Cathepsin K deficiency prevented stress-related thrombosis in a mouse FeCl3 model.

BACKGROUND: Exposure to chronic psychological stress (CPS) is a risk factor for thrombotic cardiocerebrovascular diseases (CCVDs). The expression and activity of the cysteine cathepsin K (CTSK) are upregulated in stressed cardiovascular tissues, and we investigated whether CTSK is involved in chronic stress-related thrombosis, focusing on stress serum-induced endothelial apoptosis. METHODS AND RESULTS: Eight-week-old wild-type male mice (CTSK+/+) randomly divided to non-stress and 3-week restraint stress groups received a left carotid artery iron chloride3 (FeCl3)-induced thrombosis injury for biological and morphological evaluations at specific timepoints. On day 21 post-stress/injury, the stress had enhanced the arterial thrombi weights and lengths, in addition to harmful alterations of plasma ADAMTS13, von Willebrand factor, and plasminogen activation inhibitor-1, plus injured-artery endothelial loss and CTSK protein/mRNA expression. The stressed CTSK+/+ mice had increased levels of injured arterial cleaved Notch1, Hes1, cleaved caspase8, matrix metalloproteinase-9/-2, angiotensin type 1 receptor, galactin3, p16IN4A, p22phox, gp91phox, intracellular adhesion molecule-1, TNF-α, MCP-1, and TLR-4 proteins and/or genes. Pharmacological and genetic inhibitions of CTSK ameliorated the stress-induced thrombus formation and the observed molecular and morphological changes. In cultured HUVECs, CTSK overexpression and silencing respectively increased and mitigated stressed-serum- and H2O2-induced apoptosis associated with apoptosis-related protein changes. Recombinant human CTSK degraded γ-secretase substrate in a dose-dependent manor and activated Notch1 and Hes1 expression upregulation. CONCLUSIONS: CTSK appeared to contribute to stress-related thrombosis in mice subjected to FeCl3 stress, possibly via the modulation of vascular inflammation, oxidative production and apoptosis, suggesting that CTSK could be an effective therapeutic target for CPS-related thrombotic events in patients with CCVDs.

Removal of endothelial surface-associated von villebrand factor suppresses accelerate datherosclerosis after myocardial infarction.

BACKGROUND: Thromboinflammation involving platelet adhesion to endothelial surface-associated von Willebrand factor (VWF) has been implicated in the accelerated progression of non-culprit plaques after MI. The aim of this study was to use arterial endothelial molecular imaging to mechanistically evaluate endothelial-associated VWF as a therapeutic target for reducing remote plaque activation after myocardial infarction (MI). METHODS: Hyperlipidemic mice deficient for the low-density lipoprotein receptor and Apobec-1 underwent closed-chest MI and were treated chronically with either: (i) recombinant ADAMTS13 which is responsible for proteolytic removal of VWF from the endothelial surface, (ii) N-acetylcysteine (NAC) which removes VWF by disulfide bond reduction, (iii) function-blocking anti-factor XI (FXI) antibody, or (iv) no therapy. Non-ischemic controls were also studied. At day 3 and 21, ultrasound molecular imaging was performed with probes targeted to endothelial-associated VWF A1-domain, platelet GPIbα, P-selectin and vascular cell adhesion molecule-1 (VCAM-1) at lesion-prone sites of the aorta. Histology was performed at day 21. RESULTS: Aortic signal for P-selectin, VCAM-1, VWF, and platelet-GPIbα were all increased several-fold (p < 0.01) in post-MI mice versus sham-treated animals at day 3 and 21. Treatment with NAC and ADAMTS13 significantly attenuated the post-MI increase for all four molecular targets by > 50% (p < 0.05 vs. non-treated at day 3 and 21). On aortic root histology, mice undergoing MI versus controls had 2-4 fold greater plaque size and macrophage content (p < 0.05), approximately 20-fold greater platelet adhesion (p < 0.05), and increased staining for markers of platelet transforming growth factor-ß1 signaling. Accelerated plaque growth and inflammatory activation was almost entirely prevented by ADAMTS13 and NAC. Inhibition of FXI had no significant effect on molecular imaging signal or plaque morphology. CONCLUSIONS: Plaque inflammatory activation in remote arteries after MI is strongly influenced by VWF-mediated platelet adhesion to the endothelium. These findings support investigation into new secondary preventive therapies for reducing non-culprit artery events after MI.

