Von Willebrand Factor Multimers Research Articles

Improving our understanding on the clinical role of plasmin-mediated von Willebrand factor degradation.

Von Willebrand factor (VWF) plays a pivotal role in primary hemostasis. A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif, member 13 (ADAMTS13) is primarily responsible for cleaving ultra-large VWF multimers into smaller, less adhesive forms. However, plasmin has also been shown to cleave VWF multimers. This proteolytic cleavage of VWF results in a decreased multimer size and, hence, a lower VWF activity. This review aims to present a comprehensive overview of the involvement of plasmin-mediated VWF proteolysis in (micro)thrombosis. Plasmin-mediated VWF proteolysis has been suggested to play a role in various pathologies involving microthrombosis in combination with an imbalance in VWF antigen levels and ADAMTS13 activity, as well as activation of the fibrinolytic system, but quantitative assays to demonstrate this were lacking. Recently, a VHH-based bioassay was developed designed specifically to quantify plasmin-cleaved VWF (cVWF). The novel ELISA assay holds significant promise for gaining further insights into the clinical relevance of plasmin-mediated VWF proteolysis in several pathologies. Furthermore, local plasmin activation at the site of microthrombosis has been shown to be a promising treatment strategy by degrading VWF-rich microthrombi. Plasmin-mediated proteolysis of VWF is observed during microthrombosis; however, it remains unclear whether it impacts disease severity. A novel ELISA method to detect cVWF will improve our understanding of the clinical role of plasmin-mediated VWF degradation.

Mitral regurgitation is associated with similar loss of von Willebrand factor large multimers but less anemia compared with aortic stenosis

BackgroundVarious cardiovascular diseases cause acquired von Willebrand syndrome (AVWS), which is characterized by a decrease in high-molecular-weight (large) von Willebrand factor (VWF) multimers. Mitral regurgitation (MR) has been reported as a cause of AVWS. However, much remains unclear about AVWS associated with MR. ObjectivesTo evaluate VWF multimers in MR patients and examine their impact on clinical characteristics. MethodsModerate or severe MR patients (n = 84) were enrolled. VWF parameters such as the VWF large multimer index (VWF-LMI), a quantitative value that represents the amount of VWF large multimers, and clinical data were prospectively analyzed. ResultsAt baseline, the mean hemoglobin level was 12.9 ± 1.9 g/dL and 58 patients (69.0%) showed loss of VWF large multimers defined as VWF-LMI < 80%. VWF-LMI in patients with degenerative MR was lower than in those with functional MR. VWF-LMI appeared to be restored the day after mitral valve intervention, and the improvement was maintained 1 month after the intervention. Seven patients (8.3%) had a history of bleeding, 6 (7.1%) of whom had gastrointestinal bleeding. Gastrointestinal endoscopy was performed in 23 patients (27.4%) to investigate overt gastrointestinal bleeding, anemia, etc. Angiodysplasia was detected in 2 of the 23 patients (8.7%). ConclusionModerate or severe MR is frequently associated with loss of VWF large multimers, and degenerative MR may cause more severe loss compared with functional MR. Mitral valve intervention corrects the loss of VWF large multimers. Gastrointestinal bleeding may be relatively less frequent and hemoglobin level remains stable in MR patients.

Nanobody activator improves sensitivity of the von Willebrand factor activity assay to multimer size

BackgroundThe activity of von Willebrand factor (VWF) in facilitating platelet adhesion and aggregation correlates with its multimer size. Traditional ristocetin-dependent functional assays lack sensitivity to multimer sizes. Recently, nanobodies targeting the autoinhibitory module and activating VWF were identified. ObjectivesTo develop an assay that can differentiate the platelet-binding activity of VWF multimers. MethodsAn enzyme-linked immunosorbent assay–based assay (VWF nanobody-triggered glycoprotein Ib binding assay [VWF:GPIbNab]) utilizing a VWF-activating nanobody was developed. Recombinant VWF, plasma-derived VWF (pdVWF), and selected gel-filtrated fractions of pdVWF were evaluated for VWF antigen and activity levels. A linear regression model was developed to estimate the specific activity of VWF multimers. ResultsOf the 3 activating nanobodies tested, 6C11 with the lowest activation effect exhibited the highest sensitivity for high-molecular-weight multimers (HMWMs) of VWF. VWF:GPIbNab utilizing 6C11 (VWF:GPIbNab6C11) produced significantly higher activity/antigen ratios for recombinant VWF (>2.0) and HMWM-enriched pdVWF fractions (>2.0) than for pdVWF (∼1.0) or fractions enriched with shorter multimers (<1.0). The differences were much larger than those produced by VWF:GPIbNab utilizing other nanobodies, VWF:GPIbM, VWF:GPIbR, or VWF:CB assays. Linear regression analysis of 5 pdVWF fractions of various multimer sizes produced an estimated specific activity of 2.7 for HMWMs. The analysis attributed >90% of the VWF activity measured by VWF:GPIbNab6C11 to that of HMWMs, which is significantly higher than all other activity assays tested. ConclusionThe VWF:GPIbNab6C11 assay exhibits higher sensitivity to HMWMs than ristocetin-based and collagen-binding assays. Future studies examining the application of this assay in clinical settings and any associated therapeutic benefit of doing so are warranted.

