A local drug delivery system prolongs graft survival by dampening T cell infiltration and neutrophil extracellular trap formation in vascularized composite allografts.

Introduction: The standard treatment for preventing rejection in vascularized composite allotransplantation (VCA) currently relies on systemic immunosuppression, which exposes the host to well-known side effects. Locally administered immunosuppression strategies have shown promising results to bypass this hurdle. Nevertheless, their progress has been slow, partially attributed to a limited understanding of the essential mechanisms underlying graft rejection. Recent discoveries highlight the crucial involvement of innate immune components, such as neutrophil extracellular traps (NETs), in organ transplantation. Here we aimed to prolong graft survival through a tacrolimus-based drug delivery system and to understand the role of NETs in VCA graft rejection. Methods: To prevent off-target toxicity and promote graft survival, we tested a locally administered tacrolimus-loaded on-demand drug delivery system (TGMS-TAC) in a multiple MHC-mismatched porcine VCA model. Off-target toxicity was assessed in tissue and blood. Graft rejection was evaluated macroscopically while the complement system, T cells, neutrophils and NETs were analyzed in graft tissues by immunofluorescence and/or western blot. Plasmatic levels of inflammatory cytokines were measured using a Luminex magnetic-bead porcine panel, and NETs were measured in plasma and tissue using DNA-MPO ELISA. Lastly, to evaluate the effect of tacrolimus on NET formation, NETs were induced in-vitro in porcine and human peripheral neutrophils following incubation with tacrolimus. Results: Repeated intra-graft administrations of TGMS-TAC minimized systemic toxicity and prolonged graft survival. Nevertheless, signs of rejection were observed at endpoint. Systemically, there were no increases in cytokine levels, complement anaphylatoxins, T-cell subpopulations, or neutrophils during rejection. Yet, tissue analysis showed local infiltration of T cells and neutrophils, together with neutrophil extracellular traps (NETs) in rejected grafts. Interestingly, intra-graft administration of tacrolimus contributed to a reduction in both T-cellular infiltration and NETs. In fact, in-vitro NETosis assessment showed a 62-84% reduction in NETs after stimulated neutrophils were treated with tacrolimus. Conclusion: Our data indicate that the proposed local delivery of immunosuppression avoids off-target toxicity while prolonging graft survival in a multiple MHC-mismatch VCA model. Furthermore, NETs are found to play a role in graft rejection and could therefore be a potential innovative therapeutic target.

Elevated levels of citrullinated fibrinogen in patients with cancer.

Neutrophil released peptidyl arginine deiminase 4 (PAD4) converts arginine residues on plasma proteins into citrulline. Here, we developed an assay to quantify citrullinated fibrinogen. We employed a biotin-conjugated phenylglyoxal (biotin-phenylglyoxal (PG)) compound that selectively labels citrulline. Patient samples were derived from a multicenter prospective cohort study that aimed to identify cancer patients at high risk for venous thromboembolism (VTE). Our data show that cancer patients have higher (median 2-fold increased) citrullinated fibrinogen levels when compared to normal human plasma and a cohort of healthy donors. Our results show that citrullination of fibrinogen is a common posttranslational modification in patients with cancer.

Glycocalyx dynamics and the inflammatory response of genetically modified porcine endothelial cells.

