And Metalloproteinase With Thrombospondin Motifs 13 Research Articles

Apadamtase Alfa: First Approval.

Apadamtase alfa (ADAMTS13, recombinant-krhn; ADZYNMA), a human recombinant form of a disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), is being developed by Takeda under license from KM biologics for thrombotic thrombocytopenic purpura (TTP) and sickle cell disease. On 9 November 2023, apadamtase alfa was approved in the USA for prophylactic and on-demand enzyme replacement therapy (ERT) in paediatric and adult patients with congenital TTP. Apadamtase alfa is under regulatory review for congenital TTP in the EU and Japan, and is under clinical development for immune-mediated TTP in several countries worldwide. Clinical development of apadamtase alfa for vaso-occlusive crisis related to sickle cell anaemia is underway in the USA. This article summarizes the milestones in the development of apadamtase alfa leading to this first approval in the USA for congenital TTP.

Elevated LDL Cholesterol Increases Microvascular Endothelial VWF and Thromboinflammation After Myocardial Infarction.

In reperfused myocardial infarction, VWF (von Willebrand factor)-mediated platelet adhesion contributes to impaired microvascular reflow and possibly also to postmyocardial infarction inflammation. We hypothesized that postischemic thromboinflammatory processes are worsened by elevated LDL (low-density lipoprotein) cholesterol. Myocardial ischemia-reperfusion or sham procedure was performed in wild-type mice and hyperlipidemic mice deficient for the LDL receptor and Apobec-1 (apolipoprotein-B mRNA editing enzyme catalytic polypeptide-1; DKO [double knockout]). DKO subgroups were treated with N-acetylcysteine, which inhibits pro-adhesive VWF multimers or with recombinant ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs-13), which enzymatically cleaves endothelial surface-associated VWF. Myocardial contrast echocardiography perfusion imaging and molecular imaging for VWF, platelet glycoprotein Ibα, and leukocyte CD18 (cluster of differentiation) were performed 30 minutes post-reperfusion. Histology, infarct sizing, and echocardiography were performed at 1.5 or 72 hours; late echocardiography was performed at day 21. After ischemia-reperfusion, DKO compared with wild-type mice had ≈2-fold higher (P<0.05) risk area signal for microvascular platelet adhesion, VWF, and CD18; greater impairment in microvascular reflow, and 2-fold larger infarct size. Treatment of DKO mice with N-acetylcysteine and ADAMTS13 reduced molecular imaging signal for microvascular platelet adhesion, VWF, and CD18; improved early microvascular reflow; and reduced eventual infarct size. ADAMTS13 suppressed the postmyocardial infarction neutrophil and monocyte infiltration, enhanced the time-dependent recovery of left ventricular systolic function, and prevented late left ventricular remodeling. In reperfused myocardial infarction, elevated LDL cholesterol promotes thromboinflammation through excess microvascular endothelial VWF and platelet adhesion, resulting in less microvascular reflow and larger infarct size. In the presence of elevated LDL cholesterol, therapies that suppress endothelial-associated VWF can promote recovery of left ventricular function and protect against remodeling.

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.

