Tiki1 is required for head formation via Wnt cleavage-oxidation and inactivation.

Secreted Wnt morphogens are signaling molecules essential for embryogenesis, pathogenesis, and regeneration and require distinct modifications for secretion, gradient formation, and activity. Whether Wnt proteins can be posttranslationally inactivated during development and homeostasis is unknown. Here we identify, through functional cDNA screening, a transmembrane protein Tiki1 that is expressed specifically in the dorsal Spemann-Mangold Organizer and is required for anterior development during Xenopus embryogenesis. Tiki1 antagonizes Wnt function in embryos and human cells via a TIKI homology domain that is conserved from bacteria to mammals and acts likely as a protease to cleave eight amino-terminal residues of a Wnt protein, resulting in oxidized Wnt oligomers that exhibit normal secretion but minimized receptor-binding capability. Our findings identify a Wnt-specific protease that controls head formation, reveal a mechanism for morphogen inactivation through proteolysis-induced oxidation-oligomerization, and suggest a role of the Wnt amino terminus in evasion of oxidizing inactivation. TIKI proteins may represent potential therapeutic targets.

Frizzled receptors facilitate Tiki inhibition of Wnt signaling at the cell surface.

The membrane-tethered protease Tiki antagonizes Wnt3a signaling by cleaving and inactivating Wnt3a in Wnt-producing cells. Tiki also functions in Wnt-receiving cells to antagonize Wnt signaling by an unknown mechanism. Here, we demonstrate that Tiki inhibition of Wnt signaling at the cell surface requires Frizzled (FZD) receptors. Tiki associates with the Wnt-FZD complex and cleaves the N-terminus of Wnt3a or Wnt5a, preventing the Wnt-FZD complex from recruiting and activating the coreceptor LRP6 or ROR1/2 without affecting Wnt-FZD complex stability. Intriguingly, we demonstrate that the N-terminus of Wnt3a is required for Wnt3a binding to LRP6 and activating ß-catenin signaling, while the N-terminus of Wnt5a is dispensable for recruiting and phosphorylating ROR1/2. Both Tiki enzymatic activity and its association with the Wnt-FZD complex contribute to its inhibitory function on Wnt5a. Our study uncovers the mechanism by which Tiki antagonizes Wnt signaling at the cell surface and reveals a negative role of FZDs in Wnt signaling by acting as Tiki cofactors. Our findings also reveal an unexpected role of the Wnt3a N-terminus in the engagement of the coreceptor LRP6.

Best anticoagulation strategy with and without appendage occlusion for stroke-prophylaxis in postablation atrial fibrillation patients with cardiac amyloidosis.

INTRODUCTION: Both atrial fibrillation (AF) and amyloidosis increase stroke risk. We evaluated the best anticoagulation strategy in AF patients with coexistent amyloidosis. METHODS: Consecutive AF patients with concomitant amyloidosis were divided into two groups based on the postablation stroke-prophylaxis approach; group 1: left atrial appendage occlusion (LAAO) in eligible patients and group 2: oral anticoagulation (OAC). Group 1 patients were further divided into Gr. 1A: LAAO + half-does NOAC (HD-NOAC) for 6 months followed by aspirin 81 mg/day and Gr. 1B: LAAO + HD-NOAC. In group 1 patients, with complete occlusion at the 45-day transesophageal echocardiogram, patients were switched to aspirin, 81 mg/day at 6 months. In case of leak, or dense "smoke" in the left atrium (LA) or enlarged LA, they were placed on long-term half-dose (HD) NOAC. Group 2 patients remained on full-dose NOAC during the whole study period. RESULTS: A total of 92 patients were included in the analysis; group 1: 56 and group 2: 36. After the 45-day TEE, 31 patients from group 1 remained on baby-aspirin and 25 on HD NOAC. At 1-year follow-up, four stroke, one TIA and six device-thrombus were reported in group 1A, compared to none in patients in group 1B (5/31 vs. 0/25, p = .03). No bleeding events were reported in group 1, whereas group 2 had five bleeding events (one subdural hematoma, one retinal hemorrhage, and four GI bleedings). Additionally, one stroke was reported in group 2 that happened during brief discontinuation of OAC. CONCLUSION: In patients with coexistent AF and amyloidosis, half-dose NOAC following LAAO was observed to be the safest stroke-prophylaxis strategy.

TAILS Identifies Candidate Substrates and Biomarkers of ADAMTS7, a Therapeutic Protease Target in Coronary Artery Disease.

Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease. ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating coronary artery disease; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo-N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q, or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EFEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively, our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease-related biological substrates and facilitate activity-based ADAMTS7 biomarker development.

Prevalence of atrial fibrillation and procedural outcome in patients undergoing catheter ablation for premature ventricular complexes.

INTRODUCTION: Atrial fibrillation (AF) and premature ventricular complexes (PVC) are common arrhythmias. We aimed to investigate AF prevalence in patients with PVC and its impact on PVC ablation outcomes. METHODS: Consecutive patients undergoing PVC ablation at a single institution between 2016 and 2019 were included and prospectively followed for 2 years. Patients with severe valvular heart disease, hyperthyroidism, malignancy, alcohol use disorder and advanced renal/hepatic diseases were excluded. Twelve-lead electrocardiograms were used to diagnose AF and assess PVC morphology. All PVCs were targeted for ablation using 4-mm irrigated-tip catheters at standardized radiofrequency power guided by 3-D mapping and intracardiac echocardiography. Patients were followed with remote monitoring, device interrogations and office visits every 6 months for 2 years. Detection of any PVCs in follow-up was considered as recurrence. RESULTS: A total of 394 patients underwent PVC ablation and 96 (24%) had concurrent AF. Patients with PVC and AF were significantly older (68.2 ± 10.8 vs. 58.3 ± 15.8 years, p < .001), had lower LV ejection fraction (43.3 ± 13.3% vs. 49.6 ± 12.4%, p < .001), higher CHA2 DS2 -VASc (2.8 ± 1.3 vs. 2.0 ± 1.3, p < .001) than those without. PVCs with ≥2 morphologies were detected in 60.4% and 13.7% patients with vs without AF (p < .001). At 2-year follow-up, PVC recurrence rate was significantly higher in patients with vs without AF (17.7% vs. 9.4%, p = .02). CONCLUSION: AF was documented in 1/4 of patients undergoing PVC ablation and was associated with lower procedural success at long-term follow-up. This was likely attributed to older age, worse LV function and higher prevalence of multiple PVC morphologies in patients with concurrent AF.

Catheter ablation approach and outcome in HIV+ patients with recurrent atrial fibrillation.

INTRODUCTION: Earlier studies have shown a clear association between severity of human immunodeficiency virus (HIV) infection and incident atrial fibrillation (AF). We present the long-term outcome of catheter ablation (CA) and electrophysiological characteristics in HIV+ AF patients. METHODS: This study evaluated 1438 consecutive AF patients [31 (2.15%) with HIV and 1407 (97.8%) without HIV diagnosis] undergoing their first CA at our center. A total of 31 HIV patients and 31 controls were generated by propensity matching, based on calculated risk factor scores, using a logistic model. During first procedure, all received isolation of pulmonary vein (PV) + posterior wall and superior vena cava. Non-PV triggers, defined as ectopic triggers originating from sites other than PVs, were identified at the redo ablation with high-dose isoproterenol challenge. RESULTS: Clinical characteristics were not different between the groups. When compared to the control, by the end of 5 years after the first procedure, recurrence was significantly greater in HIV group [100% vs. 54%, p < .001]. Among patients that underwent redo ablation non-PV triggers were higher in HIV group [93.5% vs. 54%, p < .001], and most frequently originated from the coronary sinus [67.7% vs. 45.2%, p < .001] and left atrial appendage [41.9% vs. 25.8%, p < .001]. After focal ablation of non-PV trigger, no difference in arrhythmia recurrence between two groups [80.6% vs. 87.1%, p = .753] at 1-year follow up was found. CONCLUSION: Our findings suggest that non-PV triggers are highly prevalent in HIV+ AF patients resulting in higher rate of the mid- and long-term arrhythmia recurrence.

Coronary Disease Association With ADAMTS7 Is Due to Protease Activity.

[Figure: see text].

Best ablation strategy in patients with premature ventricular contractions with multiple morphology: a single-centre experience.