Myosin-1C augments endothelial secretion of von Willebrand factor by linking contractile actomyosin machinery to the plasma membrane

Blood endothelial cells control the hemostatic and inflammatory response by secreting von Willebrand factor (VWF) and P-selectin from storage organelles called Weibel-Palade bodies (WPB). Actin-associated motor proteins regulate this secretory pathway at multiple points. Prior to fusion, myosin Va forms a complex that anchors WPBs to peripheral actin structures allowing maturation of content. Post-fusion, an actomyosin ring/coat is recruited and compresses the WPB to forcibly expel the largest VWF multimers. Here we provide the first evidence for the involvement of class I myosins during regulated VWF secretion. We show that the unconventional myosin-1C (Myo1c) is recruited post-fusion via its pleckstrin homology domain in an actin-independent process. This provides a link between the actin ring and phosphatidylinositol 4,5-bisphosphate (PIP2) at the membrane of the fused organelle and is necessary to ensure maximal VWF secretion. This is an active process requiring Myo1c ATPase activity as inhibition of class I myosins using the inhibitor Pentachloropseudilin or expression of an ATPase deficient Myo1c rigor mutant perturbs the expulsion of VWF and alters the kinetics of the exocytic actin ring. These data offer a novel insight into the control of an essential physiological process and provide a new way in which it can be regulated.

Optimization of plasma-based BioID identifies plasminogen as a ligand of ADAMTS13

ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13, regulates the length of Von Willebrand factor (VWF) multimers and their platelet-binding activity. ADAMTS13 is constitutively secreted as an active protease and is not inhibited by circulating protease inhibitors. Therefore, the mechanisms that regulate ADAMTS13 protease activity are unknown. We performed an unbiased proteomics screen to identify ligands of ADAMTS13 by optimizing the application of BioID to plasma. Plasma BioID identified 5 plasma proteins significantly labeled by the ADAMTS13-birA* fusion, including VWF and plasminogen. Glu-plasminogen, Lys-plasminogen, mini-plasminogen, and apo(a) bound ADAMTS13 with high affinity, whereas micro-plasminogen did not. None of the plasminogen variants or apo(a) bound to a C-terminal truncation variant of ADAMTS13 (MDTCS). The binding of plasminogen to ADAMTS13 was attenuated by tranexamic acid or ε-aminocaproic acid, and tranexamic acid protected ADAMTS13 from plasmin degradation. These data demonstrate that plasminogen is an important ligand of ADAMTS13 in plasma by binding to the C-terminus of ADAMTS13. Plasmin proteolytically degrades ADAMTS13 in a lysine-dependent manner, which may contribute to its regulation. Adapting BioID to identify protein-interaction networks in plasma provides a powerful new tool to study protease regulation in the cardiovascular system.

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

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. 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. 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. 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.

Hereditary TTP/Upshaw-Schulman syndrome: the ductus arteriosus controls newborn survival.

Hereditary TTP (hTTP), termed Upshaw-Schulman syndrome, is an ultra-rare disorder caused by a severe deficiency of plasma ADAMTS13 activity that allows circulation of ultra-large von Willebrand factor (UL-VWF) multimers. The greatest risk for hTTP is in their first days after birth, when 35-50% of patients will have severe hemolysis, jaundice, and thrombocytopenia. It is often fatal without effective treatment. In utero, fetal blood flowing from the pulmonary artery through the ductus arteriosus (DA) to the aorta is under low-shear-force. At birth, blood flow through the DA reverses to a left-to-right shunt, and the diameter of the DA begins to decrease due to hyper-oxygenated blood and decreased plasma prostaglandin E2. This causes turbulent circulation that unfolds UL-VWF, allowing platelet aggregation. If the DA closes promptly, hTTP newborns survive, but if it remains patent, turbulent circulation persists, triggering microvascular thrombosis. hTTP is commonly diagnosed as hemolytic disease of the fetus and newborn (HDFN) caused by anti-red cell antibodies and treated with exchange blood transfusion, which prevents kernicterus even when the diagnosis of hTTP is missed. The diagnosis of newborn-onset hTTP should be considered because HDFN does not cause severe thrombocytopenia, which might be effectively treated with recombinant ADAMTS13.