Xenotransplantation is a promising approach to reduce organ shortage, while genetic modification of donor pigs has significantly decreased the immunogenic burden of xenotransplants, organ rejection is still a hurdle. Genetically modified pig organs are used in xenotransplantation research, and the first clinical pig-to-human heart transplantation was performed in 2022. However, the impact of genetic modification has not been investigated on a cellular level yet. Endothelial cells (EC) and their sugar-rich surface known as the glycocalyx are the first barrier encountering the recipient's immune system, making them a target for rejection. We have previously shown that wild type venous but not arterial EC were protected against heparan sulfate (HS) shedding after activation with human serum or human tumor necrosis factor alpha (TNF𝛼). Using a 2D microfluidic system we investigated the glycocalyx dynamics of genetically modified porcine arterial and venous EC (Gal𝛼1,3 Gal knock-out, transgenic for human CD46 and thrombomodulin, GTKO/hCD46/hTM) after activation with human serum or human TNF𝛼. Interestingly, we observed that GTKO/hCD46/hTM arterial cells, additionally to venous cells, do not shed HS. Unscathed HS on GTKO/hCD46/hTM EC correlated with reduced complement deposition, suggesting that protection against complement activation contributes to maintaining an intact glycocalyx layer on arterial EC. This protection was lost on GTKO/hCD46/hTM cells after simultaneous perfusion with human serum and human TNF𝛼. HS shedding on arterial cells and increased complement deposition on both arterial and venous cells was observed. These findings suggest that GTKO/hCD46/hTM EC revert to a proinflammatory phenotype in an inflammatory xenotransplantation setting, potentially favoring transplant rejection.

Inflammatory stimuli induce shedding of heparan sulfate from arterial but not venous porcine endothelial cells leading to differential proinflammatory and procoagulant responses.

Endothelial dysfunction is an early event of vascular injury defined by a proinflammatory and procoagulant endothelial cell (EC) phenotype. Although endothelial glycocalyx disruption is associated with vascular damage, how various inflammatory stimuli affect the glycocalyx and whether arterial and venous cells respond differently is unknown. Using a 3D round-channel microfluidic system we investigated the endothelial glycocalyx, particularly heparan sulfate (HS), on porcine arterial and venous ECs. Heparan sulfate (HS)/glycocalyx expression was observed already under static conditions on venous ECs while it was flow-dependent on arterial cells. Furthermore, analysis of HS/glycocalyx response after stimulation with inflammatory cues revealed that venous, but not arterial ECs, are resistant to HS shedding. This finding was observed also on isolated porcine vessels. Persistence of HS on venous ECs prevented complement deposition and clot formation after stimulation with tumor necrosis factor α or lipopolysaccharide, whereas after xenogeneic activation no glycocalyx-mediated protection was observed. Contrarily, HS shedding on arterial cells, even without an inflammatory insult, was sufficient to induce a proinflammatory and procoagulant phenotype. Our data indicate that the dimorphic response of arterial and venous ECs is partially due to distinct HS/glycocalyx dynamics suggesting that arterial and venous thrombo-inflammatory disorders require targeted therapies.

The Endothelial Glycocalyx: A Possible Therapeutic Target in Cardiovascular Disorders.

The physiological, anti-inflammatory, and anti-coagulant properties of endothelial cells (ECs) rely on a complex carbohydrate-rich layer covering the luminal surface of ECs, called the glycocalyx. In a range of cardiovascular disorders, glycocalyx shedding causes endothelial dysfunction and inflammation, underscoring the importance of glycocalyx preservation to avoid disease initiation and progression. In this review we discuss the physiological functions of the glycocalyx with particular focus on how loss of endothelial glycocalyx integrity is linked to cardiovascular risk factors, like hypertension, aging, diabetes and obesity, and contributes to the development of thrombo-inflammatory conditions. Finally, we consider the role of glycocalyx components in regulating inflammatory responses and discuss possible therapeutic interventions aiming at preserving or restoring the endothelial glycocalyx and therefore protecting against cardiovascular disease.

The role of SERPIN citrullination in thrombosis.

Aberrant protein citrullination is associated with many pathologies; however, the specific effects of this modification remain unknown. We have previously demonstrated that serine protease inhibitors (SERPINs) are highly citrullinated in rheumatoid arthritis (RA) patients. These citrullinated SERPINs include antithrombin, antiplasmin, and t-PAI, which regulate the coagulation and fibrinolysis cascades. Notably, citrullination eliminates their inhibitory activity. Here, we demonstrate that citrullination of antithrombin and t-PAI impairs their binding to their cognate proteases. By contrast, citrullination converts antiplasmin into a substrate. We recapitulate the effects of SERPIN citrullination using in vitro plasma clotting and fibrinolysis assays. Moreover, we show that citrullinated antithrombin and antiplasmin are increased and decreased in a deep vein thrombosis (DVT) model, accounting for how SERPIN citrullination shifts the equilibrium toward thrombus formation. These data provide a direct link between increased citrullination and the risk of thrombosis in autoimmunity and indicate that aberrant SERPIN citrullination promotes pathological thrombus formation.