AIMS: This study aimed to examine the clinical benefits of targeted ablation of all Premature ventricular complex (PVC) morphologies vs. predominant PVC only. METHODS AND RESULTS: A total of 171 consecutive patients with reduced left ventricular ejection fraction (LVEF) and ≥2 PVC morphology with high burden (>10%/day) undergoing their first ablation procedure were included in the analysis. At the initial procedure, prevalent PVC alone was ablated in the majority. However, at the redo, all PVC morphologies were targeted for ablation. : At the first procedure, 152 (89%) patients received ablation of the dominant PVC only. In the remaining 19 (11%) patients, all PVC morphologies were ablated. At two years, high PVC burden was detected in 89 (52%) patients. Repeat procedure was performed in 78 of 89, where all PVC morphologies were ablated. At 5 years after the repeat procedure, 71 (91%) had PVC burden of <5% [3.8 ± 1.1% vs. 15.4 ± 4.3% in successful vs. failed subjects (P < 0.001)]. In patients with low PVC burden after the initial procedure, LVEF improved from 37.5% to 41.6% [mean difference (MD): 3.39 ± 2.9%, P < 0.001], whereas a reduction in LVEF from 39.8% to 34.5% (MD: 6.45 ± 4.7%, P < 0.001) was recorded in patients with high PVC burden. One year after the repeat procedure, LVEF improved from 36.2% to 41.7% (MD: 5.5 ± 4.3%, P < 0.001) in patients with successful ablation. CONCLUSION: In this observational series, ablation of all PVC morphologies was associated with significantly lower PVC burden and improvement of LVEF at long-term follow-up, compared with ablation of the dominant morphology only.

Half-normal saline versus normal saline for irrigation of open-irrigated radiofrequency catheters in atrial fibrillation ablation.

BACKGROUND: The creation of effective and permanent lesions is a crucial factor in determining the success rate of atrial fibrillation (AF) ablation. By increasing the efficacy of radiofrequency (RF) energy-mediated lesion formation, half-normal saline (HNS) as an irrigant for open-irrigated ablation catheters has the potential to reduce procedural times and improve acute and long-term outcomes. METHODS: This is a double-blind randomized clinical trial of 99 patients undergoing first-time RF catheter ablation for AF. Patients enrolled were randomly assigned in a 1:1 fashion to perform ablation using HNS or normal saline (NS) as an irrigant for the ablation catheter. RESULTS: The use of HNS is associated with shorter RF times (26 vs. 33 min; p = .02) with comparable procedure times (104 vs. 104 min). The rate of acute pulmonary vein reconnections (16% vs. 18%) was comparable, with a median of 1 vein reconnection in the HNS arm versus 2 in the NS arm. There was no difference in procedure-related complications, including the incidence of postprocedural hyponatremia when using HNS. Over the 1-year follow-up, there is no significant difference between the HNS and NS with respect to the recurrence of any atrial arrhythmia (off antiarrhythmic drugs [AAD]: 47% vs. 52%; hazard ratio [HR]: 1.17, 95% confidence interval [CI]: 0.66-2.06; off/on AAD: 66% vs. 66%, HR: 1.06, 95% CI: 0.53-2.12), with a potential benefit of using HNS when considering the paroxysmal AF cohort (on/off AAD 73% vs. 62%, HR: 0.72, 95% CI: 0.19-2.70). CONCLUSIONS: In a mixed cohort of patients undergoing first-time AF ablation, irrigation of open-irrigated RF ablation catheters with HNS is associated with shorter RF times, with a comparably low rate of procedure-related complications. In the long term, there is no significant difference with respect to the recurrence of any atrial arrhythmia. Larger studies with a more homogeneous population are necessary to determine whether HNS improves clinical outcomes.

Targeting non-pulmonary vein triggers in persistent atrial fibrillation: results from a prospective, multicentre, observational registry.

AIMS: We evaluated the efficacy of an ablation strategy empirically targeting pulmonary veins (PVs) and posterior wall (PW) and the prevalence and clinical impact of extrapulmonary trigger inducibility and ablation in a large cohort of patients with persistent atrial fibrillation (PerAF). METHODS AND RESULTS: A total of 1803 PerAF patients were prospectively enrolled. All patients underwent pulmonary vein antrum isolation (PVAI) extended to the entire PW. A standardized protocol was performed to confirm persistent PVAI and elicit any triggers originating from non-PV sites. All non-PV triggers initiating sustained atrial tachyarrhythmias were ablated. Ablation of non-PV sites triggering non-sustained runs (<30 s) of atrial tachyarrhythmias or promoting frequent premature atrial complexes (≥10/min) was left to operator's discretion. Overall, 1319 (73.2%) patients had documented triggers from non-PV areas. After 17.4 ± 8.5 months of follow-up, the cumulative freedom from atrial tachyarrhythmias among patients without inducible non-PV triggers (n = 484) was 70.2%. Patients with ablation of induced non-PV triggers had a significantly higher arrhythmia control than those whose triggers were not ablated (67.9% vs. 39.4%, respectively; P < 0.001). After adjusting for clinically relevant variables, patients in whom non-PV triggers were documented but not ablated had an increased risk of arrhythmia relapse (hazard ratio: 2.39; 95% confidence interval: 2.01-2.83; P < 0.001). CONCLUSION: Pulmonary vein antrum isolation extended to the entire PW might provide acceptable long-term arrhythmia-free survival in PerAF patients without inducible non-PV triggers. In our population of PerAF patients, non-PV triggers could be elicited in ∼70% of PerAF patients and their elimination significantly improved outcomes.