[ADAMTS13-Mediated Proteolytic Cleavage of Unusually Large von Willebrand Factor Polymers on Endothelial Cells in the Absence of Fluid Shear Stress].

OBJECTIVE: To investigate the molecular mechanism of proteolytic cleavage of unusually large von Willebrand Factor(ULVWF) on endothelial cells by ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) in the absence of fluid shear stress, so as to provide a theoretical basis for the pathogenesis of thrombotic thrombocytopenic purpura (TTP) and other thrombotic disorders. METHODS: The ADAMTS13-mediated proteolysis of ULVWF on the surface of endothelial cells in the absence of fluid shear stress was observed through immunofluorescence microscopy. The variation in VWF antigen levels in the conditioned media were determined by ELISA assay. The levels of VWF and the proteolytic fragments released into the conditioned media were determined by ELISA assay and Western blot in the absence and presence of fluid shear stress or FVIII. The effect of ADAMTS13-mediated ULVWF cleavage on the normal distribution of plasma VWF multimers was evaluated by multimer analysis. Histamine stimulated human umbilical vein endothelial cells (HUVECs) were incubated with ADAMTS13 and various N- and C-terminally truncated mutants. Then the ULVWF that maintained binding to the cells were observed through immunofluorescence microscopy and the soluble ULVWF released from endothelial cells was determined by ELISA, so as to demonstrate the domains of ADAMTS13 required for proteolysis of ULVWF on endothelial cells. RESULTS: The ULVWF strings on the endothelial cell surface were rapidly proteolyzed by recombinant and plasma ADAMTS13 in the absence of fluid shear stress. This proteolytic processing of ULVWF depended on incubation time and ADAMTS13 concentration, but not shear stress and FVIII. The distribution of VWF releaseded by ADAMTS13-mediated proteolysis was quite similar to that secreted by endothelial cells under histamine stimulation, suggesting the ULVWF cleavage occured at the cell surface. The proteolysis of the ULVWF on endothelial cells required the Cys-rich(CysR) and spacer domains, but not the TSP1 2-8 and CUB domains of ADAMTS13. CONCLUSION: The ULVWF polymers on endothelial cells are sensitive to ADAMTS13-mediated cleavage even in the absence of fluid shear stress. The findings provide novel insight into the molecular mechanism of ADAMTS13-mediated ULVWF cleavage at the cellular level and may contribute to understanding of the pathogenesis of TTP and other thrombotic disorders.

Persistent ADAMTS13 inhibitor delays recovery of ADAMTS13 activity in caplacizumab-treated Japanese patients with iTTP.

ABSTRACT: For patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP), caplacizumab, a nanobody against von Willebrand factor A1 domain, has become crucial. Delayed normalization of ADAMTS13 activity during caplacizumab therapy has been identified. In a retrospective analysis, we compared platelet count, ADAMTS13 activity, its inhibitor, and anti-ADAMTS13 immunoglobulin G (IgG) levels in acute iTTP cases treated with caplacizumab (n = 14) or without it (n = 16). The median time from initial therapeutic plasma exchange (TPE) to the first rituximab administration was 12 days in the caplacizumab group (n = 11) and 10 days in the group without caplacizumab (n = 13). We evaluated ADAMTS13-related parameters at onset and once a week until day 28 after the first TPE. The number of days until the platelet counts reached ≥150 × 109/L was significantly shorter in the caplacizumab group than in the non-caplacizumab group. The median ADAMTS13 activity levels on days 14, 21, and 28 were significantly lower in the caplacizumab group. The median titers of the ADAMTS13 inhibitor and anti-ADAMTS13 IgG on the same days were significantly higher in the caplacizumab group. Furthermore, the median number of days from the first TPE until finally achieving an ADAMTS13 activity of ≥10% was significantly longer in the caplacizumab group than in the non-caplacizumab group (42 vs 23 days, P = .014). We observed delayed ADAMTS13 activity recovery and continued inhibitor and anti-ADAMTS13 IgG detection in patients with acute iTTP on caplacizumab, possibly because of the decreased number of TPEs and delayed frontline rituximab.

