrk1A mice ( Figure S5) strongly support the conclusion that there is no accelerated clearance of Dyrk1A platelets.

| Mice overexpressing Dyrk1A have a reduced bleeding time
We next evaluated the consequence of Dyrk1A over-expression on bleeding. For this, we determined the tail bleeding time of the TgDyrk1A mice ( Figure 4). Mice over-expressing Dyrk1A have a significantly reduced bleeding time compared to WT mice ( Figure 4). To determine if the effects are dependent on Dyrk1A kinase activity, we treated mice with harmine, the most selective and potent inhibitor of Dyrk1A. 29,30 No effect was found in WT mice (data not shown), but harmine treatment prevents the decrease of bleeding time in TgDyrk1A mice (Figure 4). Taken together, these data indicate that increased Dyrk1A activity is associated with reduced bleeding time, which is in line with the increased level of fibrinogen observed, but not with the reduced platelet number.

| Dyrk1A was detected in platelets
As we found a discrepancy between platelet number and bleeding time in mice overexpressing Dyrk1A, we looked if we could detect the protein in platelets. We first analysed the platelet Dyrk1A protein levels in mice overexpressing Dyrk1A and found a 1.6-fold increase compared to control (WT) mice ( Figure S6). We previously detected Dyrk1A in human plasma by western blot analysis after immunoprecipitation. 23 To confirm the presence of Dyrk1A in human platelets, we used the same method with human platelets to have large volume needed. Protein extracts from human platelets were subjected to immunoprecipitation with anti-Dyrk1A antibody, and the presence of Dyrk1A in the immunoprecipitates was analysed by western blotting. Immunoblotting showed that Dyrk1A from platelets and plasma migrated at the same molecular weight (95 kD) ( Figure 5). As previously demonstrated by genome-wide platelet transcriptome and proteome analysis, 31 Dyrk1A protein is not only present in plasma but also in platelets.

| Plasma Dyrk1A results in part from platelet release
To analyse if plasma protein Dyrk1A results from platelet release, we quantified plasma protein Dyrk1A after platelet depletion in mice ( Figure 6). For this, mice were injected with the R300 antibody directed against GPIB and C301 antibody as control. Platelet depletion was confirmed by platelet count ( Figure 6A). Plasma Dyrk1A protein levels were decreased by 20% in control (WT) mice and mice F I G U R E 3 Platelet number is decreased in mice over-expressing Dyrk1A. Platelets counts (A) and volume platelet number (B) were determined in control (WT) mice and mice over-expressing Dyrk1A (TgDyrk1A). Data are presented as mean ± SEM and analysed with the Student's t-test by using Statview software. n = 16 (eight males and eight females for each group). *p < 0.05.

F I G U R E 4
Tail bleeding time is decreased in mice overexpressing Dyrk1A. Tail bleeding time was performed on male and female control (WT, n = 9 males and 9 females) or mice over-expressing Dyrk1A (TgDyrk1A), with (TgDyrk1A/Harmine, n = 9 males and 9 females) or without (TgDyrk1A/Vehicle, n = 4 males and 4 females) harmine treatment. Data are presented as mean ± SEM and analysed with the Student's t-test by using Statview software. **p < 0.005.
over-expressing (TgDyrk1A) ( Figure 6B). These results show that plasma Dyrk1A, at least in part, result from platelet release.

| DISCUSS ION
In this report, we evaluated the effect of Dyrk1A overexpression on plasma fibronectin and fibrinogen levels, platelet number and halflife, platelet receptors expression, platelet activation and bleeding in mice. Mice overexpressing Dyrk1A have a decrease in platelet count. It was previously shown that 66% of DS babies have mild to moderate thrombocytopenia. 3,4 Recent results showed that Dyrk1A kinase inhibition enhances platelet production in infantile megakaryocytes, identifying Dyrk1A as a critical morphogenetic brake in megakaryopoiesis. 32 However, another study shows that the frequency of thrombocytopenia is 2.1% and thrombocytosis is much more frequent. 33 Thus, the thrombocytopenia is far from being constant as expected from an overexpression of DyrK1A. Moreover, we only found a decrease platelet number by 25% in mice overexpressing Dyrk1A, this decrease being not due to splenomegaly, no accelerated platelet clearance with an unchanged platelet volume.
Morowski et al induced different grades of thrombocytopenia in mice and found that tail bleeding time was unaffected by reductions of platelets up to 97.5%. 34 Although thrombocytopenia is observed in some DS patients, the decreased platelet number is not sufficient to induce bleeding in DS patients. 3,4 In this sense, bleeding time, an in vivo assay designed to assess the ability of the mouse's haemostatic system to stop blood loss for a haemorrhagic problem, is reduced by 50% in mice overexpressing Dyrk1A, and harmine, a potent and selective inhibitor of Dyrk1A, 29 but also in platelet, plasma content resulting at least in part from platelet release. Dyrk1A is ubiquitously expressed in tissues with a stronger reactivity in central nervous system. 35 Dyrk1A is associated with the dysregulation of neurotrophic pathways, 36 particularly at the level of the brain-derived neurotrophic factor (BDNF). 37 In humans, BDNF has been known to accumulate in circulating platelets, and platelets can release it upon activation. 38 In agreement with these results, we previously found that human plasma BDNF level correlates positively with Dyrk1A. 39  A integrates signalling pathways between the plasma membrane and the actin cytoskeleton, platelet function being directly contingent on actin cytoskeleton integrity. 52 Previous data showed an enhancement of Filamin A level in control neonatal megakaryocytes subjected to Dyrk inhibition. 31 SRF/MKL1A is the key coactivator of many actin cytoskeletal genes, 53 and unlike the results obtained for the Filamin A level, we found no difference in βactin level. Therefore, Filamin A regulation in platelets from mice overexpressing Dyrk1A would not be link to SRF/MKL1A pathway.
Filamin A is also a key partner in Aβ and Tau pathological processes in AD. 54,55

| CON CLUS I ON S AND LIMITATI ON
Our results indicate that overexpression of Dyrk1A in mice induces decreased bleeding consistent with increased plasma fibronectin and fibrinogen levels, revealing a new role of Dyrk1A depending on its indirect interaction with these two proteins. Dyrk1A would be one of the proteins involved in hematologic disorders of DS patients, but other proteins linked with DS aneuploidy could be involved.
Hemostatic parameters will have to be therefore studied in DS people.

ACK N O WLE D G E M ENTS
This work was supported by the Fondation Jerome Lejeune (grant number 2017a-1634) and the French state funds through the 'Agence Nationale de la Recherche' program (ANR-18-CE16-0020 DYRKDOWN). We thank Claire Dumoulin for technical assistance.
We acknowledge the platform accommodation and animal testing of the animal house at the Institute Jacques-Monod (University Paris Diderot).

CO N FLI C T O F I NTER E S T S TATEM ENT
No potential conflict of interest was reported by the authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from the authors.