27: PRE-CARDIOPULMONARY BYPASS BIOMARKERS PREDICT POOR OUTCOMES IN NEONATAL CONGENITAL HEART DISEASE

Introduction: Neonates requiring cardiopulmonary bypass (CPB) for congenital heart disease have higher rates of postoperative mortality and morbidity than older children. However, identified risk factors for poor outcome to date have been limited to non-modifiable demographic or surgical factors. We aimed to identify novel biomarkers measured preoperatively that correlate with poor outcomes. Methods: Plasma samples were collected immediately prior to CPB in 65 consented subjects. Twenty-eight soluble analytes were measured via individual or multiplexed ELISA and tested for association with the validated “ICU-30” composite poor outcome, defined as (1) mortality within 30 days of CPB, (2) ICU stay >30 days, or (3) ICU readmission within 30 days. Differences in biomarker concentrations between subjects with and without ICU-30 were assessed using a Wilcoxon rank sum test. Associations of standardized biomarkers and ICU-30 was tested with logistic regression, adjusting for surgical complexity. Results: Samples for 63 patients were included for analysis (2 subjects did not undergo CPB after blood collection); 18 subjects (28%) had the ICU-30 outcome. Patients with ICU-30 were more likely to have a chromosomal abnormality, with no difference in gestational age or birth weight. There was no difference between age at CPB surgery and CPB time during operation. Seven biomarkers differed between those with and without ICU-30: angiopoietin 2, interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, growth differentiation factor-15, C-C motif ligand 2 (CCL2), and neutrophil gelatinase-associated lipocalin (NGAL) were higher in patients with ICU-30, and ADAMTS13 (A disintegrin and metalloproteinase with thrombospondin motifs 13) was lower in those with ICU-30. When controlling for STAT category, TNF-alpha (aOR 5.4, 95% CI 1.6-19.0), IL-6 (aOR 3.0, 95% CI 1.3-6.8), CCL2 (aOR 2.6, 95% CI 1.3-5.2), and NGAL (aOR 2.6, 95% CI 1.3-5.1) were associated with ICU-30. Conclusions: Four novel preoperative plasma biomarkers implicating innate immunity and renal function were associated with worse outcomes in neonates requiring CPB independent of STAT operation. Biomarkers have the potential to identify preoperative risk factors and mechanistic pathways for prognostication and targeted therapeutic interventions.

The Prognostic Value of ADAMTS-13 and von Willebrand Factor in COVID-19 Patients: Prospective Evaluation by Care Setting

Background: Endothelial dysfunction, coupled with inflammation, induces thrombo-inflammation. In COVID-19, this process is believed to be associated with clinical severity. Von Willebrand factor (VWF), and a disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS-13), are strong markers of endothelial dysfunction. We evaluated the impact of the VWF/ADAMTS-13 fraction on COVID-19 severity and prognosis. Materials and methods: A cohort study including 74 COVID-19 patients, with 22 admitted to the intensive care unit (ICU) and 52 to the medical ward (MW), was carried out. We also evaluated, in a group of 54 patients who were prospectively observed, whether variations in VWF/ADAMTS-13 correlated with the degree of severity and routine blood parameters. Results: A VWF:RCo/ADAMTS-13 fraction above 6.5 predicted in-hospital mortality in the entire cohort. At admission, a VWF:RCo/ADAMTS-13 fraction above 5.7 predicted admission to the ICU. Furthermore, the VWF:RCo/ADAMTS-13 fraction directly correlated with C-reactive protein (CRP) (Spearman r: 0.51, p < 0.0001) and D-dimer (Spearman r: 0.26, p = 0.03). In the prospective cohort, dynamic changes in VWF:RCo/ADAMTS-13 and the CRP concentration were directly correlated (Spearman r, p = 0.0014). This relationship was significant in both groups (ICU: p = 0.006; MW: p = 0.02).Conclusions: The present findings show that in COVID-19, the VWF/ADAMTS-13 fraction predicts in-hospital mortality. The VWF/ADAMTS-13 fraction may be a helpful tool to monitor COVID-19 patients throughout hospitalization.

Unfolding the pathophysiology of congenital thrombotic thrombocytopenic purpura in pregnancy: lessons from a cluster of familial cases

Thrombotic thrombocytopenic purpura (TTP), a rare, potentially life-threatening thrombotic microangiopathy, manifests either as congenital TTP or acquired forms. It is caused by the absence or severe depletion of a disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) protease, leading to the accumulation of ultra large von Willebrand factor multimers as well as extensive platelet adhesion and clumping, which can ultimately cause severe secondary end-organ damage. Pregnancy can provoke or exacerbate TTP, leading to maternal and fetal complications. In this report, we focused on pregnancy outcomes in a recently recognized cohort of congenital TTP patients of Bedouin Arab descent in southern Israel who were all homozygous for a novel c.3772delA variant of the ADAMTS13 gene, leading to the clinical manifestations of TTP largely during pregnancy. All patients presented in this study belong to 2 closely related families of Arab Bedouin descent and were found to be homozygous for a novel ADAMTS13-c.3772delA variant. The cohort consisted of 19 females; 16 of them had congenital TTP and had been pregnant and were thus included. Patient data were collected from electronic medical records. Of note, 13 women from our cohort, who delivered 14 fetuses (owing to 1 twin pregnancy), were diagnosed with congenital TTP following complicated pregnancies, which included recurrent pregnancy loss, stillbirth, early onset preeclampsia (both mild and severe), hemolysis, elevated liver enzymes and low platelet count syndrome, intrauterine growth restriction with abnormal Doppler flow, preterm premature rupture of membranes, and a total perinatal mortality rate of 30.7% (4/13). An additional 3 women, who were diagnosed owing to complications outside of pregnancy and at older ages, experienced TTP during their pregnancies, which occurred before diagnosis. Subsequent pregnancies were treated with fresh frozen plasma leading to a 100% fetal survival rate in the pregnancies that reached fetal viability. All placentas had lesions consistent with maternal vascular underperfusion. However, the severity and frequency of these lesions were lower in the 8 placentas from pregnancies treated with fresh frozen plasma. This case series details a distinctive cohort of congenital TTP patients, all homozygous for the same, novel ADAMTS13 variant, who presented with clinical complications during pregnancy and maternal vascular lesions of underperfusion in the placenta. Our findings imply that the variant identified in the ADAMTS13 gene in our cohort may have a specific functional impact on the placenta, and that treatment with fresh frozen plasma during pregnancy ameliorates the course of the disease, leading to a milder phenotype or a normal pregnancy in the majority of cases.