N-Acetylcysteine To Reduce Mortality For Patients Requiring Cardiac Catheterization or Cardiac Surgery: A Systematic Review And Meta-Analysis.

Multimers of von Willebrand factor (VWF) play a critical role in various processes inducing morbidity and mortality in cardiovascular risk patients. With the ability to reduce VWF multimers, N-acetylcysteine (NAC) could reduce mortality in patients undergoing coronary catheterization or cardiac surgery. However, its impact in perioperative period has never been studied so far in regard of its potential cardiovascular benefits. Then, four databases were searched for randomized controlled trials that compared in-hospital mortality between an experimental group, with NAC, and a control group without NAC, in patients undergoing coronary catheterization or cardiac surgery. The primary efficacy outcome was in-hospital mortality. Secondary outcomes were the occurrence of thrombotic events, major cardiovascular events, myocardial infarction, and contrast induced nephropathy. The safety outcome was occurrence of hemorrhagic events. Nineteen studies totaling 3718 patients were included. Pooled analysis demonstrated a reduction of in-hospital mortality associated with NAC: Odds Ratio (OR), 0.60; 95% CI, 0.39-0.92; P=0.02. The occurrence of secondary outcomes was not significantly reduced with NAC except for contrast-induced nephropathy. No difference was reported for hemorrhagic events. Subgroup analyses revealed a life-saving effect of NAC in a dose-dependent manner with reduction of in-hospital mortality for the NAC high-dose group, but not for the NAC standard-dose (<3500 mg) group. In conclusion, without being able to conclude on the nature of the mechanism involved, our review suggests a benefit of NAC in cardiovascular risk patients in perioperative period in terms of mortality and supports prospective confirmatory studies.

A career in solving clinical-pathological conundrums: Heyde syndrome, anti-platelet factor 4 disorders, and microvascular limb ischemic necrosis.

Hematology is a clinical specialty with strong roots in the laboratory; accordingly, the lab can help solve perplexing clinical problems. This review highlights clinical-pathological conundrums addressed during my 35-year hematology career at McMaster University. Heyde syndrome is the association between aortic stenosis and bleeding gastrointestinal (GI) angiodysplasia where the bleeding is usually cured by aortic valve replacement; the chance reading of a neonatal study showing reversible deficiency of high-molecular-weight (HMW) multimers of von Willebrand factor (vWF) following surgical correction of congenital heart disease provided the key insight that a subtle deficiency of HMW multimers of vWF explains Heyde syndrome. The unusual immunobiology of heparin-induced thrombocytopenia (HIT)-a highly prothrombotic, antibody-mediated, anti-platelet factor 4 (PF4) disorder featuring rapid appearance and then disappearance (seroreversion) of the pathological heparin-dependent platelet-activating antibodies-permitted identification of key clinical features that informed development of a scoring system (4Ts) to aid in HIT diagnosis. Atypical clinical presentations of HIT prompted identification of heparin-independent anti-PF4 antibodies, now recognized as the explanation for vaccine-induced immune thrombotic thrombocytopenia (VITT), as well as VITT-like disorders triggered by adenovirus infection. Another unusual feature of HIT is its strong association with limb ischemia, including limb necrosis secondary to deep-vein/microvascular thrombosis (venous limb gangrene). The remarkable observation that supratherapeutic warfarin anticoagulation predisposes to HIT- and cancer-associated venous limb gangrene provided insight into disturbed procoagulant/anticoagulant balance; these concepts are relevant to microvascular thrombosis in critical illness (symmetrical peripheral gangrene), including a pathophysiological role for proximate "shock liver" (impaired hepatic synthesis of natural anticoagulants).

A noncanonical splicing variant c.875-5 T > G in von Willebrand factor causes in-frame exon skipping and type 2A von Willebrand disease

IntroductionA novel variant involving noncanonical splicing acceptor site (c.875-5 T > G) in propeptide coding region of von Willebrand factor (VWF) was identified in a patient with type 2A von Willebrand disease (VWD), who co-inherited with a null variant (p.Tyr271*) and presented characteristic discrepancy of plasma level of VWF antigen and activity, and a selective reduction of both intermediate-molecular-weight (IMWMs) and high-molecular-weight VWF multimers (HMWMs). Materials and methodsVWF mRNA transcripts obtained from peripheral leukocytes and platelets of the patients were investigated to analyze the consequence of c.875-5 T > G on splicing. The impact of the variant on expression and multimer assembly was further analyzed by in vitro expression studies in AtT-20 cells. The intracellular processing of VWF mutant and the Weibel-Palade bodies (WPBs) formation was evaluated by immunofluorescence staining and electron microscopy. ResultsThe mRNA transcript analysis revealed that c.875-5 T > G variant led to exon 8 skipping and an in-frame deletion of 41 amino acids in the D1 domain of VWF (p.Ser292_Glu333delinsLys), yielding a truncated propeptide. Consistent with the patient's laboratory manifestations, the AtT-20 cells transfected with mutant secreted less VWF, with the VWF antigen level in conditioned medium 47 % of wild-type. A slight retention in the endoplasmic reticulum was observed for the mutant. Almost complete loss of IMWMs and HMWMs in the medium and impaired WPBs formation in the cell, indicating truncated VWF propeptide lost its chaperon-like function for VWF multimerization and tubular storage. ConclusionsThe VWF splicing site variant (c.875-5 T > G) causes propeptide truncation, severely compromising VWF multimer assembly and tubular storage.