NLRP3 Inflammasome Assembly in Neutrophils Is Supported by PAD4 and Promotes NETosis Under Sterile Conditions.

Neutrophil extracellular trap formation (NETosis) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome assembly are associated with a similar spectrum of human disorders. While NETosis is known to be regulated by peptidylarginine deiminase 4 (PAD4), the role of the NLRP3 inflammasome in NETosis was not addressed. Here, we establish that under sterile conditions the cannonical NLRP3 inflammasome participates in NETosis. We show apoptosis-associated speck-like protein containing a CARD (ASC) speck assembly and caspase-1 cleavage in stimulated mouse neutrophils without LPS priming. PAD4 was needed for optimal NLRP3 inflammasome assembly by regulating NLRP3 and ASC protein levels post-transcriptionally. Genetic ablation of NLRP3 signaling resulted in impaired NET formation, because NLRP3 supported both nuclear envelope and plasma membrane rupture. Pharmacological inhibition of NLRP3 in either mouse or human neutrophils also diminished NETosis. Finally, NLRP3 deficiency resulted in a lower density of NETs in thrombi produced by a stenosis-induced mouse model of deep vein thrombosis. Altogether, our results indicate a PAD4-dependent formation of the NLRP3 inflammasome in neutrophils and implicate NLRP3 in NETosis under noninfectious conditions in vitro and in vivo.

Impact of porcine cytomegalovirus on long-term orthotopic cardiac xenotransplant survival.

Xenotransplantation using pig organs has achieved survival times up to 195 days in pig orthotopic heart transplantation into baboons. Here we demonstrate that in addition to an improved immunosuppressive regimen, non-ischaemic preservation with continuous perfusion and control of post-transplantation growth of the transplant, prevention of transmission of the porcine cytomegalovirus (PCMV) plays an important role in achieving long survival times. For the first time we demonstrate that PCMV transmission in orthotopic pig heart xenotransplantation was associated with a reduced survival time of the transplant and increased levels of IL-6 and TNFα were found in the transplanted baboon. Furthermore, high levels of tPA-PAI-1 complexes were found, suggesting a complete loss of the pro-fibrinolytic properties of the endothelial cells. These data show that PCMV has an important impact on transplant survival and call for elimination of PCMV from donor pigs.

Extracellular DNA NET-Works With Dire Consequences for Health.

Neutrophils play a central role in innate immune defense. Advances in neutrophil biology have brought to light the capacity of neutrophils to release their decondensed chromatin and form large extracellular DNA networks called neutrophil extracellular traps (NETs). NETs are produced in response to many infectious and noninfectious stimuli and, together with fibrin, block the invasion of pathogens. However, their formation in inflamed blood vessels produces a scaffold that supports thrombosis, generates neo-antigens favoring autoimmunity, and aggravates damage in ischemia/reperfusion injury. NET formation can also be induced by cancer and promotes tumor progression. Formation of NETs within organs can be immediately detrimental, such as in lung alveoli, where they affect respiration, or they can be harmful over longer periods of time. For example, NETs initiate excessive deposition of collagen, resulting in fibrosis, thus likely contributing to heart failure. Here, we summarize the latest knowledge on NET generation and discuss how excessive NET formation mediates propagation of thrombosis and inflammation and, thereby, contributes to various diseases. There are many ways in which NET formation could be averted or NETs neutralized to prevent their detrimental consequences, and we will provide an overview of these possibilities.

Plasma Peptidylarginine Deiminase IV Promotes VWF-Platelet String Formation and Accelerates Thrombosis After Vessel Injury.

