Disulfide bond reduction of von Willebrand factor by ADAMTS‐13

See also Lenting PJ, Rastegarlari G. ADAMTS‐13: double trouble for von Willebrand factor. This issue, pp 2775–7. Summary. Background: von Willebrand factor (VWF) released from endothelial cells is rich in ultra‐large (UL) multimers that are intrinsically active in binding platelets, whereas plasma‐type VWF multimers require shear stress to be activated. This functional difference may be attributed to thiols exposed on the surface of plasma‐type VWF multimers, but not on ULVWF multimers. Shear stress induces the exposed thiols to form disulfide bonds between laterally apposed plasma‐type VWF multimers, leading to enhanced VWF binding to platelets. Objectives: We tested a hypothesis that ADAMTS‐13 has a disulfide bond reducing activity that regulates shear‐induced thiol‐disulfide exchange of VWF. Methods: Thiol blocking agents and active thiol bead capturing were used to identify and locate this activity, along with truncated ADAMTS‐13 mutants. Results: ADAMTS‐13 contains a disulfide bond reducing activity that primarily targets disulfide bonds in plasma‐type VWF multimers induced by high shear stress or formed with thiol beads, but not disulfide bonds in native multimeric structures. Cysteine thiols targeted by this activity are in the VWF C‐domain and are known to participate in shear‐induced thiol‐disulfide exchange. ADAMTS‐13 contains cysteine thiols that remain exposed after being subjected to hydrodynamic forces. Blocking these active thiols eliminates this reducing activity and moderately decreases ADAMTS‐13 activity in cleaving ULVWF strings anchored to endothelial cells under flow conditions, but not under static conditions. This activity is located in this C‐terminal region of ADAMTS‐13. Conclusions: This novel disulfide‐bond‐reducing activity of ADAMTS‐13 may prevent covalent lateral association and increased platelet adherence of plasma‐type VWF multimers induced by high fluid shear stress.     

Use of a mouse model to elucidate the phenotypic effects of the von Willebrand factor cleavage mutants,Y1605A/M1606A and R1597W

Background:  von Willebrand Factor (VWF) is tightly regulated by the metalloproteinase ADAMTS13, which cleaves VWF to reduce VWF multimer size and binding affinity for collagen and platelets. Objective:  This study examines two VWF mutations, R1597W (enhanced cleavage) and Y1605A‐M1606A (decreased cleavage), to determine their impact on VWF, in addition to ADAMTS13‐mediated cleavage. Methods:  In vitro mouse ADAMTS13 digestions were performed on recombinant proteins. VWF knockout mice received hydrodynamic injections of mouse Vwf cDNA, following which VWF antigen, multimer profile and VWF propeptide levels were determined. A ferric chloride injury model of thrombosis was also evaluated. Results:  In vitro ADAMTS13 digestion of full‐length mouse VWF required > 97‐fold higher ADAMTS13 levels for Y1605A/M1606A, and 68% lower ADAMTS13 levels for R1597W compared with wild type. In vivo, R1597W had reduced VWF:Ag and both mutations exhibited increased VWF propeptide/VWF:Ag ratios. R1597W multimers show a lower molecular weight profile compared with wild type and Y1605A/M1606A mice. When co‐injected with Adamts13 cDNA, Y1605A/M1606A multimers were larger compared with wild type, and R1597W showed only a single multimer band and decreased clearance via VWFpp/VWF:Ag ratio. R1597W was associated with reduced thrombus formation but normal platelet accumulation in a ferric chloride injury model while Y1605A/M1606A had a loss of occlusive thrombi but increased platelet accumulation compared with wild type. Conclusions:  This study demonstrates that mutations that alter ADAMTS13 cleavage also can affect VWF clearance, VWF antigen level, multimer structure and thrombotic potential in the VWF knockout hydrodynamic injection model.     