Haemostaseological Changes of VWF and FVIII during Pregnancy and the Oestrus Cycle in a Porcine Model of Von Willebrand Disease

Pregnancy and the oestrus cycle are challenging for female patients suffering from von Willebrand disease (VWD). Therefore, our study aimed to investigate the changes in von Willebrand factor (VWF) and factor VIII (FVIII) during pregnancy and the oestrus cycle in our porcine model of von Willebrand disease compared with the wild-type. Plasma analyses regarding primary hemostasis, secondary hemostasis, and VWF multimers, as well as immunohistochemistry analyses of VWF in the uterus and ovary, were performed. For levels of VWF and FVIII activities, significant elevations were seen in the last trimester. Primary hemostasis improved towards the end of pregnancy. In the oestrus cycle, significantly lower VWF values can be seen in the immunohistochemistry of the ovaries during the oestrus, while values were highest in the metoestrus. VWF multimer patterns in pigs were similar to the ones in human VWD patients. In summary, the course of VWF and FVIII during pregnancy and the oestrus cycle in porcine VWD were investigated for the first time. The porcine model seems to be suitable for haemostaseological studies on VWD. This provides an advantage for investigating reproduction-related bleeding and understanding the underlying mechanisms of post-partum hemorrhage or miscarriage in women with VWD.

Influence of Hematocrit Level and Integrin αIIbβIII Function on vWF-Mediated Platelet Adhesion and Shear-Induced Platelet Aggregation in a Sudden Expansion.

Shear-mediated thrombosis is a clinically relevant phenomenon that underlies excessive arterial thrombosis and device-induced thrombosis. Red blood cells are known to mechanically contribute to physiological hemostasis through margination of platelets and vWF, facilitating the unfurling of vWF multimers, and increasing the fraction of thrombus-contacting platelets. Shear also plays a role in this phenomenon, increasing both the degree of margination and the near-wall forces experienced by vWF and platelets leading to unfurling and activation. Despite this, the contribution of red blood cells in shear-induced platelet aggregation has not been fully investigated-specifically the effect of elevated hematocrit has not yet been demonstrated. Here, a microfluidic model of a sudden expansion is presented as a platform for investigating platelet adhesion at hematocrits ranging from 0 to 60% and shear rates ranging from 1000 to 10,000 s-1. The sudden expansion geometry models nonphysiological flow separation characteristic to mechanical circulatory support devices, and the validatory framework of the FDA benchmark nozzle. PDMS microchannels were fabricated and coated with human collagen. Platelets were fluorescently tagged, and blood was reconstituted at variable hematocrit prior to perfusion experiments. Integrin function of selected blood samples was inhibited by a blocking antibody, and platelet adhesion and aggregation over the course of perfusion was monitored. Increasing shear rates at physiological and elevated hematocrit levels facilitate robust platelet adhesion and formation of large aggregates. Shear-induced platelet aggregation is demonstrated to be dependent on both αIIbβIII function and the presence of red blood cells. Inhibition of αIIbβIII results in an 86.4% reduction in overall platelet adhesion and an 85.7% reduction in thrombus size at 20-60% hematocrit. Hematocrit levels of 20% are inadequate for effective platelet margination and subsequent vWF tethering, resulting in notable decreases in platelet adhesion at 5000 and 10,000 s-1 compared to 40% and 60%. Inhibition of αIIbβIII triggered dramatic reductions in overall thrombus coverage and large aggregate formation. Stability of platelets tethered by vWF are demonstrated to be αIIbβIII-dependent, as adhesion of single platelets treated with A2A9, an anti-αIIbβIII blocking antibody, is transient and did not lead to sustained thrombus formation. This study highlights driving factors in vWF-mediated platelet adhesion that are relevant to clinical suppression of shear-induced thrombosis and in vitro assays of platelet adhesion. Primarily, increasing hematocrit promotes platelet margination, permitting shear-induced platelet aggregation through αIIbβIII-mediated adhesion at supraphysiological shear rates. The online version contains supplementary material available at 10.1007/s12195-024-00796-0.