Arrhythmia profile and ablation-outcome in elderly women with atrial fibrillation undergoing first catheter ablation.

BACKGROUND: This study evaluated the arrhythmia profile and ablation outcome in women with atrial fibrillation (AF) aged ≥75 years. METHODS: A total of 573 consecutive female patients undergoing first AF ablation were classified into group 1: ≥75 years (n = 221) and group 2: < 75 years (n = 352). Isolation of PVs, posterior wall and superior vena cava was performed in all. Non-PV triggers from other areas were ablated based on operator's discretion. RESULTS: Group 1 had higher prevalence of hypertension (154 (69.7%) vs. 188 (53.4%), p < .001) and non-paroxysmal AF (136 (61.5%) vs. 126 (35.8%), p < .001). Non-PV triggers were detected in 194 (87.8%) patients from group 1 and 143 (40.6%) from group 2 (p < .001) and were ablated in 152 (68.8%) and 114 (32.4%) from group 1 and 2 respectively. Remaining patients (group 1: 69/221 and group 2: 238/352) received no additional ablation. At 4 years, 109 (49.3%) and 185 (52.6%) from group 1 and 2, respectively, were arrhythmia-free, p = .69. When stratified by ablation-strategy, success-rate was similar across groups in patients receiving non-PV trigger ablation (96 (63.2%) in group 1 and 76 (66.7%) in group 2, p = .61), whereas it was significantly lower in group 1 patients not receiving additional ablation compared to those from group 2 (13 (18.8%) vs. 109 (45.8%), p < .001). CONCLUSION: Non-paroxysmal AF was more common in women aged ≥75 years. Furthermore, significantly higher number of non-PV triggers were detected in elderly women and ablation of those provided similar ablation success as that in women aged < 75 years.

A simple method to detect leaks after left atrial appendage occlusion with Watchman.

BACKGROUND: We evaluated the efficacy of a new method in identifying peri-device leak (PDL) using morphology of the thrombus formed inside the left atrial appendage (LAA) as seen on follow-up transesophageal echo (TEE). METHOD: A total of 291 consecutive patients undergoing Watchman procedure were included in this analysis. TEE was performed at 45 days postprocedure. Based on the presence of the thrombus inside the LAA behind the device, patients were grouped as (1) white (W) group: LAA completely filled with thrombus (n = 101), 2) nonwhite (NW) group: LAA completely black or mixed (part black and part white; n = 190). Follow-up TEE was repeated at 6 and 12 months. RESULTS: Baseline characteristics were comparable between groups except the device size, number of patients with chicken-wing morphology, and prevalence of left atrial "smoke" that were significantly higher in the NW group. Detection of black appearance was comparable between the pre-coil closure image and the NW population (26/36 [72.2%] vs 99/154 [64.3%], p = .37). After adjusting for clinically relevant covariates, NW appearance of the LAA was associated with the presence of significant leak (odds ratio: 47.96, 95% confidence interval: 2.91-790.2, p < .001). The 11 patients with mixed appearance at the 6-month TEE remained unchanged (part black and part white) at the 12-month TEE. LAA appearance was white in all 36 patients following coil closure. CONCLUSION: Our findings demonstrated white and nonwhite appearance of the appendage on TEE to be reliable markers of complete closure and leak respectively, following LAA occlusion with the Watchman device.

Prevention, diagnosis, and management of atrioesophageal fistula.

Atrioesophageal fistula (AEF) is a rare but deadly complication of radiofrequency ablation for atrial fibrillation. Given its rarity, the diagnosis of AEF is usually delayed with a wide variation in management strategies, which contribute to the high mortality associated with AEF. As such, the most important step of AEF management is prevention, whereas early diagnosis and treatment are crucial to reduce its associated high mortality.