Correction of ADAMTS13 Deficiency by In Utero Gene Transfer of Lentiviral Vector encoding ADAMTS13 Genes

Deficiency of A Disintegrin And Metalloprotease with ThromboSpondin (ADAMTS13) results in thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective treatment to date. We show in this study that an administration of a self-inactivating lentiviral vector encoding human full-length ADAMTS13 and a variant truncated after the spacer domain (MDTCS) in mice by in utero injection at embryonic days 8 and 14 resulted in detectable plasma proteolytic activity (~5–70%), which persisted for the length of the study (up to 24 weeks). Intravascular injection via a vitelline vein at E14 was associated with significantly lower rate of fetal loss than intra-amniotic injection, suggesting that the administration of vector at E14 may be a preferred gestational age for vector delivery. The mice expressing ADAMTS13 and MDTCS exhibited reduced sizes of von Willebrand factor (vWF) compared to the Adamts13^−/− mice expressing enhanced green fluorescent protein (eGFP). Moreover, the mice expressing both ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride–induced carotid arterial occlusion time as compared to the Adamts13^−/− expressing eGFP. The data demonstrate the successful correction of the prothrombotic phenotypes in Adamts13^−/− mice by a single in utero injection of lentiviral vectors encoding human ADAMTS13 genes, providing the basis for developing a gene therapy for hereditary TTP in humans.     

ADAMTS13 reduces VWF‐mediated acute inflammation following focal cerebral ischemia in mice

Summary. Background: ADAMTS13 cleaves hyperactive ultra‐large von Willebrand factor (ULVWF) multimers into smaller and less active forms. It remains unknown whether VWF‐mediated inflammatory processes play a role in the enhanced brain injury due to ADAMTS13 deficiency. Objective: We tested the hypothesis that the deleterious effect of ADAMTS13 deficiency on ischemic brain injury is mediated through VWF‐dependent enhanced vascular inflammation. Methods: Transient focal cerebral ischemia was induced by 60 min of occlusion of the right middle cerebral artery. Myeloperoxidase (MPO) activity and inflammatory cytokines in the infarcted region were evaluated 23 h after reperfusion injury. Neutrophil infiltration within the infarct and surrounding areas was quantitated by immunohistochemistry. Results: We report that ADAMTS13‐deficient mice exhibited significantly enlarged infarct size, concordant with increased myeloperoxidase (MPO) activity, neutrophil infiltration and expression of the pro‐inflammatory cytokines interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNF‐α). In contrast, VWF‐deficient mice exhibited significantly reduced MPO activity, neutrophil infiltration and inflammatory cytokine induction, demonstrating a role of VWF in these inflammatory processes. Mice deficient for both ADAMTS13 and VWF exhibited an identical reduction of the same inflammatory parameters, demonstrating that the increased inflammation observed in ADAMTS13‐deficient mice is VWF dependent. Finally, the increased infarct size observed in ADAMTS13‐deficient mice was completely abrogated by prior immunodepletion of neutrophils, demonstrating a causal role for acute inflammation in the enhanced brain injury that occurs in the setting of ADAMTS13 deficiency. Conclusion: These findings provide new evidence for ADAMTS13 in reducing VWF‐mediated acute cerebral inflammation following ischemic stroke.     