Histone content, and thus DNA content, is associated with differential in vitro lysis of acute ischemic stroke clots.

BACKGROUND: Fibrin, von Willebrand factor, and extracellular DNA from neutrophil extracellular traps all contribute to acute ischemic stroke thrombus integrity. OBJECTIVES: In this study, we explored how the proteomic composition of retrieved thromboemboli relates to susceptibility to lysis with distinct thrombolytics. METHODS: Twenty-six retrieved stroke thromboemboli were portioned into 4 segments, with each subjected to 1 hour of in vitro lysis at 37 °C in 1 of 4 solutions: tissue plasminogen activator (tPA), tPA + von Willebrand factor-cleaving ADAMTS-13, tPA + DNA-cleaving deoxyribonuclease (DNase) I, and all 3 enzymes. Lysis, characterized by the percent change in prelysis and postlysis weight, was compared across the solutions and related to the corresponding abundance of proteins identified on mass spectrometry for each of the thromboemboli used in lysis. RESULTS: Solutions containing DNase resulted in approximately 3-fold greater thrombolysis than that with the standard-of-care tPA solution (post hoc Tukey, P < .01 for all). DNA content was directly related to lysis in solutions containing DNase (Spearman's ρ > 0.39 and P < .05 for all significant histones) and inversely related to lysis in solutions without DNase (Spearman's ρ < -0.40 and P < .05 for all significant histones). Functional analysis suggests distinct pathways associated with susceptibility to thrombolysis with tPA (platelet-mediated) or DNase (innate immune system-mediated). CONCLUSION: This study demonstrates synergy of DNase and tPA in thrombolysis of stroke emboli and points to DNase as a potential adjunct to our currently limited selection of thrombolytics in treating acute ischemic stroke.

ADAMTS13 recovery in acute thrombotic thrombocytopenic purpura after caplacizumab therapy.

ABSTRACT: Caplacizumab prevents the interaction between von Willebrand factor and platelets and is used to treat immune thrombotic thrombocytopenic purpura (iTTP). Its administration has been associated with a delay in ADAMTS13 activity restoration after plasma exchange (PEX) suspension. We analyzed the outcomes of 113 iTTP episodes, 75 of which were treated with caplacizumab, in 108 patients from the Spanish Registry of Thrombotic Thrombocytopenic Purpura. Caplacizumab shortened the time to platelet count normalization and reduced PEX requirement, exacerbations, and relapses. There was no difference in the time to achieve ADAMTS13 activity ≥20% after PEX end between caplacizumab-treated and nontreated episodes (median [interquartile range], 14.5 [7.7-27.2] vs 13.0 [8.0-29.0] days, P = .653). However, considering the 36 episodes in which caplacizumab was started ≤3 days after iTTP diagnosis, the time for ADAMTS13 restoration from PEX end was higher than in those episodes in which caplacizumab was started >3 days after iTTP diagnosis (20.0 [12.0-43.0] vs 11.0 [3.5-20.0] days, P = .003) or than in non-caplacizumab-treated episodes (P = .033). This finding could be related to a significantly shorter duration of PEX in early caplacizumab-treated episodes than in late caplacizumab-treated episodes (5.5 [4.0-9.0] vs 15.0 [11.0-21.5] days, P < .001) or non-caplacizumab-treated episodes (11.0 [6.0-26.0] days, P < .001). There were no differences in time to ADAMTS-13 restoration from PEX start (28.0 [17.2-47.5], 27.0 [19.0-37.5] and 29.5 [15.2-45.0] days in early caplacizumab-treated, late caplacizumab-treated and non-caplacizumab-treated episodes). Early administered caplacizumab does not prevent the requirement for immunosuppression but has beneficial effects by shortening PEX requirement without major safety concerns.

ADAMTS13 regulates angiogenic markers via Ephrin/Eph signaling in human mesenchymal stem cells under serum-deprivation stress.