The HLA Variant rs6903608 Is Associated with Disease Onset and Relapse of Immune-Mediated Thrombotic Thrombocytopenic Purpura in Caucasians.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare, life-threatening thrombotic microangiopathy caused by severe ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs 13) deficiency, recurring in 30–50% of patients. The common human leukocyte antigen (HLA) variant rs6903608 was found to be associated with prevalent iTTP, but whether this variant is associated with disease relapse is unknown. To estimate the impact of rs6903608 on iTTP onset and relapse, we performed a case-control and cohort study in 161 Italian patients with a first iTTP episode between 2002 and 2018, and in 456 Italian controls. Variation in rs6903608 was strongly associated with iTTP onset (homozygotes odds ratio (OR) 4.68 (95% confidence interval (CI) 2.67 to 8.23); heterozygotes OR 1.64 (95%CI 0.95 to 2.83)), which occurred over three years earlier for each extra risk allele (β −3.34, 95%CI −6.69 to 0.02). Of 153 survivors (median follow-up 4.9 years (95%CI 3.7 to 6.1)), 44 (29%) relapsed. The risk allele homozygotes had a 46% (95%CI 36 to 57%) absolute risk of relapse by year 6, which was significantly higher than both heterozygotes (22% (95%CI 16 to 29%)) and reference allele homozygotes (30% (95%CI 23 to 39%)). In conclusion, HLA variant rs6903608 is a risk factor for both iTTP onset and relapse. This newly identified biomarker may help with recognizing patients at high risk of relapse, who would benefit from close monitoring or intensified immunosuppressive therapy.

Tryptophan 387 and 390 residues in ADAMTS13 are crucial to the ability of vascular tube formation and cell migration of endothelial cells.

A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) was mainly generated and secreted from endothelial cells (ECs). Our previous study showed that tryptophan (Trp) residues at 387 and 390 in ADAMTS13 are required for its secretion and enzymatic activity. However, the effects on its host cell as well as the potential mechanism have not been clear. The aim of the study was to examine the effects of Trp residues 387 and 390 of ADAMTS13 on the biological processes of ECs. Herein, Trp was substituted with alanine in ADAMTS13 to generate ADAMTS13 mutants at 387 (W387A), 390 (W390A), and double mutants at 387 and 390 (2WA), respectively. We found that substitution mutation impaired vascular endothelial growth factor (VEGF) secretion and the downstream JAK1/STAT3 activation, the binding ability to Von Willebrand factor, cell proliferation, migration, and vascular tube formation. Overall, our study concluded that Trp387 and Trp390 of ADAMTS13 play vital roles in the biological function of ECs.