RATIONALE: PAD4 (peptidylarginine deiminase type IV), an enzyme essential for neutrophil extracellular trap formation (NETosis), is released together with neutrophil extracellular traps into the extracellular milieu. It citrullinates histones and holds the potential to citrullinate other protein targets. While NETosis is implicated in thrombosis, the impact of the released PAD4 is unknown. OBJECTIVE: This study tests the hypothesis that extracellular PAD4, released during inflammatory responses, citrullinates plasma proteins, thus affecting thrombus formation. METHODS AND RESULTS: Here, we show that injection of r-huPAD4 in vivo induces the formation of VWF (von Willebrand factor)-platelet strings in mesenteric venules and that this is dependent on PAD4 enzymatic activity. VWF-platelet strings are naturally cleaved by ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type-1 motif-13). We detected a reduction of endogenous ADAMTS13 activity in the plasma of wild-type mice injected with r-huPAD4. Using mass spectrometry and in vitro studies, we found that r-huPAD4 citrullinates ADAMTS13 on specific arginine residues and that this modification dramatically inhibits ADAMTS13 enzymatic activity. Elevated citrullination of ADAMTS13 was observed in plasma samples of patients with sepsis or noninfected patients who were elderly (eg, age >65 years) and had underlying comorbidities (eg, diabetes mellitus and hypertension) as compared with healthy donors. This shows that ADAMTS13 is citrullinated in vivo. VWF-platelet strings that form on venules of Adamts13-/- mice were immediately cleared after injection of r-huADAMTS13, while they persisted in vessels of mice injected with citrullinated r-huADAMTS13. Next, we assessed the effect of extracellular PAD4 on platelet-plug formation after ferric chloride-induced injury of mesenteric venules. Administration of r-huPAD4 decreased time to vessel occlusion and significantly reduced thrombus embolization. CONCLUSIONS: Our data indicate that PAD4 in circulation reduces VWF-platelet string clearance and accelerates the formation of a stable platelet plug after vessel injury. We propose that this effect is, at least in part, due to ADAMTS13 inhibition.

Recombinant Human ADAMTS13 Treatment Improves Myocardial Remodeling and Functionality After Pressure Overload Injury in Mice.

BACKGROUND: A disintegrin-like metalloproteinase with thrombospondin motif type 1 member 13 (ADAMTS13), the von Willebrand factor-cleaving enzyme, decreases leukocyte and platelet recruitment and, thus, reduces thrombosis and inflammation. Recombinant human ADAMTS13 (rhADAMTS13) is a novel drug candidate for ischemia/reperfusion injury and has shown short-term benefits in mouse models of myocardial injury, but long-term outcome has not been investigated. METHODS AND RESULTS: We evaluated the impact of rhADAMTS13 on cardiac remodeling, scarring, and contractile function, under chronic left ventricular pressure overload. The role of von Willebrand factor and the effect of rhADAMTS13 treatment were studied. This model of heart failure, based on ascending aortic constriction, produces a coronary inflammatory response and microvascular dysfunction, resulting in fibrotic remodeling and cardiac failure. Mice were treated with either rhADAMTS13 or vehicle and assessed for coronary vascular inflammation and ventricular function at several postsurgical time points, as well as for cardiac fibrosis after 4 weeks. Early upon induction of pressure overload under rhADAMTS13 treatment, we detected less endothelial-lumen-associated von Willebrand factor, fewer platelet aggregates, and decreased activated transforming growth factor-ß1 levels than in vehicle-treated mice. We observed significant preservation of cardiac function and decrease in fibrotic remodeling as a result of rhADAMTS13 administration. CONCLUSIONS: Herein, we show that rhADAMTS13 decreases coronary vascular dysfunction and improves cardiac remodeling after left ventricular pressure overload in mice. We propose that this effect may, at least in part, be the result of decreased von Willebrand factor-mediated recruitment of platelets, a major source of the activated profibrotic cytokine transforming growth factor-ß1. Our study further supports the therapeutic potential of rhADAMTS13 for conditions characterized by inflammatory cardiac damage that results in fibrosis.