Mesenchymal stem cells (MSCs) are known to facilitate angiogenesis and promote neo-vascularization via secretion of trophic factors. Here, we explored the molecular mechanism adopted by ADAMTS13 in modulating the expression of some key angiogenic markers in human umbilical cord-derived MSCs under serum-deprivation stress. Wharton's jelly MSCs (WJ-MSCs) were isolated from the perivascular region of human umbilical cords by explant culture. ADAMTS13 was upregulated at both mRNA and protein levels in WJ-MSCs under serum-deprivation stress. Correspondingly, some key angiogenic markers were also seen to be upregulated. By screening signaling pathways, p38 and JNK pathways were identified as negative and positive regulators for expression of ADAMTS13, and the angiogenic markers, respectively. Our results also indicated the Notch pathway and p53 as other probable partners modulating the expression of ADAMTS13 and the angiogenic markers. Knockdown of ADAMTS13 using siRNA led to reversal in the expression of these angiogenic markers. Further, ADAMTS13 was shown to act via the EphrinB2/EphB4 axis followed by ERK signaling to control expression of the angiogenic markers. Interestingly, stronger expression levels were noted for ADAMTS13, VEGF and PDGF under a more stringent nutrient stress condition. Thus, we highlight a novel role of ADAMTS13 in WJ-MSCs under nutrient stress condition.

ADAMTS13 inhibits H2O2-induced human venous endothelial cell injury to attenuate deep-vein thrombosis by blocking the p38/ERK signaling pathway.

Deep vein thrombosis (DVT) is a common complication in hematologic malignancies and immunologic disorders. Endothelial cell injury and dysfunction comprise the critical contributor for the development of DVT. A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), a plasma metalloprotease that cleaves von Willebrand factor, acts as a critical regulator in normal hemostasis. This study was aimed to explore the role of ADAMTS13 in endothelial cell injury during DVT and the possible mechanism. First, human umbilical vein endothelial cells (HUVECs) were exposed to hydrogen peroxide (H2O2). Then, the mRNA and protein expressions of ADAMTS13 were evaluated with the reverse transcription-quantitative polymerase chain reaction and western blot. After treatment with recombinant ADAMTS13 (rADAMTS13; rA13), the viability and apoptosis of H2O2-induced HUVECs were assessed by cell counting kit-8 assay and terminal-deoxynucleoitidyl transferase-mediated nick end labeling staining. In addition, the levels of prostaglandin F1-alpha, endothelin-1, and reactive oxygen species were detected using the enzyme-linked immunosorbent assay and dichloro-dihydro-fluorescein diacetate assay. The expressions of proteins related to p38/extracellular signal-regulated kinase (ERK) signaling pathway were estimated with the western blot. Then, p79350 (p38 agonist) was used to pretreat cells to analyze the regulatory effects of rA13 on p38/ERK signaling in H2O2-induced HUVEC injury. The results revealed that ADAMTS13 expression was significantly downregulated in H2O2-induced HUVECs. The reduced viability and increased apoptosis of HUVECs induced by H2O2 were revived by ADAMTS13. ADAMTS13 also suppressed the oxidative stress in HUVECs after H2O2 treatment. Besides, ADAMTS13 was found to block p38/ERK signaling pathway, and p79350 reversed the impacts of ADAMTS13 on the damage of HUVECs induced by H2O2. To sum up, ADAMTS13 could alleviate H2O2-induced HUVEC injury through the inhibition of p38/ERK signaling pathway.

ADAMTS-13 and HMGB1-induced oxidative stress in Taenia multiceps-infected animals.

