Clusterin knockdown has effects on intracellular and secreted von Willebrand factor in human umbilical vein endothelial cells.

Alterations in von Willebrand factor (VWF) have an important role in human health and disease. Deficiency of VWF is associated with symptoms of bleeding and excesses of VWF are associated with thrombotic outcomes. Understanding the mechanisms that drive VWF regulation can lead to a better understanding of modulation of VWF levels in humans. We identified clusterin (CLU) as a potential candidate regulator of VWF based on a single cell RNA sequencing (scRNA-seq) analysis in control endothelial cells (ECs) and von Willebrand disease (VWD) endothelial colony-forming-cells (ECFCs). We found that patients with deficiencies of VWF (von Willebrand disease, VWD) had decreased CLU expression and ECs with low VWF expression also had low CLU expression. Based on these findings, we sought to evaluate the role of CLU in the regulation of VWF, specifically as it relates to VWD. As CLU is primarily thought to be a golgi protein involved in protein chaperoning, we hypothesized that knockdown of CLU would lead to decreases in VWF and alterations in Weibel-Palade bodies (WPBs). We used both siRNA- and CRISPR-Cas9-based approaches to modulate CLU in human umbilical vein endothelial cells (HUVECs) and evaluated VWF protein levels, VWF mRNA copy number, and WPB quantity and size. We demonstrated that siRNA-based knockdown of CLU resulted in decreases in VWF content in cellular lysates and supernatants, but no significant change in WPB quantity or size. A CRISPR-Cas9-based knockdown of CLU demonstrated similar findings of decreases in intracellular VWF content but no significant change in WPB quantity or size. Our data suggests that CLU knockdown is associated with decreases in cellular VWF content but does not affect VWF mRNA levels or WPB quantity or size.

Temporal and anatomic relationship between superficial and deep vein thromboses in hospitalized children.

BACKGROUND: Recent publications have increasingly demonstrated a link between superficial-vein thrombosis (SVT) and deep-vein thrombosis (DVT) in the adult population and have led to changes in SVT treatment considerations. A similar relationship between SVT and DVT in pediatric populations, however, is not currently well established. OBJECTIVES: We sought to evaluate the temporal and anatomic relationship between SVT and DVT among pediatric inpatients in order to determine to what degree SVT is associated with DVT. METHODS: We first retrospectively reviewed our institution's local prospective hospital-acquired VTE (HA-VTE) registry to identify all children (age 0-21 years, inclusive) admitted to Children's Hospital Colorado for more than 48 h between January 2012 and September 2017 who developed a DVT while hospitalized. We then reviewed each patient's electronic health record for evidence of SVT to identify SVT + DVT cases. Afterwards, we utilized a list of ICD codes to identify all patients during this time frame who developed an SVT and removed patients whom we previously identified as SVT + DVT cases to obtain the number of patients with isolated SVT. RESULTS: Of 59,910 patients admitted during the study period, 438 (0.7%) developed a thrombosis while hospitalized - 197 (0.3%) with isolated SVT, 161 (0.3%) with isolated DVT, and 80 (0.1%) with both SVT + DVT. These 80 SVT + DVT patients represent 33% of the 241 total DVT patients and 29% of the 277 total SVT patients. Of the 12 SVT + DVT patients in whom the SVT was diagnosed before the DVT, the subsequent DVT occurred within a mean of 6.4 (range 1-22) days and at the same anatomic site in 6 (50%). The age breakdown for this cohort was: 0 (0%) 0-1 months, 2 (17%) 1 month-2 years, 3 (25%) 2-12 years, 3 (25%) 12-16 years, and 4 (33%) 16-21 years. CONCLUSIONS: Our results indicate a temporal and anatomic relationship between SVT and DVT in hospitalized children, particularly those with central venous catheters.

Pathologic Shear and Elongation Rates Do Not Cause Cleavage of Von Willebrand Factor by ADAMTS13 in a Purified System.

INTRODUCTION: Pathological flows in patients with severe aortic stenosis are associated with acquired von Willebrand syndrome. This syndrome is characterized by excessive cleavage of von Willebrand factor by its main protease, A Disintegrin and Metalloproteinase with a Thrombospondin Type 1 Motif, Member 13 (ADAMTS13) leading to decreased VWF function and mucocutaneous bleeding. Aortic valve replacement and correction of the flow behavior to physiological levels reverses the syndrome, supporting the association between pathological flow and acquired von Willebrand syndrome. We investigated the effects of shear and elongational rates on von Willebrand factor cleavage in the presence of ADAMTS13. METHODS: We identified acquired von Willebrand syndrome in five patients with severe aortic stenosis. Doppler echography values from these patients were used to develop three computational fluid dynamic (CFD) aortic valve models (normal, mild and severe stenosis). Shear, elongational rates and exposure times identified in the CFD simulations were used as parameters for the design of microfluidic devices to test the effects of pathologic shear and elongational rates on the structure and function of von Willebrand factor. RESULTS: The shear rates (0-10,000s-1), elongational rates (0-1000 s-1) and exposure times (1-180 ms) tested in our microfluidic designs mimicked the flow features identified in patients with aortic stenosis. The shear and elongational rates tested in vitro did not lead to excessive cleavage or decreased function of von Willebrand factor in the presence of the protease. CONCLUSIONS: High shear and elongational rates in the presence of ADAMTS13 are not sufficient for excessive cleavage of von Willebrand Factor.