SARS-CoV-2 S protein activates NLRP3 inflammasome and deregulates coagulation factors in endothelial and immune cells

BackgroundHyperinflammation, hypercoagulation and endothelial injury are major findings in acute and post-COVID-19. The SARS-CoV-2 S protein has been detected as an isolated element in human tissues reservoirs and is the main product of mRNA COVID-19 vaccines. We investigated whether the S protein alone triggers pro-inflammatory and pro-coagulant responses in primary cultures of two cell types deeply affected by SARS-CoV-2, such are monocytes and endothelial cells.MethodsIn human umbilical vein endothelial cells (HUVEC) and monocytes, the components of NF-κB and the NLRP3 inflammasome system, as well as coagulation regulators, were assessed by qRT-PCR, Western blot, flow cytometry, or indirect immunofluorescence.ResultsS protein activated NF-κB, promoted pro-inflammatory cytokines release, and triggered the priming and activation of the NLRP3 inflammasome system resulting in mature IL-1β formation in both cell types. This was paralleled by enhanced production of coagulation factors such as von Willebrand factor (vWF), factor VIII or tissue factor, that was mediated, at least in part, by IL-1β. Additionally, S protein failed to enhance ADAMTS-13 levels to counteract the pro-coagulant activity of vWF multimers. Monocytes and HUVEC barely expressed angiotensin-converting enzyme-2. Pharmacological approaches and gene silencing showed that TLR4 receptors mediated the effects of S protein in monocytes, but not in HUVEC.ConclusionS protein behaves both as a pro-inflammatory and pro-coagulant stimulus in human monocytes and endothelial cells. Interfering with the receptors or signaling pathways evoked by the S protein may help preventing immune and vascular complications driven by such an isolated viral element.BJi2XdMkuptrjYtS_YhKHeVideo

Numerical study of ultra-large von Willebrand factor multimers in coagulopathy.

An excessive von Willebrand factor (VWF) secretion, coupled with a moderate to severe deficiency of ADAMTS13 activity, serves as a linking mechanism between inflammation to thrombosis. The former facilitates platelet adhesion to the vessel wall and the latter is required to cleave VWF multimers. As a result, the ultra-large VWF (UL-VWF) multimers released by Weibel-Palade bodies remain uncleaved. In this study, using a computational model based on first principles, we quantitatively show how the uncleaved UL-VWF multimers interact with the blood cells to initiate microthrombosis. We observed that platelets first adhere to unfolded and stretched uncleaved UL-VWF multimers anchored to the microvessel wall. By the end of this initial adhesion phase, the UL-VWF multimers and platelets make a mesh-like trap in which the red blood cells increasingly accumulate to initiate a gradually growing microthrombosis. Although high-shear rate and blood flow velocity are required to activate platelets and unfold the UL-VWFs, during the initial adhesion phase, the blood velocity drastically drops after thrombosis, and as a result, the wall shear stress is elevated near UL-VWF roots, and the pressure drops up to 6 times of the healthy condition. As the time passes, these trends progressively continue until the microthrombosis fully develops and the effective size of the microthrombosis and these flow quantities remain almost constant. Our findings quantitatively demonstrate the potential role of UL-VWF in coagulopathy.

First 24 Hour-Long Intensive Care Unit Testing of a Clinical-Scale Microfluidic Oxygenator in Swine: A Safety and Feasibility Study.

Microfluidic membrane oxygenators are designed to mimic branching vasculature of the native lung during extracorporeal lung support. To date, scaling of such devices to achieve clinically relevant blood flow and lung support has been a limitation. We evaluated a novel multilayer microfluidic blood oxygenator (BLOx) capable of supporting 750-800 ml/min blood flow versus a standard hollow fiber membrane oxygenator (HFMO) in vivo during veno-venous extracorporeal life support for 24 hours in anesthetized, mechanically ventilated uninjured swine (n = 3/group). The objective was to assess feasibility, safety, and biocompatibility. Circuits remained patent and operated with stable pressures throughout 24 hours. No group differences in vital signs or evidence of end-organ damage occurred. No change in plasma free hemoglobin and von Willebrand factor multimer size distribution were observed. Platelet count decreased in BLOx at 6 hours (37% dec, P = 0.03), but not in HFMO; however, thrombin generation potential was elevated in HFMO (596 ± 81 nM·min) versus BLOx (323 ± 39 nM·min) at 24 hours (P = 0.04). Other coagulation and inflammatory mediator results were unremarkable. BLOx required higher mechanical ventilator settings and showed lower gas transfer efficiency versus HFMO, but the stable device performance indicates that this technology is ready for further performance scaling and testing in lung injury models and during longer use conditions.