This study investigated the cytotoxic effects of oxidative stress (OS), high mobility group box 1 (HMGB1), ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs), and neuropathology associated with coenurus cerebralis (Taenia multiceps). ADAMTS-13, HMGB1, glutathione reductase (GR), copper/zinc superoxide dismutase (Cu/Zn SOD), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression levels were studied. The study found that ADAMTS-13 (P < 0.005), HMGB1 (P < 0.005), GR (P < 0.005), Cu/Zn SOD (P < 0.005), and 8-OHdG (P < 0.005) levels were significantly higher in T. multiceps (c. cerebralis)-infected animals compared to healthy control animals. This study's most important finding was that HMGB1 up-regulation in neurons, endothelial cells, and glial cells can directly cause brain parenchymal destruction and that HMGB1-mediated oxidative stress plays a crucial role in the neuropathogenesis of coenurosis. The results also showed that increased levels of ADAMTS-13 may play a pivotal role in regulating and protecting the blood-brain barrier integrity and neuroprotection. These findings also suggest that ADAMTS-13 and HMGB1 compete in the prevention or formation of microthrombi, which was regarded as a remarkable finding. ADAMTS-13 and HMGB1 are valuable biomarkers for disease risk assessment, estimating host neuropathy following T. multiceps (c. cerebralis) exposure, and providing a new therapeutic target. This is the first study to show that HMGB1 and ADAMTS-13 are expressed in reactive cells and are associated with neuroimmunopathology in coenurosis.

Regulation of von Willebrand factor by ADAMTS13 ameliorates lipopolysaccharide-induced lung injury in mice.

The relationship between von Willebrand factor (VWF) and inflammation has attracted considerable attention in recent years. VWF, which is stored in the Weibel-Palade bodies (WPBs) of endothelial cells (ECs), is released from WPBs in response to inflammatory stimuli and is thought to contribute to inflammation by promoting leukocyte extravasation. In this study, lung injury model mice were produced by intratracheal injection with lipopolysaccharides. The severity of lung inflammation was evaluated in mice with different genotypes (wild-type, Vwf-/-, Adamts13-/-) and mice treated with drugs that inhibit VWF function. Lung inflammation was significantly ameliorated in Vwf-/- mice compared with wild-type mice. Furthermore, inflammation was significantly suppressed in wild-type mice treated with anti-VWF A1 antibody or recombinant human ADAMTS13 compared with the untreated control group. The underlying mechanism appears to be an increased VWF/ADAMTS13 ratio at the site of inflammation and the interaction between blood cell components, such as leukocytes and platelets, and the VWF A1 domain, which promotes leukocyte infiltration into the lung. This study suggested that ADAMTS13 protein and other VWF-targeting agents may be a novel therapeutic option for treatment of pulmonary inflammatory diseases.

Diagnostic and treatment guidelines for thrombotic thrombocytopenic purpura (TTP) in Japan 2023.

Thrombotic thrombocytopenic purpura (TTP) can rapidly become a life-threatening condition, and the importance of its appropriate diagnosis and treatment cannot be overstated. Until recently, TTP has mainly been diagnosed by clinical findings such as thrombocytopenia and hemolytic anemia. In addition to these clinical findings, however, reduced activity of a disintegrin-like and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13) below 10% has become internationally accepted as a diagnostic criterion for TTP. TTP is classified as immune-mediated TTP (iTTP) if the patient is positive for anti-ADAMTS13 autoantibodies, and as congenital TTP (cTTP) if ADAMTS13 gene abnormalities are detected. Fresh frozen plasma (FFP) transfusion is performed in patients with cTTP to supplement ADAMTS13. Plasma exchange therapy using FFP is conducted in patients with iTTP to supplement ADAMTS13 and to remove both anti-ADAMTS13 autoantibodies and unusually large von Willebrand factor (VWF) multimers. To suppress autoantibody production, corticosteroid therapy is administered in conjunction with plasma exchange. The monoclonal anti-CD-20 antibody rituximab is effective in patients with iTTP. In addition, caplacizumab, an anti-VWF A1 domain nanobody, has a novel mechanism of action, involving direct inhibition of platelet glycoprotein Ib-VWF binding. The recommended first-line treatments of iTTP in Japan are plasma exchange and corticosteroids, as well as caplacizumab.

Hidden behind thromboinflammation: revealing the roles of von Willebrand factor in sickle cell disease pathophysiology.