Abstract WP58: ADAMTS13/vWF Regulates the Efficacy of tPA in Ischemic Stroke Patients

Background: Intravenous tissue plasminogen activator (tPA), an efficacious thrombolytic, is sometimes ineffective due to various clinical factors and high clot burden. Recent animal data show that elevated von Willebrand Factor (vWF) content in thrombi may impair the recanalization efficacy of tPA, which can be improved by administering ADAMTS13 (A Disintegrin and Metalloproteinase with Thrombospondin motifs 13), a vWF-cleaving protease. In the current study, we investigated the role of ADAMTS13 and vWF in regulating the early response to tPA in ischemic stroke patients. Method: 138 consecutive tPA-treated ischemic stroke patients were prospectively recruited in accordance with IRB protocol. Peripheral venous blood was sampled at 12, 24 and 72hr post tPA administration. tPA responders are defined as patients with rapid clinical improvement (NIHSS≥4) within 7 days post tPA treatment and with mRankins ≤ 2 at 3 months, while non-responders show no improvement and remain disabled (mRankins &gt; 2) after 3 months. ADAMTS13 levels and vWF activity were measured by ELISA. Result: ADAMTS13 and vWF are inversely correlated with each other, consistent with a role of ADAMTS13 in degrading vWF multimer (Figure 1A). tPA responders demonstrated significantly elevated level of ADAMTS13 (p&lt;0.001) and reduced vWF activity (p=0.007) as early as 12 hours post tPA treatment, while in non-responders, ADAMTS13 deficiency correlated to delayed vWF reduction after 72hr (Figure 1B). Conclusion: Our results point to a crucial role of ADAMTS13 in regulating hyperacute-stage tPA response, and suggest the potential of ADAMTS13 as an adjunct therapy to tPA. Studies in a larger patient cohort with extended time course are still ongoing.

Abstract WP5: The Age Effect on tPA Response Was Regulated by ADAMTS13/vWF

Background: Although growing evidence has suggested that elderly stroke patients can benefit from intravenous tissue plasminogen activator (tPA), impaired functional outcome and increased risk of hemorrhagic transformation have also been frequently observed in those patients after tPA treatment. By cleaving von Willebrand factor (vWF), ADAMTS13 (A Disintegrin and Metalloproteinase with Thrombospondin motifs 13) is involved in arterial thrombosis and tPA regulation. Recent studies have identified decreased ADAMTS13 activity and increased vWF levels with aging and vascular cognitive impairment, which may put elderly individuals in a prothrombotic status at baseline. We therefore hypothesized that the ADAMTS13/vWF axis may also play a role in regulating the age effect on tPA response. Method: Consecutive tPA-treated ischemic stroke patients were prospectively recruited in accordance with IRB protocol. Peripheral venous blood was sampled at 12, 24 and 72hr post tPA administration. Result: A total of 173 patients were recruited, of whom 128 were younger than 70 years and 45 were older than 70 years. Impaired tPA response (mRankins &gt; 2 at 3 months) was observed in patients ≥70 years compared to patients &lt;70 years. Increased age was associated with decreased ADAMTS13 level and enhanced vWF (Figure 1A and 1B). Post tPA treatment, patients &lt;70 years old had rapid and persistently elevated ADAMTS13 levels, which may enhance thrombolysis by cleaving vWF multimers (Figure 1C). However, in patients ≥70 years old , ADAMTS13 levels was lower and also rose slowly, which may have resulted in delayed vWF degradation and impaired thrombolytic efficacy (Figure 1C). Conclusion: Our results suggest that the ADAMTS13/vWF axis may contribute to impaired tPA response in older stroke patients and that ADAMTS13 may be a potential adjunct therapy to enhance the tPA efficacy. This proof of concept data may pave the way for future clinical trials.

Caplacizumab to treat immune-mediated thrombotic thrombocytopenic purpura

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare and life-threatening disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and multiorgan failure, resulting from autoantibody-mediated severe A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) deficiency. In spite of treatment with plasma exchange and immunosuppression, patients remain at risk of exacerbations, refractoriness and death. Caplacizumab (Cablivi; Ablynx, a Sanofi company), a nanobody targeting von Willebrand factor (vWF), has been recently approved in the E.U. and the U.S. as the first therapeutic specifically indicated for the treatment of adults experiencing an episode of iTTP. Caplacizumab blocks the interaction of all multimers with platelets and, therefore, has an immediate effect on platelet aggregation and the ensuing formation and accumulation of platelet-rich microthrombi. This immediate effect of caplacizumab has the potential to protect the patient from tissue ischemia and organ dysfunction while the underlying disease process resolves. We detail here the preclinical and clinical data on caplacizumab for iTTP, including the recent studies that led to approval by the U.S. Food and Drug Administration (FDA) in 2019.

Thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP; also known as Moschcowitz disease) is characterized by the concomitant occurrence of often severe thrombocytopenia, microangiopathic haemolytic anaemia and a variable degree of ischaemic organ damage, particularly affecting the brain, heart and kidneys. Acute TTP was almost universally fatal until the introduction of plasma therapy, which improved survival from <10% to 80-90%. However, patients who survive an acute episode are at high risk of relapse and of long-term morbidity. A timely diagnosis is vital but challenging, as TTP shares symptoms and clinical presentation with numerous conditions, including, for example, haemolytic uraemic syndrome and other thrombotic microangiopathies. The underlying pathophysiology is a severe deficiency of the activity of a disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), the protease that cleaves von Willebrand factor (vWF) multimeric strings. Ultra-large vWF strings remain uncleaved after endothelial cell secretion and anchorage, bind to platelets and form microthrombi, leading to the clinical manifestations of TTP. Congenital TTP (Upshaw-Schulman syndrome) is the result of homozygous or compound heterozygous mutations in ADAMTS13, whereas acquired TTP is an autoimmune disorder caused by circulating anti-ADAMTS13 autoantibodies, which inhibit the enzyme or increase its clearance. Consequently, immunosuppressive drugs, such as corticosteroids and often rituximab, supplement plasma exchange therapy in patients with acquired TTP.

Genetic variants in the ADAMTS13 and SUPT3H genes are associated with ADAMTS13 activity

AbstractA disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) cleaves von Willebrand factor, reducing its prothrombotic activity. The genetic determinants of ADAMTS13 activity remain unclear. We performed a genome-wide association study of ADAMTS13 activity in the Rotterdam Study, a population-based cohort study. We used imputed genotypes of common variants in a discovery sample of 3443 individuals and replication sample of 2025 individuals. We examined rare exonic variant associations in ADAMTS13 in 1609 individuals using an exome array. rs41314453 in ADAMTS13 was associated with ADAMTS13 activity in both our discovery (β, −20.2%; P = 1.3 × 10−33) and replication sample (P = 3.3 × 10−34), and explained 3.6% to 6.5% of the variance. In the combined analysis of our discovery and replication samples, there were 2 further independent associations at the ADAMTS13 locus: rs3118667 (β, 3.0; P = 9.6 × 10−21) and rs139911703 (β, −11.6; P = 3.6 × 10−8). In addition, rs10456544 in SUPT3H was associated with a 4.2 increase in ADAMTS13 activity (P = 1.13.6 × 10−8). Finally, we found 3 independent associations with rare coding variants in ADAMTS13: rs148312697 (β, −32.2%; P = 3.7 × 10−6), rs142572218 (β, −46.0%; P = 3.9 × 10−5), and rs36222275 (β, −13.9%; P = 2.9 × 10−3). In conclusion, we identified rs41314453 as the main genetic determinant of ADAMTS13 activity, and we present preliminary findings for further associations at the ADAMTS13 and SUPT3H loci.

Preserved Expression of mRNA Coding von Willebrand Factor-Cleaving Protease ADAMTS13 by Selenite and Activated Protein C.

In sepsis, the severity-dependent decrease of von Willebrand factor (VWF)-inactivating protease, a disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), results in platelet aggregation and consumption, leading to sepsis-associated thrombotic microangiopathy (TMA) and organ failure. Previous reports assessing its functional deficiency have pinpointed involvement of autoantibodies or mutations to propagate thrombotic thrombocytopenic purpura (TTP). However, mechanisms of acquired ADAMTS13 deficiency during host response remain unclear. To enhance understanding of ADAMTS13 deficiency in sepsis, we evaluated changes in expression of mRNA coding ADAMTS13 during septic conditions using primary cellular sources of the protease. We hypothesized that proinflammatory cytokines and constituents of serum from septic patients affect the transcriptional level of ADAMTS13 in vitro, and previously recommended therapeutic agents as adjunctive therapy for sepsis interact therewith. Cultured hepatic stellate cells (HSCs), endothelial cells (HMEC) and human precision-cut liver slices as an ex vivo model were stimulated with sepsis prototypic cytokines, bacterial endotoxin and pooled serum obtained from septic patients. Stimulation resulted in a significant decrease in ADAMTS13 mRNA between 10% and 80% of basal transcriptional rates. Costimulation of selenite or recombinant activated protein C (APC) with serum prevented ADAMTS13 decrease in HSCs and increased ADAMTS13 transcripts in HMEC. In archived clinical samples, the activity of ADAMTS13 in septic patients treated with APC (n = 5) increased with an accompanying decrease in VWF propeptide as surrogate for improved endothelial function. In conclusion, proinflammatory conditions of sepsis repress mRNA coding ADAMTS13 and the ameliorating effect by selenite and APC may support the concept for identification of beneficial mechanisms triggered by these drugs at a molecular level.