Thrombospondin-1 is an endogenous substrate of cereblon responsible for immunomodulatory drug–induced thromboembolism

AbstractImmunomodulatory drugs (IMiDs) are key drugs for treating multiple myeloma and myelodysplastic syndrome with chromosome 5q deletion. IMiDs exert their pleiotropic effects through the interaction between cell-specific substrates and cereblon, a substrate receptor of the E3 ubiquitin ligase complex. Thus, identification of cell-specific substrates is important for understanding the effects of IMiDs. IMiDs increase the risk of thromboembolism, which sometimes results in fatal clinical outcomes. In this study, we sought to clarify the molecular mechanisms underlying IMiDs-induced thrombosis. We investigated cereblon substrates in human megakaryocytes using liquid chromatography–mass spectrometry and found that thrombospondin-1 (THBS-1), which is an inhibitor of a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13, functions as an endogenous substrate in human megakaryocytes. IMiDs inhibited the proteasomal degradation of THBS-1 by impairing the recruitment of cereblon to THBS-1, leading to aberrant accumulation of THBS-1. We observed a significant increase in THBS-1 in peripheral blood mononuclear cells as well as larger von Willebrand factor multimers in the plasma of patients with myeloma, who were treated with IMiDs. These results collectively suggest that THBS-1 represents an endogenous substrate of cereblon. This pairing is disrupted by IMiDs, and the aberrant accumulation of THBS-1 plays an important role in the pathogenesis of IMiDs-induced thromboembolism.

Von Willebrand factor Ristocetin co-factor activity to von Willebrand factor antigen level ratio for diagnosis of acquired von Willebrand syndrome caused by aortic stenosis

BackgroundSevere aortic stenosis (AS) causes acquired von Willebrand syndrome by the excessive shear stress–dependent cleavage of high molecular weight multimers of von Willebrand factor (VWF). While the current standard diagnostic method is so-called VWF multimer analysis that is western blotting under nonreducing conditions, it remains unclear whether a ratio of VWF Ristocetin co-factor activity (VWF:RCo) to VWF antigen levels (VWF:Ag) of <0.7, which can be measured with an automated coagulation analyzer in clinical laboratories and is used for the diagnosis of hereditary von Willebrand disease. ObjectivesTo evaluated whether the VWF:RCo/VWF:Ag is useful for the diagnosis of AS-induced acquired von Willebrand syndrome. MethodsVWF:RCo and VWF:Ag were evaluated with the VWF large multimer index as a reference, which represents the percentage of a patient’s VWF high molecular weight multimer ratio to that of standard plasma in the VWF multimer analysis. ResultsWe analyzed 382 patients with AS having transaortic valve maximal pressure gradients of >30 mmHg, 27 patients with peripheral artery disease, and 46 control patients free of cardiovascular disease with osteoarthritis, diabetes, and so on. We assumed a large multimer index of <80% as loss of VWF large multimers since 59.0% of patients with severe AS had the indices of <80%, while no control patients or patients with peripheral artery disease, except for 2 patients, exhibited the indices of <80%. The VWF:RCo/VWF:Ag ratios, measured using an automated blood coagulation analyzer, were correlated with the indices (rs = 0.470, P < .001). When the ratio of <0.7 was used as a cut-off point, the sensitivity and specificity to VWF large multimer indices of <80% were 0.437 and 0.826, respectively. ConclusionVWF:RCo/VWF:Ag ratios of <0.7 may indicate loss of VWF large multimers with high specificity, but low sensitivity. VWF:RCo/VWF:Ag ratios in patients with AS having a ratio of <0.7 may be useful for monitoring the loss of VWF large multimers during their clinical courses.

A Mutation in Complement Factor H (W1183R) Enhances VWF Proteolysis By ADAMTS13 Under Shear