PURPOSE OF REVIEW: This review provides an update on the pathophysiology of sickle cell disease (SCD) with a particular focus on the dysregulation of the von Willebrand factor (VWF) - ADAMTS13 axis that contributes to its pathogenesis. In discussing recent developments, we hope to encourage new and ongoing discussions surrounding therapeutic targets for SCD. RECENT FINDINGS: Within the last 5 years, the role of VWF in the pathophysiology of SCD has been further elucidated and is now a target of study in ongoing clinical trials. SUMMARY: The pathophysiology of SCD is multifaceted, as it involves systemwide vascular activation, altered blood rheology, and the activation of immune responses and coagulative pathways. The presence of VWF in excess in SCD, particularly in its largest multimeric form, greatly contributes to its pathogenesis. Understanding the molecular mechanisms that underly the presence of large VWF multimers in SCD will provide further insight into the pathogenesis of SCD and provide specific targets for therapy.

Impact of the von Willebrand factor-ADAMTS-13 axis on the risk of future venous thromboembolism.

BACKGROUND: von Willebrand factor (VWF) and its cleaving protease, ADAMTS-13, form a pivotal axis that regulates hemostasis. However, the role of the VWF-ADAMTS-13 axis in the risk of future venous thromboembolism (VTE) is unknown. OBJECTIVES: To investigate whether plasma ADAMTS-13 levels and an imbalance with VWF levels, assessed as the VWF/ADAMTS-13 ratio, are associated with the risk of future VTE. PATIENTS/METHODS: A population-based nested case-control study, comprising 383 incident VTE cases and 780 age- and sex-matched controls, was derived from the Tromsø study cohort (1994-2007). Antigen levels of ADAMTS-13 and VWF were measured in plasma samples obtained at cohort baseline. Odds ratios (ORs) with 95% CIs were estimated according to quartile cutoffs of ADAMTS-13 and VWF/ADAMTS-13 ratio determined in controls. RESULTS: In age- and sex-adjusted analysis, ADAMTS-13 levels were inversely associated with the VTE risk, with an OR of 1.40 (95% CI, 0.99-1.99) for the lowest vs highest quartiles. The VWF/ADAMTS-13 ratio was linearly associated with the VTE risk (P for trend = .001), with an OR of 1.70 (95% CI, 1.19-2.43) for the highest vs lowest quartiles, and the association was particularly pronounced for unprovoked VTE (OR, 2.81; 95% CI, 1.65-4.81). The ORs were only slightly attenuated after additional adjustments for body mass index and C-reactive protein. CONCLUSIONS: Lowered ADAMTS-13 levels and an imbalance between ADAMTS-13 and VWF levels, reflected by an increased VWF/ADAMTS-13 ratio, were associated with an increased risk of future VTE. Our findings suggest that the VWF-ADAMTS-13 axis is involved in the pathogenesis of VTE.

Overlapping ADAMTS13 peptide binding profiles of DRB1∗08:03 and DRB1∗11:01 suggest a common etiology of immune-mediated thrombotic thrombocytopenic purpura.

BACKGROUND: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an ultra-rare autoimmune disorder caused by autoantibodies against ADAMTS13. A strong association of DRB1∗11 with iTTP and DRB1∗11-restricted T-cell epitopes in ADAMTS13 have been reported in Europeans, whereas we previously found DRB1∗08:03 as a susceptible allele in Japanese. OBJECTIVES: The limited information is available regarding a susceptible allele and its T-cell epitopes in Japanese patients with iTTP. MATERIALS AND METHODS: We conducted a reanalysis on iTTP-predisposing alleles using 3 distinct Japanese control groups. Subsequently, a novel human leukocyte antigen (HLA)-peptide expression assay (MHC-density assay) was used to identify the presentation of 24 ADAMTS13-derived peptides, including the regions that were identified previously by MHC-peptidome analysis and/or T-cell assays or predicted by NetMHCIIpan-4.0, to DRB1∗08:03 and DRB1∗11:01. RESULTS: We reconfirmed the strong association of DRB1∗08:03 with iTTP, as well as the absence of the secondary risk alleles and protective alleles in Japanese iTTP, which altogether reveal that the HLA association pattern is completely different between the European and Japanese iTTP. MHC-density assay found the 3 ADAMTS13-derived peptides in the spacer domain as a potential strong binder to DRB1∗08:03. Moreover, 6 peptides in the metalloprotease, spacer, sixth thrombospondin-1 repeat, and CUB domains in ADAMTS13 showed increased presentation by both DRB1∗08:03 and DRB1∗11:01. CONCLUSION: Altogether, the findings of distinct HLA-DR association with iTTP across populations and the presentation of common peptides by DRB1∗08:03 and DRB1∗11:01 suggest that the same ADAMTS13-derived peptides might be presented and trigger the activation of autoreactive CD4+ T cells, leading to production of anti-ADAMTS13 autoantibodies by autoreactive B cells.