Hypermethioninemia Leads to Fatal Bleeding and Increased Mortality in a Transgenic I278T Mouse Model of Homocystinuria.

Severely elevated plasma homocysteine and methionine lead to thromboembolic events and strokes in homocystinuric (HCU) patients. Mouse models of HCU failed to exhibit prothrombotic phenotype, presumably due to lack of hypermethioninemia. We evaluated the impact of hypermethioninemia together with hyperhomocysteinemia on murine HCU phenotype and compared the efficacy of the current and novel therapies for HCU. High methionine intake decreased survival of I278T mice, which died from intestinal bleeding with hepatic and pancreatic failure. I278T mice on normal or increased methionine intake developed endothelial dysfunction, but paradoxically demonstrated delayed occlusion in an induced arterial thrombosis model. RNA-seq analysis suggested that expression of coagulation factor XI (FXI) is downregulated in livers of I278T mice. Indeed, plasma concentrations of FXI were decreased in I278T mice on normal diet and further reduced by increased methionine intake. Dietary methionine restriction normalized the observed phenotype. Similarly, treatment with OT-58, a novel enzyme therapy for HCU, corrected the phenotype in I278T mice regardless of their dietary methionine intake. Hypermethioninemia does not contribute to prothrombotic phenotype in murine HCU. Downregulation of FXI may contribute to the lack of prothrombotic tendency in I278T mice. Methionine restriction or treatment with OT-58 corrects vascular disease in the I278T mouse model of HCU.

Behavior, body composition, and vascular phenotype of homocystinuric mice on methionine-restricted diet or enzyme replacement therapy.

Classic homocystinuria (HCU) is an inherited disorder characterized by elevated homocysteine (Hcy) in plasma and tissues resulting from cystathionine ß-synthase (CBS) deficiency. There is no cure, and patients are predominantly managed by methionine-restricted diet (MRD) to limit the production of Hcy. In this study, we used the I278T mouse model of HCU to evaluate the long-term impact of a novel enzyme replacement therapy [truncated human CBS C15S mutant modified with linear 20-kDa N-hydroxysuccinimide ester polyethylene glycol (OT-58)] on clinical end points relevant to human patients with HCU. In addition, we compared its efficacy on a background of either MRD or normal methionine intake [regular diet (REG)] to that of MRD alone. We found that, compared with untreated I278T mice, OT-58 treatment of I278T mice fed with the REG diet resulted in a 90% decrease in plasma Hcy concentrations and correction of learning/cognition, endothelial dysfunction, hemostasis, bone mineralization, and body composition. On background of the MRD, OT-58 performed equally well with plasma Hcy entirely normalized. The MRD alone decreased plasma Hcy by 67% and corrected the HCU phenotype in I278T mice. However, the MRD increased anxiety and reduced bone mineral content in both I278T mice and wild-type controls. This study shows that OT-58 is a highly efficacious novel treatment for HCU on the background of either normal or restricted methionine intake.-Majtan, T., Park, I., Cox, A., Branchford, B. R., di Paola, J., Bublil, E. M., Kraus, J. P. Behavior, body composition, and vascular phenotype of homocystinuric mice on methionine-restricted diet or enzyme replacement therapy.

TNF-α-driven inflammation and mitochondrial dysfunction define the platelet hyperreactivity of aging.

Aging and chronic inflammation are independent risk factors for the development of atherothrombosis and cardiovascular disease. We hypothesized that aging-associated inflammation promotes the development of platelet hyperreactivity and increases thrombotic risk during aging. Functional platelet studies in aged-frail adults and old mice demonstrated that their platelets are hyperreactive and form larger thrombi. We identified tumor necrosis factor α (TNF-α) as the key aging-associated proinflammatory cytokine responsible for platelet hyperreactivity. We further showed that platelet hyperreactivity is neutralized by abrogating signaling through TNF-α receptors in vivo in a mouse model of aging. Analysis of the bone marrow compartments showed significant platelet-biased hematopoiesis in old mice reflected by increased megakaryocyte-committed progenitor cells, megakaryocyte ploidy status, and thrombocytosis. Single-cell RNA-sequencing analysis of native mouse megakaryocytes showed significant reprogramming of inflammatory, metabolic, and mitochondrial gene pathways in old mice that appeared to play a significant role in determining platelet hyperreactivity. Platelets from old mice (where TNF-α was endogenously increased) and from young mice exposed to exogenous TNF-α exhibited significant mitochondrial changes characterized by elevated mitochondrial mass and increased oxygen consumption during activation. These mitochondrial changes were mitigated upon TNF-α blockade. Similar increases in platelet mitochondrial mass were seen in platelets from patients with myeloproliferative neoplasms, where TNF-α levels are also increased. Furthermore, metabolomics studies of platelets from young and old mice demonstrated age-dependent metabolic profiles that may differentially poise platelets for activation. Altogether, we present previously unrecognized evidence that TNF-α critically regulates megakaryocytes resident in the bone marrow niche and aging-associated platelet hyperreactivity and thrombosis.