Platelet--vessel wall interactions

The vascular endothelium is a thromboresistant surface, allowing the free flowing of blood cell elements. Platelets are predominantly involved in the rapid response to a vascular lesion, exposing the underlying thrombogenic subendothelium and leading to initiation of thrombus formation. Thrombus growth requires on the one hand, the recruitment of circulating platelets to the luminal side of the thrombus and on the other hand, the assembly of the proteins of the blood coagulation cascade on the platelet catalytic surface leading to thrombin formation. High shear forces are necessary for the dual role of von Willebrand factor (VWF) in the initiation of platelet thrombus formation and in its growth and stabilization. In hemodynamic conditions, platelet adhesion depends on the interaction between VWF and the platelet receptor glycoprotein Ib (GPIb). This interaction is the only one able to resist to the high shear rates that prevail in arterioles, the microcirculation or stenosed arteries. Thereafter, the interaction between VWF and the alphaIIbbeta3 integrin allows the definitive arrest of platelets and induces thrombus formation. Thus, high shear forces by themselves are able to induce platelet activation/aggregation, without added exogenous agonist. VWF is synthesised by endothelial cells as a series of multimers of different sizes. The multimers with the highest molecular weight, the so-called ultra-large multimers, are strongly thrombogenic by their increased ability to bind platelet GPIb and to induce the formation of circulating aggregates. These ultra-large multimers are normally cleaved by the ADAMTS13 metalloprotease into smaller multimers which are also less thrombogenic. The in vivo proteolysis of VWF by ADAMTS13 depends on the high shear rates, which increase the opening of multimers anchored to the endothelial cell layer and the exposure of the cleavage site of VWF by ADAMTS13. An ADAMTS13 deficiency thus likely would result in the accumulation of ultra-large multimers on the endothelial surface, which retains platelets on the activated endothelium and results in micro-thrombi formation, as seen in thrombotic thrombocytopenic purpura. Platelet-VWF interactions are also involved in inflammation. Activation of endothelial cells induces the release of VWF from the Weibel-Palade bodies as well as the surface expression of VWF and P-selectin. These molecules allow leukocyte and platelet rolling on endothelial cells, and expression of E-selectin, VCAM-1 and other adhesion molecules. Recently, it has been shown that activated platelets allow transient activation of intact, non stimulated endothelial cells, thus increasing the inflammation process. VWF and platelet P-selectin have been shown to be essential to this process. Thus, platelet--vessel wall interactions are involved in thrombosis and inflammation essentially via VWF.     

Platelet rescue by macrophage depletion in obese ADAMTS‐13‐deficient mice at risk of thrombotic thrombocytopenic purpura

Essentials

• Obesity is a potential risk factor for development of thrombotic thrombocytopenic purpura (TTP).
• Obese ADAMTS‐13‐deficient mice were triggered with von Willebrand factor (VWF).
• Depletion of hepatic and splenic macrophages protects against thrombocytopenia in this model.
• VWF enhances phagocytosis of platelets by macrophages, dose‐dependently.

Summary

Background

Thrombotic thrombocytopenic purpura (TTP) is caused by the absence of ADAMTS‐13 activity. Thrombocytopenia is presumably related to the formation of microthrombi rich in von Willebrand factor (VWF) and platelets. Obesity may be a risk factor for TTP; it is associated with abundance of macrophages that may phagocytose platelets.

Objectives

To evaluate the role of obesity and ADAMTS‐13 deficiency in TTP, and to establish whether macrophages contribute to thrombocytopenia.

Methods

Lean or obese ADAMTS‐13‐deficient (Adamts‐13^?/?) and wild‐type (WT) mice were injected with 250 U kg^?1 of recombinant human VWF (rVWF), and TTP characteristics were evaluated 24 h later. In separate experiments, macrophages were depleted in the liver and spleen of lean and obese WT or Adamts‐13^?/? mice by injection of clodronate‐liposomes, 48 h before injection of rVWF.

Results

Obese Adamts‐13^?/? mice had a lower platelet count than their lean counterparts, suggesting that they might be more susceptible to TTP development. Lean Adamts‐13^?/? mice triggered with a threshold dose of rVWF did not develop TTP, whereas typical TTP symptoms developed in obese Adamts‐13^?/? mice, including severe thrombocytopenia and higher lactate dehydrogenase (LDH) levels. Removal of hepatic and splenic macrophages by clodronate injection in obese Adamts‐13^?/? mice before treatment with rVWF preserved the platelet counts measured 24 h after the trigger. In vitro experiments with cultured macrophages confirmed a VWF dose‐dependent increase of platelet phagocytosis.

Conclusions

Obese Adamts‐13^?/? mice are more susceptible to the induction of TTP‐related thrombocytopenia than lean mice. Phagocytosis of platelets by macrophages contributes to thrombocytopenia after rVWF injection in this model.