Effect of tranexamic acid on endothelial von Willebrand factor/ADAMTS-13 response to in vitro shock conditions.

BACKGROUND: Traumatic/hemorrhagic shock, sepsis and other inflammatory processes lead to endothelial activation and a loss of the endothelial glycocalyx. von Willebrand factor (vWF) is an acute phase reactant that is released from endothelial cells and megakaryocytes. Stimulated but not basal vWF leads to significant formation of ultralarge multimers (ultralarge vWF [ULvWF]) and risk for thrombotic complications. Ultralarge vWF is cleaved by a disintegrin and metalloproteinase with a thrombospondin type motif 13 (ADAMTS 13); alterations in ULvWF/ADAMTS 13 ratio may contribute to trauma-induced coagulopathy. Salutary effects of tranexamic acid (TXA) on trauma-induced coagulopathy have been described. These effects appear apart from antifibrinolytic actions of TXA and include protection of the endothelial glycocalyx. Ultralarge vWF is in part anchored to the glycocalyx layer of the endothelium. Tranexamic acid protected the endothelial glycocalyx layer from degradation using a microfluidic model of the microcirculation subjected to hypoxia-reoxygenation and catecholamine excess. We hypothesized that TXA administration following shock conditions would impact the vWF-ADAMTS-13 axis by protecting the glycocalyx from degradation. This was studied in a endothelial microfluidic flow study. METHODS: Human umbilical vein endothelial cells were established under flow conditions and subjected to biomimetic shock. Tranexamic acid was added after 90 minutes of perfusion. von Willebrand factor antigen and ADAMTS-13 activity were measured. Western blot analysis was performed for vWF characterization from perfusion media. RESULTS: Shock conditions increased vWF antigen and decreased ADAMTS 13 activity. Tranexamic acid ameliorated shock induced cleavage in the ADAMTS 13-vWF axis with a reduction of the thrombogenic ULvWF. CONCLUSION: These results suggest another mechanism whereby administration of TXA early following traumatic/hemorrhagic shock mitigates microvascular perfusion abnormalities and subsequent organ failure. The resultant effects on platelet adhesion and aggregation require further study.

ADAMTS13 conformation and immunoprofiles in Japanese patients with immune-mediated thrombotic thrombocytopenic purpura.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an ultrarare thrombotic disease caused by autoantibody-induced ADAMTS13 deficiency. Open ADAMST13 conformation, induced by autoantibodies, was identified as a novel biomarker for iTTP. Determining immunoprofiles in patients with iTTP has been shown to guide the development of novel targeted therapies. However, these studies were done in mainly Caucasian iTTP cohorts. To validate those findings across other ethnic cohorts, we investigated 195 acute TTP plasma samples from the Japanese iTTP registry. Seventy-six of the 195 samples had detectable ADAMTS13 antigen levels, of which 94.7% were shown to have an open ADAMTS13 conformation. A positive correlation was observed between ADAMTS13 inhibitor titers (a diagnostic parameter in Japan) and anti-ADAMTS13 immunoglobulin G autoantibody titers. Studying anti-M, anti-DT, anti-CS, anti-T2-T5, anti-T6-T8, anti-CUB1-2 autoantibodies and the corresponding immunoprofile showed that 73% of the patients had anti-CS autoantibodies and 25.8% had anti-M autoantibodies, with the latter being higher than in Caucasians. Stratifying patients according to their immunoprofiles revealed that the profile with only anti-CS autoantibodies was the most common immunoprofile similar to that in Caucasians (28.9%). Although this profile did not affect the 1-year TTP-related mortality rate, patients with autoantibodies against all 6 ADAMTS13 fragments had a higher risk for TTP-related death than other patients (P = .02). We here validated open ADAMTS13 as a novel biomarker for acute iTTP and determined the dominant immunoprofiling in the Japanese cohort, contributing to setting up the diagnosis and managing guidelines across different ethnic cohorts and developing ADAMTS13 variants that do not bind to the anti-CS autoantibodies.