Pelvic modelling and the comparison between plate position for double pelvic osteotomy using artificial cancellous bone and finite element analysis.

BACKGROUND: Finite element analysis was used to compare fixation methods for double pelvic osteotomy (DPO). Using 3D scanning a stereolithography (stl) image was produced of a canine pelvis and this was subsequently refined in computer aided design (CAD). Using the CAD files, the images were imported in MSC Marc software to produce a working finite element (FE) model with 3 dimensional tetrahedral elements with linear shaped functions. The dimensions of a precontoured pelvic osteotomy plate with eight screws and a twisted seven screw straight plate were used to build the 2 fixations implants for the FE models. An equivalent load of 300 N was applied progressively on all FE models in order to facilitate its convergence. The load was applied in a distributed manner on the femur-hip joint contact area in order to simulate the actual behavior of the joint. The aim of the present study was to analyze the difference in stiffness and behavior under loading between a lateral vs ventral plate fixation, with unlocked screws and different gap scenarios, for stabilization of a pelvic osteotomy using finite element analysis. RESULTS: From both configurations the maximum displacement of the ventral plate with 7 screws without gap had a value of 1.988 mm, while in the DPO plate had a maximum displacement of 2.191 mm. The load applied for each of the different configurations studied when a gap of 1° was considered and also when a condition of no gap was considered. The ventral plate was stiffer than the lateral plate when a gap was not present. When the gap was closed in the ventral plate, the stiffness increased until a point that remained constant. CONCLUSIONS: Ventral plate fixation can be as or more stiff as lateral plate fixation and provides flexible fixation. This behavior should reduce screw loosening. Using ventral plate fixation is recommended to reduce screw loosening or failure.

Characterization of Tiki, a New Family of Wnt-specific Metalloproteases.

The Wnt family of secreted glycolipoproteins plays pivotal roles in development and human diseases. Tiki family proteins were identified as novel Wnt inhibitors that act by cleaving the Wnt amino-terminal region to inactivate specific Wnt ligands. Tiki represents a new metalloprotease family that is dependent on Mn(2+)/Co(2+) but lacks known metalloprotease motifs. The Tiki extracellular domain shares homology with bacterial TraB/PrgY proteins, known for their roles in the inhibition of mating pheromones. The TIKI/TraB fold is predicted to be distantly related to structures of additional bacterial proteins and may use a core ß-sheet within an α+ß-fold to coordinate conserved residues for catalysis. In this study, using assays for Wnt3a cleavage and signaling inhibition, we performed mutagenesis analyses of human TIKI2 to examine the structural prediction and identify the active site residues. We also established an in vitro assay for TIKI2 protease activity using FRET peptide substrates derived from the cleavage motifs of Wnt3a and Xenopus wnt8 (Xwnt8). We further identified two pairs of potential disulfide bonds that reside outside the ß-sheet catalytic core but likely assist the folding of the TIKI domain. Finally, we systematically analyzed TIKI2 cleavage of the 19 human WNT proteins, of which we identified 10 as potential TIKI2 substrates, revealing the hydrophobic nature of Tiki cleavage sites. Our study provides insights into the Tiki family of proteases and its Wnt substrates.

Somatic mutation as a mechanism of Wnt/ß-catenin pathway activation in CLL.

One major goal of cancer genome sequencing is to identify key genes and pathways that drive tumor pathogenesis. Although many studies have identified candidate driver genes based on recurrence of mutations in individual genes, subsets of genes with nonrecurrent mutations may also be defined as putative drivers if they affect a single biological pathway. In this fashion, we previously identified Wnt signaling as significantly mutated through large-scale massively parallel DNA sequencing of chronic lymphocytic leukemia (CLL). Here, we use a novel method of biomolecule delivery, vertical silicon nanowires, to efficiently introduce small interfering RNAs into CLL cells, and interrogate the effects of 8 of 15 mutated Wnt pathway members identified across 91 CLLs. In HEK293T cells, mutations in 2 genes did not generate functional changes, 3 led to dysregulated pathway activation, and 3 led to further activation or loss of repression of pathway activation. Silencing 4 of 8 mutated genes in CLL samples harboring the mutated alleles resulted in reduced viability compared with leukemia samples with wild-type alleles. We demonstrate that somatic mutations in CLL can generate dependence on this pathway for survival. These findings support the notion that nonrecurrent mutations at different nodes of the Wnt pathway can contribute to leukemogenesis.

Dkk1 in the peri-cloaca mesenchyme regulates formation of anorectal and genitourinary tracts.