     

Formation of platelet strings and microthrombi in the presence of ADAMTS-13 inhibitor does not require P-selectin or β3 integrin

BACKGROUND: Ultra-large von Willebrand factor (ULVWF) and the receptor P-selectin are released from endothelial Weibel-Palade bodies during injury or inflammation. VWF mediates platelet adhesion and P-selectin promotes leukocyte rolling. ADAMTS-13 limits the duration of platelet adhesion by cleaving the ULVWF. In the absence of ADAMTS-13, long VWF filaments decorated with platelets form. Recent in vitro studies suggested that P-selectin might anchor these platelet strings to endothelium, but whether the same mechanism exists in vivo remains to be elucidated. METHODS: We address the role of P-selectin and beta(3) integrin in platelet string formation in vivo using intravital microscopy by infusing inhibitory ADAMTS-13 antibody in P-selectin-/- and beta(3)-deficient mice and activating the endothelium by injecting histamine. RESULTS: We show that inhibition of ADAMTS-13 combined with endothelial activation leads to similar extents of platelet string formation in wild-type, P-selectin- and integrin beta(3)-deficient mice. Further, in venules the platelet strings can coalesce into VWF-platelet aggregates. This process utilizes neither the platelet beta(3) integrin nor P-selectin. We also show in vitro that platelets can act as a bridge between the VWF fibers and that VWF can self-associate even in areas devoid of platelets. CONCLUSIONS: The formation or retention of the platelet strings does not require P-selectin or the endothelial VWF receptor alpha(v)beta(3). Furthermore, in the presence of low ADAMTS-13 activity, VWF-dependent and alpha(IIb)beta(3)-independent platelet clustering occurs in veins, as has been shown at high arterial shear rates. Our study further supports the importance of regulation of VWF multimer size upon secretion from Weibel-Palade bodies.     

Critical Points of Tumor Necrosis Factor Action in Central Nervous System Autoimmune Inflammation Defined by Gene Targeting

Tumor necrosis factor (TNF)–dependent sites of action in the generation of autoimmune inflammation have been defined by targeted disruption of TNF in the C57BL/6 mouse strain. C57BL/6 mice are susceptible to an inflammatory, demyelinating form of experimental autoimmune encephalomyelitis (EAE) induced by the 35–55 peptide of myelin oligodendrocyte glycoprotein. Direct targeting of a strain in which EAE was inducible was necessary, as the location of the TNF gene renders segregation of the mutated allele from the original major histocompatibility complex by backcrossing virtually impossible. In this way a single gene effect was studied. We show here that TNF is obligatory for normal initiation of the neurological deficit, as demonstrated by a significant (6 d) delay in disease in its absence relative to wild-type (WT) mice. During this delay, comparable numbers of leukocytes were isolated from the perfused central nervous system (CNS) of WT and TNF^−/− mice. However, in the TNF^−/− mice, immunohistological analysis of CNS tissue indicated that leukocytes failed to form the typical mature perivascular cuffs observed in WT mice at this same time point. Severe EAE, including paralysis and widespread CNS perivascular inflammation, eventually developed without TNF. TNF^−/− and WT mice recovered from the acute illness at the same time, such that the overall disease course in TNF^−/− mice was only 60% of the course in control mice. Primary demyelination occurred in both WT and TNF^−/− mice, although it was of variable magnitude. These results are consistent with the TNF dependence of processes controlling initial leukocyte movement within the CNS. Nevertheless, potent alternative mechanisms exist to mediate all other phases of EAE.     

Systemic antithrombotic effects of ADAMTS13

The metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats 13) cleaves highly adhesive large von Willebrand factor (VWF) multimers after their release from the endothelium. ADAMTS13 deficiency is linked to a life-threatening disorder, thrombotic thrombocytopenic purpura (TTP), characterized by platelet-rich thrombi in the microvasculature. Here, we show spontaneous thrombus formation in activated microvenules of Adamts13-/- mice by intravital microscopy. Strikingly, we found that ADAMTS13 down-regulates both platelet adhesion to exposed subendothelium and thrombus formation in injured arterioles. An inhibitory antibody to ADAMTS13 infused in wild-type mice prolonged adhesion of platelets to endothelium and induced thrombi formation with embolization in the activated microvenules. Absence of ADAMTS13 did not promote thrombi formation in alphaIIbbeta3 integrin-inhibited blood. Recombinant ADAMTS13 reduced platelet adhesion and aggregation in histamine-activated venules and promoted thrombus dissolution in injured arterioles. Our findings reveal that ADAMTS13 has a powerful natural antithrombotic activity and recombinant ADAMTS13 could be used as an antithrombotic agent.     