Analysis of the HLA-DR peptidome from human dendritic cells reveals high affinity repertoires and nonconventional pathways of peptide generation.

Dendritic cells (DCs) are the major professional APCs of the immune system; however, their MHC-II-associated peptide repertoires have been hard to analyze, mostly because of their scarce presence in blood and tissues. In vitro matured human monocyte-derived DCs (MoDCs) are widely used as professional APCs in experimental systems. In this work, we have applied mass spectrometry to identify the HLA-DR-associated self-peptide repertoires from small numbers of mature MoDCs (∼5 × 106 cells), derived from 7 different donors. Repertoires of 9 different HLA-DR alleles were defined from analysis of 1319 peptides, showing the expected characteristics of MHC-II-associated peptides. Most peptides identified were predicted high binders for their respective allele, formed nested sets, and belonged to endo-lysosomal pathway-degraded proteins. Approximately 20% of the peptides were derived from cytosolic and nuclear proteins, a recurrent finding in HLA-DR peptide repertoires. Of interest, most of these peptides corresponded to single sequences, did not form nested sets, and were located at the C terminus of the parental protein, which suggested alternative processing. Analysis of cleavage patterns for terminal peptides predominantly showed aspartic acid before the cleavage site of both C- and N-terminal peptides and proline immediately after the cleavage site in C-terminal peptides. Proline was also frequent next to the cut sites of internal peptides. These data provide new insights into the Ag processing capabilities of DCs. The relevance of these processing pathways and their contribution to response to infection, tolerance induction, or autoimmunity deserve further analysis.

The class I scavenger receptor CD163 promotes internalization of ADAMTS13 by macrophages.

Internalization of ADAMTS13 by macrophages may contribute to its clearance from the circulation. Here we investigated endocytic mechanisms that contribute to the uptake of ADAMTS13 by macrophages. Human monocyte-derived macrophages were used to monitor the uptake of fluorescently labeled recombinant ADAMTS13 by flow cytometry. Internalization of ADAMTS13 was blocked upon addition of the cell-permeable dynamin inhibitor dynasore. Partial blocking of ADAMTS13 uptake was observed by using mannan; however, uptake was not affected by an antibody that blocked binding to the macrophage mannose receptor CD206, which suggests that other endocytic receptors contribute to the internalization of ADAMTS13 by macrophages. A pull-down with ADAMTS13 and subsequent mass spectrometric analysis identified the class I scavenger receptor CD163 as a candidate receptor for ADAMTS13. Blocking experiments with monoclonal anti-CD163 antibody EDHu-1 resulted in decreased ADAMTS13 internalization by macrophages. Pronounced inhibition of ADAMTS13 uptake by EDHu-1 was observed in CD163 high-expressing macrophages. In agreement with these findings, CD163-expressing Chinese hamster ovary cells were capable of rapidly internalizing ADAMTS13. Surface plasmon resonance revealed binding of ADAMTS13 to scavenger receptor cysteine-rich domains 1-9 and 1-5 of CD163. Taken together, our data identify CD163 as a major endocytic receptor for ADAMTS13 on macrophages.

CD4+ T cells from patients with acquired thrombotic thrombocytopenic purpura recognize CUB2 domain-derived peptides.

Acquired thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder resulting from the development of autoantibodies against ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). HLA-DRB1*11 provides a risk factor for developing acquired TTP. Pulsing of antigen-presenting cells from HLA-DRB1*11- and HLA-DRB1*03-positive individuals with ADAMTS13 resulted in presentation of peptides derived from the CUB2 domain of ADAMTS13 with core sequences FINVAPHAR or ASYILIRD. Here, we assessed whether FINVAPHAR- or ASYILIRD-reactive CD4(+)T cells are present in peripheral blood mononuclear cells from HLA-DRB1*11 and HLA-DRB1*03-positive subjects with acquired TTP. The presence of ADAMTS13-reactive CD4(+)T cells was addressed by flow cytometry and the expression of activation marker CD40 ligand by CD4(+)T cells. FINVAPHAR-reactive CD4(+)T cells were identified in an HLA-DRB1*11-positive patient during the acute phase of the disease whereas ASYILIRD-positive CD4(+)T cells were identified in a DRB1*03-positive patient with acquired TTP. Frequencies of CUB2 domain-reactive CD4(+)T cells ranged from 3.3% to 4.5%. Control peptides in which the anchor residues were modified did not induce activation of CD4(+)T cells. Taken together, our data provide evidence for the involvement of CUB2 domain-reactive CD4(+)T cells in the etiology of acquired TTP.