Anorectal malformation (ARM) is a common birth defect but the developmental history and the underlying molecular mechanism are poorly understood. Using murine genetic models, we report here that a signaling molecule Dickkopf-1 (Dkk1) is a critical regulator. The anorectal and genitourinary tracts are major derivatives of caudal hindgut, or the cloaca.Dkk1 is highly expressed in the dorsal peri-cloacal mesenchymal (dPCM) progenitors. We show that the deletion of Dkk1 causes the imperforate anus with rectourinary fistula. Mutant genital tubercles exhibit a preputial hypospadias phenotype and premature urethral canalization.Dkk1 mutants have an ectopic expansion of the dPCM tissue, which correlates with an aberrant increase of cell proliferation and survival. This ectopic tissue is detectable before the earliest sign of the anus formation, suggesting that it is most likely the primary or early cause of the defect. Deletion of Dkk1 results in an elevation of the Wnt/ß-catenin activity. Signaling molecules Shh, Fgf8 and Bmp4 are also upregulated. Furthermore, genetic hyperactivation of Wnt/ß-catenin signal pathway in the cloacal mesenchyme partially recapitulates Dkk1 mutant phenotypes. Together, these findings underscore the importance ofDKK1 in regulating behavior of dPCM progenitors, and suggest that formation of anus and urethral depends on Dkk1-mediated dynamic inhibition of the canonical Wnt/ß-catenin signal pathway.

Disulfide bond requirements for active Wnt ligands.

Secreted Wnt lipoproteins are cysteine-rich and lipid-modified morphogens that bind to the Frizzled (FZD) receptor and LDL receptor-related protein 6 (LRP6). Wnt engages FZD through protruding thumb and index finger domains, which are each assembled from paired ß strands secured by disulfide bonds and grasp two sides of the FZD ectodomain. The importance of Wnt disulfide bonds has been assumed but uncharacterized. We systematically analyzed cysteines and associated disulfide bonds in the prototypic Wnt3a. Our data show that mutation of any individual cysteine of Wnt3a results in covalent Wnt oligomers through ectopic intermolecular disulfide bond formation and diminishes/abolishes Wnt signaling. Although individual cysteine mutations in the amino part of the saposin-like domain and in the base of the index finger are better tolerated and permit residual Wnt3a secretion/activity, those in the amino terminus, the thumb, and at the tip of the index finger are incompatible with secretion and/or activity. A few select double cysteine mutants based on the disulfide bond pattern restore Wnt secretion/activity. Further, a double cysteine mutation at the index finger tip results in a Wnt3a with normal secretion but minimal FZD binding and dominant negative properties. Our results experimentally validate predictions from the Wnt crystal structure and highlight critical but different roles of the saposin-like and cytokine-like domains, including the thumb and the index finger in Wnt folding/secretion and FZD binding. Finally, we modified existing expression vectors for 19 epitope-tagged human WNT proteins by removal of a tag-supplied ectopic cysteine, thereby generating tagged WNT ligands active in canonical and non-canonical signaling.

Canonical Wnt signaling in megakaryocytes regulates proplatelet formation.

Wnt signaling is involved in numerous aspects of vertebrate development and homeostasis, including the formation and function of blood cells. Here, we show that canonical and noncanonical Wnt signaling pathways are present and functional in megakaryocytes (MKs), with several Wnt effectors displaying MK-restricted expression. Using the CHRF288-11 cell line as a model for human MKs, the canonical Wnt3a signal was found to induce a time and dose-dependent increase in ß-catenin expression. ß-catenin accumulation was inhibited by the canonical antagonist dickkopf-1 (DKK1) and by the noncanonical agonist Wnt5a. Whole genome expression analysis demonstrated that Wnt3a and Wnt5a regulated distinct patterns of gene expression in MKs, and revealed a further interplay between canonical and noncanonical Wnt pathways. Fetal liver cells derived from low-density-lipoprotein receptor-related protein 6-deficient mice (LRP6(-/-)), generated dramatically reduced numbers of MKs in culture of lower ploidy (2N and 4N) than wild-type controls, implicating LRP6-dependent Wnt signaling in MK proliferation and maturation. Finally, in wild-type mature murine fetal liver-derived MKs, Wnt3a potently induced proplatelet formation, an effect that could be completely abrogated by DKK1. These data identify novel extrinsic regulators of proplatelet formation, and reveal a profound role for Wnt signaling in platelet production.