Introduction: Complement factor H (CFH) is the primary negative regulator of complement activation in the alternative pathway in both fluidic phase and on cell surface. Loss of function of CFH such as W1183R is associated with atypical hemolytic uremic syndrome (aHUS). Homozygous mutation W1206R in the murine CFH, corresponding to W1183R in human CFH, results in acute aHUS. Interestingly, in these mice, while plasma VWF antigen levels are significantly increased, plasma VWF multimer size is markedly reduced in ADAMTS13-depedent manner. However, the molecular mechanism underlying such a dramatic effect of the mutant CFH (W1206R) on VWF multimer distribution is not known. The goal of this study is to determine how wild type CFH and mutant CFH W1183R affects VWF proteolysis under shear, and the mechanism of action. Methods: Recombinant WT CFH, CFH W1183R, ADAMTS13, and VWF fragments were expressed in mammalian cells and purified to homogeneity. The effects of WT CFH or CFH W1183R on proteolytic cleavage of VWF73 peptide and full-length multimeric VWF were determined. Additionally, the binding and unfolding events were determined by micro scale thermophoresis (MST) and optical tweezer, respectively. Results : WT CFH showed no effect on the cleavage of peptide VWF73 substrate, but CFH W1183R enhanced the proteolytic cleavage of VWF73 by ADAMTS13 in a dose-dependent manner ( Fig. 1). Counterintuitively, WT CFH appeared to inhibit the proteolytic cleavage of multimeric VWF by ADAMTS13 under arterial shear, but CFH W1183R exhibited an opposite effect, modestly enhancing the proteolytic cleavage of multimeric VWF by ADAMTS13 under the same conditions. The MST binding assay demonstrated that both WT CFH and CFH W1183R bind to a short A1, a long AIM-A1, and A1-A2-A3 fragments with a dissociation constant ( K D) of 6.4 x 10 -7, 3.0 x 10 -9 M, and 3.3 x10 -9 mol/L, respectively. These results suggest that CFH may bind to the AIM, A1-A2 junction, and A2 domain. To further assess the impact of CFH binding to A2, we performed optical tweezer experiments. The results showed that both WT CFH (Lc: from 27.4 nm at 12 nM to 40 nm at 1.0 mM) and CFH W1183R (Lc: from 26 nm at 12 nM to 31.8 nm at 1.0 mM) resulted in significantly extended contour length (Lc) of VWF-A2 domain in a concentration-dependent manner ( Fig. 2), suggesting a dramatic conformational change in the A2 domain upon binding by WT CFH or CFH W1183R. Conclusions : Our results demonstrate that WT CFH at physiological concentrations may exhibit a modest inhibitory activity towards cleavage of VWF by ADAMTS13 under shear, but such an inhibitory activity disappears as the result of a point mutation in the C-terminal end of CFH (e.g., W1183R). Instead, the mutant CFH W1183R appears to enhance the VWF cleavage by ADAMTS13, likely through direct binding to the AIM, A1-A2 junction, and A2 domain of VWF, resulting in conformational changes in the central A2 domain.

Proof-of-Concept in a Murine Model of Treatment of Thrombotic Thrombocytopenic Purpura Using Engineered Red Blood Cells Expressing Fragments of ADAMTS13 on Their Membrane

Thrombotic Thrombocytopenic Purpura (TTP) is due to congenital or acquired deficiency in A Disintegrin and Metalloproteinase with a Thrombospondin Type 1 Motif, Member 13 (ADAMTS13), a metalloproteinase responsible for the cleavage of the pro-coagulant glycoprotein von Willebrand Factor (vWF). Lack of ADAMTS13 activity causes induction of systemic platelet aggregation and microthrombi in small blood vessels by vWF multimers. Current treatment of TTP consists of daily plasma exchange and immune suppression but is costly and associated with high morbidity. A recombinant ADAMTS13 treatment for the congenital form is in advanced clinical trials but alternative treatment methodologies remain needed. We have developed methods to generate genetically engineered cultured red blood cells (cRBCs) from iPSCs which can be used as drug delivery vectors. We hypothesize that generating cRBCs with membrane-bound variants of ADAMTS13 could be a potential method of treatment for TTP. We have inserted at AAVS1, in human erythroid cells, constructs that contain globin-LCR driven MDTCS ADAMTS13 fusion transgenes with or without gain-of function mutations that confer resistance to ADAMTS13 auto-antibodies. We have shown using flow cytometry and a FRET assay that these erythroid cells express these fusion proteins at high levels and that membrane bound ADAMTS13 is enzymatically active. We have also shown that, as expected, the enzymatic activity of erythroid cells carrying the MDTCS fragment of ADAMTS13 with gain-of-function mutations is resistant to inhibition by plasma from TTP patients, while the wildtype version of the same fragment is not. To obtain an in vivo proof-of-concept that such erythroid cells can prevent TTP, we have generated rosa26 knock-ins and transgenic mice expressing the wildtype or gain-of-function variants of MDTCS ADAMTS13 and observed that RBCs from these animals have normal half-lives, and, as shown via FACS and FRET assays, express significant amounts of catalytically active ADAMTS13 on their RBC membranes. ADAMTS13 knock-out mice inoculated with large amounts of recombinant human vWF (rhvWF) have been shown to exhibit typical TTP symptoms including platelet drops, schistocytes formation and cardiac abnormalities providing a useful murine model for TTP. To determine if ADAMTS13 expressing RBCs could be used to prevent TTP symptoms, we transfused ADAMTS13 knock-out mice with either wild-type or ADAMTS13-RBCs up to 24 hours prior to inoculation with rhvWF. Mice transfused with ADAMTS13-RBCs displayed a significant reduction of ADAMTS13 symptoms including drastically reduced platelet drops, lack of schistocytes formation and absent cardiac abnormalities. We conclude that the membrane-bound MDTCS fragment of ADAMTS13 is capable of cleaving the full-length rhvWF in the presence of flow, in vivo. The above data provide a proof-of-concept that RBCs expressing membrane-bound ADAMTS13 fusion proteins can be used as a therapeutic for congenital and acquired TTP. Experiments are in progress to determine in vivo, the TTP antibody resistance of these cells in the presence of anti-ADAMTS13 auto-antibodies from TTP patients.