Ultralarge von Willebrand Factor Fibers Mediate Luminal Staphylococcus aureus Adhesion to an Intact Endothelial Cell Layer Under Shear Stress

During pathogenesis of infective endocarditis, Staphylococcus aureus adherence often occurs without identifiable preexisting heart disease. However, molecular mechanisms mediating initial bacterial adhesion to morphologically intact endocardium are largely unknown. Perfusion of activated human endothelial cells with fluorescent bacteria under high-shear-rate conditions revealed 95% attachment of the S aureus by ultralarge von Willebrand factor (ULVWF). Flow experiments with VWF deletion mutants and heparin indicate a contribution of the A-type domains of VWF to bacterial binding. In this context, analyses of different bacterial deletion mutants suggest the involvement of wall teichoic acid but not of staphylococcal protein A. The presence of inactivated platelets and serum increased significantly ULVWF-mediated bacterial adherence. ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs 13) caused a dose-dependent reduction of bacterial binding and a reduced length of ULVWF, but single cocci were still tethered by ULVWF at physiological levels of ADAMTS13. To further prove the role of VWF in vivo, we compared wild-type mice with VWF knockout mice. Binding of fluorescent bacteria was followed in tumor necrosis factor-α-stimulated tissue by intravital microscopy applying the dorsal skinfold chamber model. Compared with wild-type mice (n=6), we found less bacteria in postcapillary (60±6 versus 32±5 bacteria) and collecting venules (48±5 versus 18±4 bacteria; P<0.05) of VWF knockout mice (n=5). Our data provide the first evidence that ULVWF contributes to the initial pathogenic step of S aureus-induced endocarditis in patients with an apparently intact endothelium. An intervention reducing the ULVWF formation with heparin or ADAMTS13 suggests novel therapeutic options to prevent infective endocarditis.

Von Willebrand factor and angiopoietin-2

von Willebrand factor and angiopoietin-2

Oxidation of Met1606 in von Willebrand factor is a risk factor for thrombotic and septic complications in chronic renal failure

CKD (chronic kidney disease) is a life-threatening pathology, often requiring HD (haemodialysis) and characterized by high OS (oxidative stress), inflammation and perturbation of vascular endothelium. HD patients have increased levels of vWF (von Willebrand factor), a large protein (~240kDa) released as UL-vWF (ultra large-vWF polymers, molecular mass ~20000-50000kDa) from vascular endothelial cells and megakaryocytes, and responsible for the initiation of primary haemostasis. The pro-haemostatic potential of vWF increases with its length, which is proteolytically regulated by ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin motifs 13), a zinc-protease cleaving vWF at the single Tyr1605-Met1606 bond, and by LSPs (leucocyte serine proteases), released by activated PMNs (polymorphonuclear cells) during bacterial infections. Previous studies have shown that in vitro oxidation of Met1606 hinders vWF cleavage by ADAMTS-13, resulting in the accumulation of UL-vWF that are not only more pro-thrombotic than shorter vWF oligomers, but also more efficient in binding to bacterial adhesins during sepsis. Notably, HD patients have increased risk of developing dramatic cardiovascular and septic complications, whose underlying mechanisms are largely unknown. In the present study, we first purified vWF from HD patients and then chemically characterized its oxidative state. Interestingly, HD-vWF contains high carbonyl levels and increased proportion of UL-vWF polymers that are also more resistant to ADAMTS-13. Using TMS (targeted MS) techniques, we estimated that HD-vWF contains >10% of Met1606 in the sulfoxide form. We conclude that oxidation of Met1606, impairing ADAMTS-13 cleavage, results in the accumulation of UL-vWF polymers, which recruit and activate platelets more efficiently and bind more tightly to bacterial adhesins, thus contributing to the development of thrombotic and septic complications in CKD.