Oxidized von Willebrand factor is efficiently cleaved by serine proteases from primary granules of leukocytes: divergence from ADAMTS‐13

To cite this article: Lancellotti S, De Filippis V, Pozzi N, Oggianu L, Rutella S, Scaglione GL, Maset F, Peyvandi F, Mannucci PM, De Cristofaro R. Oxidized von Willebrand factor is efficiently cleaved by serine proteases from primary granules of leukocytes: divergence from ADAMTS‐13. J Thromb Haemost 2011; 9 : 1620–7. Summary. Background: The leukocyte serine proteases (LSPs) elastase, proteinase 3 and cathepsin G cleave von Willebrand factor (VWF) near or at the same cleavage site (Tyr1605–Met1606) as ADAMTS‐13, the metalloprotease that specifically controls the proteolytic processing of VWF. Recent studies have shown that oxidation of VWF at Met1606 with formation of methionine sulfoxide (MetSO) severely impairs its proteolysis by ADAMTS‐13. Methods: This study was aimed at assessing whether or not oxidation of VWF by reactive oxygen species (ROS) can also affect its cleavage by elastase, proteinase 3, and cathepsin G. In this study, the catalytic specificity of hydrolysis by LSPs of the VWF peptide substrate VWF74 and full‐length VWF, both unaltered and in the oxidized form, was measured by RP‐HPLC, electrophoretic and mass spectrometry methods. Results: LSPs cleaved both VWF multimers and VWF74 near or at the same peptide bond as is cleaved by ADAMTS‐13, with k_cat/K_m values similar to those of the metalloprotease. However, unlike ADAMTS‐13, cathepsin G cleaved VWF74 containing a MetSO residue at position 1606 with a k_cat/K_m value higher than that for VWF74, whereas the catalytic efficiencies of both elastase and proteinase 3 were unaffected by the replacement of Met1606 with MetSO. Likewise, oxidation of VWF multimers by hypochlorous acid and ROS, produced by activated leukocytes, improved their hydrolysis by LSPs. Conclusions: Oxidation by leukocyte ROS has a net positive effect on the cleavage of VWF multimers by LSPs, under conditions where high concentrations of oxidant species would severely reduce the proteolytic efficiency of ADAMTS‐13.     

Interleukin-10 Is a Natural Suppressor of Cytokine Production and Inflammation in a Murine Model of Allergic Bronchopulmonary Aspergillosis

We have used interleukin-10 (IL-10) gene knockout mice (IL-10^−/−) to examine the role of endogenous IL-10 in allergic lung responses to Aspergillus fumigatus Ag. In vitro restimulated lung cells from sensitized IL-10^−/− mice produced exaggerated amounts of IL-4, IL-5, and interferon-γ (IFN-γ) compared with wild-type (WT) lung cells. In vivo, the significance of IL-10 in regulating responses to repeated A. fumigatus inhalation was strikingly revealed in IL-10^−/− outbred mice that had a 50–60% mortality rate, while mortality was rare in similarly treated WT mice. Furthermore, IL-10^−/− outbred mice exhibited exaggerated airway inflammation and heightened levels of IL-5 and IFN-γ in bronchoalveolar lavage (BAL) fluids. In contrast, the magnitude of the allergic lung response was similar in intranasally (i.n.) sensitized IL-10^−/− and wild-type mice from a different strain (C57BL/6). Using a different route of priming (intraperitoneal) followed by one i.n. challenge we found that IL-10^−/− C57BL/6 mice had heightened eosinophilic airway inflammation, BAL–IL-5 levels, and numbers of αβT cells in the lung tissues compared with WT mice. We conclude that IL-10 can suppress inflammatory Th2-like lung responses as well as Th1-like responses given the constraints of genetic background and route of priming.     