von Willebrand factor binds to the surface of dendritic cells and modulates peptide presentation of factor VIII.

It has been proposed that von Willebrand factor might affect factor VIII immunogenicity by reducing factor VIII uptake by antigen presenting cells. Here we investigate the interaction of recombinant von Willebrand factor with immature monocyte-derived dendritic cells using flow cytometry and confocal microscopy. Surprisingly, von Willebrand factor was not internalized by immature dendritic cells, but remained bound to the cell surface. As von Willebrand factor reduces the uptake of factor VIII, we investigated the repertoire of factor VIII presented peptides when in complex with von Willebrand factor. Interestingly, factor VIII-derived peptides were still abundantly presented on major histocompatibility complex class II molecules, even though a reduction of factor VIII uptake by immature dendritic cells was observed. Inspection of peptide profiles from 5 different donors showed that different core factor VIII peptide sequences were presented upon incubation with factor VIII/von Willebrand factor complex when compared to factor VIII alone. No von Willebrand factor peptides were detected when immature dendritic cells were pulsed with different concentrations of von Willebrand factor, confirming lack of von Willebrand factor endocytosis. Several von Willebrand factor derived peptides were recovered when cells were pulsed with von Willebrand factor/factor VIII complex, suggesting that factor VIII promotes endocytosis of small amounts of von Willebrand factor by immature dendritic cells. Taken together, our results establish that von Willebrand factor is poorly internalized by immature dendritic cells. We also show that von Willebrand factor modulates the internalization and presentation of factor VIII-derived peptides on major histocompatibility complex class II.

Acquired TTP: ADAMTS13 meets the immune system.

The majority of the patients affected by acquired thrombotic thrombocytopenic purpura (TTP) develop autoantibodies directed towards ADAMTS13 that interfere with its von Willebrand Factor (VWF) processing activity. B cell responses have been shown to primarily target the spacer domain of ADAMTS13 thereby prohibiting the binding of ADAMTS13 to the VWF A2 domain. In this review we summarize recent knowledge gained on the immune recognition and processing of ADAMTS13 by antigen-presenting cells (APCs). HLA-DRB1*11 has been identified as a risk factor for acquired TTP. Analysis of MHC class II/peptide complexes of ADAMTS13 pulsed dendritic cells have shown that the CUB2 domain derived peptide FINVAPHAR is preferentially presented on HLA-DRB1*11. Based on these findings we propose a model for the initiation of the autoimmune reactivity against ADAMTS13 in previously healthy individuals. We hypothesize that mimicry between a pathogen-derived peptide and the CUB2 derived FINVAPHAR-peptide might contribute to the onset of acquired TTP.

Preferential HLA-DRB1*11-dependent presentation of CUB2-derived peptides by ADAMTS13-pulsed dendritic cells.