A nanobody against the VWF A3 domain detects ADAMTS13-induced proteolysis in congenital and acquired VWD.

von Willebrand factor (VWF) is a multimeric protein, the size of which is regulated via ADAMTS13-mediated proteolysis within the A2 domain. We aimed to isolate nanobodies distinguishing between proteolyzed and non-proteolyzed VWF, leading to the identification of a nanobody (designated KB-VWF-D3.1) targeting the A3 domain, the epitope of which overlaps the collagen-binding site. Although KB-VWF-D3.1 binds with similar efficiency to dimeric and multimeric derivatives of VWF, binding to VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2 domain modulates exposure of its epitope in the A3 domain. We therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact VWF could be detected using this nanobody. By comparing plasma from volunteers to that from congenital von Willebrand disease (VWD) patients, intact-VWF levels were significantly reduced for all VWD types, and most severely in VWD type 2A-group 2, in which mutations promote ADAMTS13-mediated proteolysis. Unexpectedly, we also observed increased proteolysis in some patients with VWD type 1 and VWD type 2M. A significant correlation (r = 0.51, P < .0001) between the relative amount of high-molecular weight multimers and levels of intact VWF was observed. Reduced levels of intact VWF were further found in plasmas from patients with severe aortic stenosis and patients receiving mechanical circulatory support. KB-VWF-D3.1 is thus a nanobody that detects changes in the exposure of its epitope within the collagen-binding site of the A3 domain. In view of its unique characteristics, it has the potential to be used as a diagnostic tool to investigate whether a loss of larger multimers is due to ADAMTS13-mediated proteolysis.

Mechanisms of ADAMTS13 regulation.

Recombinant ADAMTS13 is currently undergoing clinical trials as a treatment for hereditary thrombotic thrombocytopenic purpura, a lethal microvascular condition resulting from ADAMTS13 deficiency. Preclinical studies have also demonstrated its efficacy in treating arterial thrombosis and inflammation without causing bleeding, suggesting that recombinant ADAMTS13 may have broad applicability as an antithrombotic agent. Despite this progress, we currently do not understand the mechanisms that regulate ADAMTS13 activity in vivo. ADAMTS13 evades canonical means of protease regulation because it is secreted as an active enzyme and has a long half-life in circulation, suggesting that it is not inhibited by natural protease inhibitors. Although shear can spatially and temporally activate von Willebrand factor to capture circulating platelets, it is also required for cleavage by ADAMTS13. Therefore, spatial and temporal regulation of ADAMTS13 activity may be required to stabilize von Willebrand factor-platelet strings at sites of vascular injury. This review outlines potential mechanisms that regulate ADAMTS13 in vivo including shear-dependency, local inactivation, and biochemical and structural regulation of substrate binding. Recently published structural data of ADAMTS13 is discussed, which may help to generate novel hypotheses for future research.

Endothelial VWF is critical for the pathogenesis of vaso-occlusive episode in a mouse model of sickle cell disease.

Vaso-occlusive episode (VOE) is a common and critical complication of sickle cell disease (SCD). Its pathogenesis is incompletely understood. von Willebrand factor (VWF), a multimeric plasma hemostatic protein synthesized and secreted by endothelial cells and platelets, is increased during a VOE. However, whether and how VWF contributes to the pathogenesis of VOE is not fully understood. In this study, we found increased VWF levels during tumor necrosis factor (TNF)-induced VOE in a humanized mouse model of SCD. Deletion of endothelial VWF decreased hemolysis, vascular occlusion, and organ damage caused by TNF-induced VOE in SCD mice. Moreover, administering ADAMTS13, the VWF-cleaving plasma protease, reduced plasma VWF levels, decreased inflammation and vaso-occlusion, and alleviated organ damage during VOE. These data suggest that promoting VWF cleavage via ADAMTS13 may be an effective treatment for reducing hemolysis, inflammation, and vaso-occlusion during VOE.