Neutrophil Emigration in the Skin, Lungs, and Peritoneum: Different Requirements for CD11/CD18 Revealed by CD18-deficient Mice

To determine the role of CD11/CD18 complexes in neutrophil emigration, inflammation was induced in the skin, lungs, or peritoneum of mutant mice deficient in CD18 (CD18^−/− mutants). Peripheral blood of CD18^−/− mutants contained 11-fold more neutrophils than did blood of wild-type (WT) mice. During irritant dermatitis induced by topical application of croton oil, the number of emigrated neutrophils in histological sections of dermis was 98% less in CD18^−/− mutants than in WT mice. During Streptococcus pneumoniae pneumonia, neutrophil emigration in CD18^−/− mutants was not reduced. These data are consistent with expectations based on studies using blocking antibodies to inhibit CD11/CD18 complexes, and on observations of humans lacking CD11/CD18 complexes. The number of emigrated neutrophils in lung sections during Escherichia coli pneumonia, or in peritoneal lavage fluid after 4 h of S. pneumoniae peritonitis, was not reduced in CD18^−/− mutants, but rather was greater than the WT values (240 ± 30 and 220 ± 30% WT, respectively). Also, there was no inhibition of neutrophil emigration during sterile peritonitis induced by intraperitoneal injection of thioglycollate (90 ± 20% WT). These data contrast with expectations. Whereas CD11/CD18 complexes are essential to the dermal emigration of neutrophils during acute dermatitis, CD18^−/− mutant mice demonstrate surprising alternative pathways for neutrophil emigration during pneumonia or peritonitis.     

The Functional Paradox of CD43 in Leukocyte Recruitment: A Study Using CD43-deficient Mice

Although there is considerable evidence implicating a role for CD43 (leukosialin) in leukocyte cell–cell interactions, its precise function remains uncertain. Using CD43-deficient mice (CD43^−/−) and intravital microscopy to directly visualize leukocyte interactions in vivo, we investigated the role of CD43 in leukocyte–endothelial cell interactions within the cremasteric microcirculation under flow conditions. Our studies demonstrated significantly enhanced leukocyte rolling and adhesion after chemotactic stimuli in CD43^−/− mice compared with wild type mice. Using an in vitro flow chamber, we established that the enhanced rolling interactions of CD43^−/− leukocytes, primarily neutrophils, were also observed using immobilized E-selectin as a substrate, suggesting that passive processes related to steric hindrance or charge repulsion were likely mechanisms. Despite increased adhesion and rolling interactions by CD43^−/− leukocytes, we uncovered a previously unrecognized impairment of CD43^−/− leukocytes to infiltrate tissues. Oyster glycogen–induced neutrophil and monocyte infiltration into the peritoneum was significantly reduced in CD43^−/− mice. In response to platelet activating factor, CD43^−/− leukocytes were impaired in their ability to emigrate out of the vasculature. These results suggest that leukocyte CD43 has a dual function in leukocyte–endothelial interactions. In addition to its role as a passive nonspecific functional barrier, CD43 also facilitates emigration of leukocytes into tissues.     

Covalent regulation of ULVWF string formation and elongation on endothelial cells under flow conditions

Summary.  Background and Objectives:  The adhesion ligand von Willebrand factor (VWF) is a multimeric glycoprotein that mediates platelet adhesion to exposed subendothelium. On endothelial cells, freshly released ultra-large (UL) VWF multimers form long string-like structures to which platelets adhere. Methods:  The formation and elongation of ULVWF strings were studied in the presence of the thiol-blocking N-ethylmaleimide (NEM). The presence of thiols in ULVWF and plasma VWF multimers was determined by maleimide-PEO₂-Biotin labeling and thiol-chromatography. Finally, covalent re-multimerization of ULVWF was examined in a cell- and enzyme-free system. Results:  We found that purified plasma VWF multimers adhere to and elongate ULVWF strings under flow conditions. The formation and propagation of ULVWF strings were dose-dependently reduced by blocking thiols on VWF with NEM, indicating that ULVWF strings are formed by the covalent association of perfused VWF to ULVWF anchored to endothelial cells. The association is made possible by the presence of free thiols in VWF multimers and by the ability of (UL) VWF to covalently re-multimerize. Conclusion:  The data provide a mechanism by which the thrombogenic ULVWF strings are formed and elongated on endothelial cells. This mechanism suggests that the thiol-disulfide state of ULVWF regulates the adhesion properties of strings on endothelial cells.     

Cloning, expression and functional characterization of the full-length murine ADAMTS13.