Autoantibodies directed against ADAMTS13 prohibit the processing of von Willebrand factor multimers, initiating a rare and life-threatening disorder called acquired thrombotic thrombocytopenic purpura (TTP). Recently, HLA-DRB1*11 has been identified as a risk factor for the development of acquired TTP. Here, we identified ADAMTS13-derived peptides presented on MHC class II alleles from 17 healthy donors. Dendritic cells from a panel of both HLA-DRB1*11-positive and -negative donors were pulsed with ADAMTS13, and the HLA-DR-presented peptide repertoire was analyzed by mass spectrometry. Interestingly, at low antigen concentrations, HLA-DRB1*11- or DRB1*03-positive donors presented a limited number of CUB2-derived peptides. Pulsing of dendritic cells using higher concentrations of ADAMTS13 resulted in the presentation of larger numbers of ADAMTS13-derived peptides by both HLA-DRB1*11-positive and -negative donors. Although the presented peptides were derived from several ADAMTS13 domains, inspection of the peptide profiles revealed that CUB2 domain-derived peptides were presented with a higher efficiency when compared with other peptides. Remarkably, dendritic cells from DRB1*11 donors pulsed with higher concentrations of ADAMTS13-present derivatives of a single CUB2-derived peptide. We hypothesize that functional presentation of CUB2-derived peptides on HLA-DRB1*11 contributes to the onset of acquired TTP by stimulating low-affinity, self-reactive CD4+ T cells.

The macrophage mannose receptor promotes uptake of ADAMTS13 by dendritic cells.

ADAMTS13 is a plasma metalloproteinase that regulates platelet adhesion and aggregation by cleaving ultra-large VWF multimers on the surfaces of endothelial cells. Autoantibodies directed against ADAMTS13 prohibit the processing of VWF multimers, initiating a rare and life-threatening disorder called acquired thrombotic thrombocytopenic purpura. The formation of autoantibodies depends on the activation of CD4(+) T cells. This process requires immune recognition, endocytosis, and subsequent processing of ADAMTS13 into peptides that are presented on MHC class II molecules to CD4(+) T cells by dendritic cells (DCs). In the present study, we investigated endocytosis of recombinant ADAMTS13 by immature monocyte-derived DCs using flow cytometry and confocal microscopy. After incubation of fluorescently labeled ADAMTS13 with DCs, significant uptake of ADAMTS13 was observed. Endocytosis of ADAMTS13 was completely blocked by the addition of EGTA and mannan. ADAMTS13 endocytosis was decreased in the presence of a blocking mAb directed toward the macrophage mannose receptor (MR). Furthermore, siRNA silencing of MR reduced the uptake of ADAMTS13 by DCs. In addition, in vitro binding studies confirmed the interaction of ADAMTS13 with the carbohydrate recognition domains of MR. The results of the present study indicate that sugar moieties on ADAMTS13 interact with MR, thereby promoting its endocytosis by APCs.

Essential domains of a disintegrin and metalloprotease with thrombospondin type 1 repeats-13 metalloprotease required for modulation of arterial thrombosis.

OBJECTIVE: A disintegrin and metalloprotease with thrombospondin type 1 repeats-13 (ADAMTS13) inhibits platelet aggregation and arterial thrombosis by cleavage of von Willebrand factor. However, the structural components of ADAMTS13 required for inhibition of arterial thrombosis are not fully defined. METHODS AND RESULTS: Using recombinant proteins and a murine model, we demonstrated that an ADAMTS13 variant truncated after either the eighth thrombospondin type 1 repeat or the spacer domain inhibits ferric chloride-induced arterial thrombosis in ADAMTS13(-/-) mice with efficacy similar to that of full-length ADAMTS13. The results obtained from monitoring thrombus formation in carotid and mesenteric arteries were highly concordant. Further analyses by site-directed mutagenesis and human monoclonal antibody inhibition assay revealed that the Cys-rich and spacer domains of ADAMTS13, particularly the amino acid residues between Arg559 and Glu664 in the spacer domain, may be critical for modulation of arterial thrombosis in vivo. Finally, the thrombosis-modulating function of ADAMTS13 and variants/mutants was highly correlated with the von Willebrand factor-cleavage activity under fluid shear stress. CONCLUSIONS: Our results suggest that the amino terminus of ADAMTS13, specifically the variable region of the spacer domain, is crucial for modulation of arterial thromboses under (patho)physiological conditions. These findings shed more light on the structure-function relationship of ADAMTS13 in vivo and may be applicable for rational design of protein- or gene-based therapy of arterial thromboses.