Caplacizumab Frontline Added to Therapeutic Plasma Exchange and Immunosuppression Prevents Unfavorable Outcomes in Immune-Mediated TTP: An International Real-World Study of the TTP-IWG (The Capla 500 Project)

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) results from autoantibody-mediated severe deficiency of ADAMTS13, the Von Willebrand factor (VWF)-cleaving protease. In this context, ultra-large VWF multimers accumulate in the circulation, leading to increased platelet clumping, with subsequent severe thrombocytopenia, microangiopathic hemolytic anemia and multiorgan failure. The anti-VWF nanobody caplacizumab is licensed for adults with iTTP. Prospective controlled trials and national real-world studies have provided evidence that caplacizumab improved outcome of the acute phase of the disease. However, whether caplacizumab decreases mortality, and the optimal timing of caplacizumab initiation, remain to be determined. To address these questions, an international survey, the Capla 500+ project, has been conducted. An academic call for observations involving experienced teams from 10 worldwide countries recruited 942 adult patients treated with a regimen of daily therapeutic plasma exchange (TPE), caplacizumab and immunosuppression with corticosteroids ± rituximab (caplacizumab group). Cases were compared to historic controls (control group) randomly selected from 2015 to 2018 by 4 teams in a 2:1 ratio (N=495), treated with TPE and immunosuppression. Primary outcome was 3-month survival post-first TPE. Key secondary outcomes were refractoriness and exacerbations, time to clinical response, number of TPE to achieve clinical response, time to ADAMTS13 activity recovery ≥20%, and caplacizumab-related adverse events. Clinical presentation and severity were comparable between groups ( Table). All patients received TPE in both caplacizumab and control groups, in association with corticosteroids (99% and 93%, respectively), and rituximab (92% and 71%, respectively) frontline or as salvage therapy. Caplacizumab was started within 3 days (715 patients), or delayed by≥4 days from first TPE (218 patients); for those with delayed initiation, caplacizumab was added for management of exacerbation or refractoriness (63 cases) or a slow improvement to TPE/immunosuppression (155 cases). The 3-month survival post-first TPE in the caplacizumab group was 98.6% vs 93.3% in the control group (p&amp;lt;0.0001). Failure to achieve clinical response was 5-times less likely in the caplacizumab group as compared to the control group (95% confidence interval, 0.1-0.37, p&amp;lt;0.0001). Deaths in the caplacizumab group occurred after a median of 4.5 days (interquartile range [IQR], 3-5), mostly due to uncontrolled iTTP. Patients receiving caplacizumab experienced less refractoriness and exacerbations, and required less TPE to achieve clinical remission than controls (p&amp;lt;0.0001 for all), regardless of the use of rituximab. Forty-six patients experienced an exacerbation following caplacizumab interruption while ADAMTS13 activity was still undetectable. Time to ADAMTS13 activity ≥20% was shorter in the caplacizumab group (p=0.01) ( Table), where rituximab was more systematically used. In patients with delayed caplacizumab initiation, clinical response was obtained after a median of 4 days (IQR, 2-8) following caplacizumab introduction. This result was comparable to the time to clinical response obtained in patients who initiated caplacizumab within 3 days of the first TPE (median, 4 days [IQR, 3-6], p=0.8). In patients with early caplacizumab initiation, unfavorable outcomes were particularly infrequent (death, 1%; refractoriness, 1.1%; exacerbations, 5.3%),andtime to clinical response in survivors was half the time compared to control group survivors (p&amp;lt;0.0001) ( Figure). Caplacizumab-related adverse events occurred in 220 patients (23%). They consisted of major bleeding (N=19, 2.2%), including: digestive tract bleeding (N=8), intracranial hemorrhage, severe central venous catheter insertion bleeding, metrorrhagia (N=2 cases each). Clinically relevant non-major bleeding (N=34, 3.7%), non-clinically relevant non-major bleeding (N=114, 14%), and inflammatory reaction at the injection site (N=35, 4.5%) were also reported. This unprecedented, international academic effort on such a rare disease provides convincing evidence that caplacizumab added to TPE and immunosuppression prevents unfavorable outcomes during the acute phase of iTTP, including mortality, and alleviates the burden of care at the potential expense of rare, major bleeding events.