Functional deficiency or absence of the human von Willebrand factor (VWF)-cleaving protease (VWF-cp), recently termed ADAMTS13, has been shown to cause acquired and congenital thrombotic thrombocytopenic purpura (TTP), respectively. As a first step towards developing a small animal model of TTP, we have cloned the complete (non-truncated) murine Adamts13 gene from BALB/c mice liver poly A+ mRNA. Murine ADAMTS13 is a 1426-amino-acid protein with a high homology and similar structural organization to the human ortholog. Transient expression of the murine Adamts13 cDNA in HEK 293 cells yielded a protein with a molecular weight of approximately 180 kDa which degraded recombinant murine VWF (rVWF) in a dose-dependent manner. The cleavage products of murine rVWF had the expected size of 140 and 170 kDa. Murine ADAMTS13 was inhibited by EDTA and the plasma from a TTP patient.     

JAM-A regulates permeability and inflammation in the intestine in vivo

Recent evidence has linked intestinal permeability to mucosal inflammation, but molecular studies are lacking. Candidate regulatory molecules localized within the tight junction (TJ) include Junctional Adhesion Molecule (JAM-A), which has been implicated in the regulation of barrier function and leukocyte migration. Thus, we analyzed the intestinal mucosa of JAM-A–deficient (JAM-A^−/−) mice for evidence of enhanced permeability and inflammation. Colonic mucosa from JAM-A^−/− mice had normal epithelial architecture but increased polymorphonuclear leukocyte infiltration and large lymphoid aggregates not seen in wild-type controls. Barrier function experiments revealed increased mucosal permeability, as indicated by enhanced dextran flux, and decreased transepithelial electrical resistance in JAM-A^−/− mice. The in vivo observations were epithelial specific, because monolayers of JAM-A^−/− epithelial cells also demonstrated increased permeability. Analyses of other TJ components revealed increased expression of claudin-10 and -15 in the colonic mucosa of JAM-A^−/− mice and in JAM-A small interfering RNA–treated epithelial cells. Given the observed increase in colonic inflammation and permeability, we assessed the susceptibility of JAM-A^−/− mice to the induction of colitis with dextran sulfate sodium (DSS). Although DSS-treated JAM-A^−/− animals had increased clinical disease compared with controls, colonic mucosa showed less injury and increased epithelial proliferation. These findings demonstrate a complex role of JAM-A in intestinal homeostasis by regulating epithelial permeability, inflammation, and proliferation.     

The active conformation of von Willebrand factor in patients with thrombotic thrombocytopenic purpura in remission

Summary.  Background:  Functional deficiency of ADAMTS13 in thrombotic thrombocytopenic purpura (TTP) patients is associated with circulating ultralarge von Willebrand factor (VWF) molecules that display spontaneous platelet-binding capacities. Upon remission, however, ADAMTS13 activity does not always return to baseline. Objective:  To study ADAMTS13 and VWF-related features in TTP patients in remission. Methods:  ADAMTS13 activity, anti-ADAMTS13 antibodies, VWF antigen, ultralarge VWF and levels of VWF that circulate in a glycoprotein Ibα-binding conformation were determined in plasma samples of 22 acquired TTP patients in remission between 1 month and 6 years after achieving remission. The composition of active multimers was investigated with a novel immunoprecipitation assay based on monoclonal antibody AU/VWF-a12, which specifically recognizes the active conformation of VWF. Results:  ADAMTS13 activity was undetectable in 23% of the patients, even years after they had achieved remission, and lack of ADAMTS13 activity was associated with increased active VWF levels and the presence of ultralarge VWF multimers. Active VWF levels and ultralarge VWF were also associated with blood groups. Results from immunoprecipitation experiments revealed the full range of multimers to be present. Conclusion : ADAMTS13 deficiency and the concurrent presence of ultralarge VWF and increased active VWF levels can be detected in TTP patients for years after they have achieved remission. Immunoprecipitation results suggest that the active conformation of VWF may be present in the lower molecular weight multimers, but future studies are necessary to confirm our findings.     

Modulation of the Intestinal Microbiota Alters Colitis-Associated Colorectal Cancer Susceptibility

It is well established that the intestinal microbiota plays a key role in the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC) collectively referred to as inflammatory bowel disease (IBD). Epidemiological studies have provided strong evidence that IBD patients bear increased risk for the development of colorectal cancer (CRC). However, the impact of the microbiota on the development of colitis-associated cancer (CAC) remains largely unknown. In this study, we established a new model of CAC using azoxymethane (AOM)-exposed, conventionalized-Il10^−/− mice and have explored the contribution of the host intestinal microbiota and MyD88 signaling to the development of CAC. We show that 8/13 (62%) of AOM-Il10^−/− mice developed colon tumors compared to only 3/15 (20%) of AOM- wild-type (WT) mice. Conventionalized AOM-Il10^−/− mice developed spontaneous colitis and colorectal carcinomas while AOM-WT mice were colitis-free and developed only rare adenomas. Importantly, tumor multiplicity directly correlated with the presence of colitis. Il10^−/− mice mono-associated with the mildly colitogenic bacterium Bacteroides vulgatus displayed significantly reduced colitis and colorectal tumor multiplicity compared to Il10^−/− mice. Germ-free AOM-treated Il10^−/− mice showed normal colon histology and were devoid of tumors. Il10^−/−; Myd88^−/− mice treated with AOM displayed reduced expression of Il12p40 and Tnfα mRNA and showed no signs of tumor development. We present the first direct demonstration that manipulation of the intestinal microbiota alters the development of CAC. The TLR/MyD88 pathway is essential for microbiota-induced development of CAC. Unlike findings obtained using the AOM/DSS model, we demonstrate that the severity of chronic colitis directly correlates to colorectal tumor development and that bacterial-induced inflammation drives progression from adenoma to invasive carcinoma.     

The role of ADAMTS‐13 in the coagulopathy of sepsis

Summary

The interaction between platelets and the vessel wall is mediated by various receptors and adhesive proteins, of which von Willebrand factor (VWF) is the most prominent. The multimeric size of VWF is an important determinant of a more intense platelet–vessel wall interaction, and is regulated by the VWF‐cleaving protease ADAMTS‐13. A deficiency in ADAMTS‐13 leads to higher concentrations of ultralarge VWF multimers and pathological platelet–vessel wall interactions, in its most typical and extreme form leading to thrombocytopenic thrombotic purpura, a thrombotic microangiopathy characterized by thrombocytopenia, non‐immune hemolysis, and organ dysfunction. Thrombotic microangiopathy associated with low levels of ADAMTS‐13 may be a component of the coagulopathy observed in patients with sepsis. Here, we review the potential role of ADAMTS‐13 deficiency and ultralarge VWF multimers in sepsis, and their relationship with sepsis severity and prognosis. In addition, we discuss the possible benefit of restoring ADAMTS‐13 levels or reducing the effect of ultralarge VWF as an adjunctive treatment in patients with sepsis.

     

A Critical Role for Lymphotoxin in Experimental Allergic Encephalomyelitis

The lymphotoxin (LT)/tumor necrosis factor (TNF) family has been implicated in the neurologic inflammatory diseases multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). To determine the role of individual family members in EAE, C57BL/6 mice, LT-α–deficient (LT-α^−/− mice), or LT-β–deficient (LT-β^−/− mice), and their wild-type (WT) littermates were immunized with rat myelin oligodendrocyte glycoprotein (MOG) peptide 35-55. C57BL/6 and WT mice developed chronic, sustained paralytic disease with average maximum clinical scores of 3.5 and disease indices (a measure of day of onset and sustained disease scores) ranging from 367 to 663 with central nervous system (CNS) inflammation and demyelination. LT-α^−/− mice were primed so that their splenic lymphocytes proliferated in response to MOG 35-55 and the mice produced anti-MOG antibody. However, LT-α^−/− mice were quite resistant to EAE with low average clinical scores (<1), an average disease index of 61, and the negligible CNS inflammation and demyelination. WT T cells transferred EAE to LT-α^−/− recipients. LT-β^−/− mice were susceptible to EAE, though less than WT, with an average maximum clinical score of 1.9 and disease index of 312. These data implicate T cell production of LT-α in MOG EAE and support a major role for LT-α3, a minor role for the LT-α/β complex, and by inference, no role for TNF-α.