Shear‐induced unfolding activates von Willebrand factor A2 domain for proteolysis

Summary. Background: To avoid pathological platelet aggregation by von Willebrand factor (VWF), VWF multimers are regulated in size and reactivity for adhesion by ADAMTS13‐mediated proteolysis in a shear flow dependent manner. Objective and methods: We examined whether tensile stress in VWF under shear flow activates the VWF A2 domain for cleavage by ADAMTS13 using molecular dynamics simulations. We generated a full length mutant VWF featuring a homologous disulfide bond in A2 (N1493C and C1670S), in an attempt to lock A2 against unfolding. Results: We indeed observed stepwise unfolding of A2 and exposure of its deeply buried ADAMTS13 cleavage site. Interestingly, disulfide bonds in the adjacent and highly homologous VWF A1 and A3 domains obstruct their mechanical unfolding. We find this mutant A2 (N1493C and C1670S) to feature ADAMTS13‐resistant behavior in vitro. Conclusions: Our results yield molecular‐detail evidence for the force‐sensing function of VWF A2, by revealing how tension in VWF due to shear flow selectively exposes the A2 proteolysis site to ADAMTS13 for cleavage while keeping the folded remainder of A2 intact and functional. We find the unconventional ‘knotted’ Rossmann fold of A2 to be the key to this mechanical response, tailored for regulating VWF size and activity. Based on our model we discuss the pathomechanism of some natural mutations in the VWF A2 domain that significantly increase the cleavage by ADAMTS13 without shearing or chemical denaturation, and provide with the cleavage‐activated A2 conformation a structural basis for the design of inhibitors for VWF type 2 diseases.     

Multi‐step binding of ADAMTS‐13 to von Willebrand factor

Summary. Background: ADAMTS‐13 proteolytic activity is controlled by the conformation of its substrate, von Willebrand factor (VWF), and changes in the secondary structure of VWF are essential for efficient cleavage. Substrate recognition is mediated through several non‐catalytic domains in ADAMTS‐13 distant from the active site. Objectives: We hypothesized that not all binding sites for ADAMTS‐13 in VWF are cryptic and analyzed binding of native VWF to ADAMTS‐13. Methods: Immunoprecipiation of VWF–ADAMTS‐13 complexes using anti‐VWF antibodies and magnetic beads was used. Binding was assessed by Western blotting and immunosorbent assays. Results: Co‐immunoprecipitation demonstrated that ADAMTS‐13 binds to native multimeric VWF (K_d of 79 ± 11 nmol L^?1) with no measurable proteolysis. Upon shear‐induced unfolding of VWF, binding increased 3‐fold and VWF was cleaved. Binding to native VWF was saturable, time dependent, reversible and did not vary with ionic strength (I of 50–200). Moreover, results with ADAMTS‐13 deletion mutants indicated that binding to native VWF is mediated through domains distal to the ADAMTS‐13 spacer, probably thrombospondin‐1 repeats. Interestingly, this interaction occurs in normal human plasma with an ADAMTS‐13 to VWF stoichiometry of 0.0040 ± 0.0004 (mean ± SEM, n = 10). Conclusions: ADAMTS‐13 binds to circulating VWF and may therefore be incorporated into a platelet‐rich thrombus, where it can immediately cleave VWF that is unfolded by fluid shear stress.     

ADAMTS13: a new link between thrombosis and inflammation

von Willebrand factor (VWF) levels are elevated and a disintegrin-like and metalloprotease with thrombospondin type I repeats–13 (ADAMTS13) activity is decreased in both acute and chronic inflammation. We hypothesized that by cleaving hyperactive ultralarge VWF (ULVWF) multimers, ADAMTS13 down-regulates both thrombosis and inflammation. Using intravital microscopy, we show that ADAMTS13 deficiency results in increased leukocyte rolling on unstimulated veins and increased leukocyte adhesion in inflamed veins. Both processes were dependent on the presence of VWF. Depletion of platelets in Adamts13^−/− mice reduced leukocyte rolling, suggesting that platelet interaction with ULVWF contributes to this process. Increased levels of endothelial P-selectin and plasma VWF in Adamts13^−/− compared with wild-type (WT) mice indicated an elevated release of Weibel-Palade bodies. ULVWF multimers released upon stimulation with histamine, a secretagogue of Weibel-Palade bodies, slowed down leukocyte rolling in Adamts13^−/− but not in WT mice. Furthermore, in inflammatory models, ADAMTS13 deficiency resulted in enhanced extravasation of neutrophils, and this process was also dependent on VWF. Our findings reveal an important role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade body secretion, and in the regulation of leukocyte adhesion and extravasation during inflammation.     

N-acetylcysteine reduces the size and activity of von Willebrand factor in human plasma and mice

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterized by systemic microvascular thrombosis caused by adhesion of platelets to ultra-large vWF (ULVWF) multimers. These multimers accumulate because of a deficiency of the processing enzyme ADAMTS13. vWF protein forms long multimers from homodimers that first form through C-terminal disulfide bonds and then join through their N termini by further disulfide bonding. N-acetylcysteine (NAC) is an FDA-approved drug that has long been used to treat chronic obstructive lung disease and acetaminophen toxicity and is known to function in the former disorder by reducing mucin multimers. Here, we examined whether NAC could reduce vWF multimers, which polymerize in a manner similar to mucins. In vitro, NAC reduced soluble plasma-type vWF multimers in a concentration-dependent manner and rapidly degraded ULVWF multimer strings extruded from activated ECs. The effect was preceded by reduction of the intrachain disulfide bond encompassing the platelet-binding A1 domain. NAC also inhibited vWF-dependent platelet aggregation and collagen binding. Injection of NAC into ADAMTS13-deficient mice led to the rapid resolution of thrombi produced by ionophore treatment of the mesenteric venules and reduced plasma vWF multimers. These results suggest that NAC may be a rapid and effective treatment for patients with TTP.     

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.     

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.     

The distal carboxyterminal domains of murine ADAMTS13 influence proteolysis of platelet‐decorated VWF strings in vivo

Summary. Background: The multidomain metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers upon their release from endothelial cells. How the different domains in ADAMTS13 control VWF proteolysis in vivo remains largely unidentified. Methods: Seven C‐terminally truncated murine ADAMTS13 (mADAMTS13) mutants were constructed and characterized in vitro. Their ability to cleave VWF strings in vivo was studied in the ADAMTS13^?/? mouse. Results: Murine MDTCS (devoid of T2‐8 and CUB domains) retained full enzyme activity in vitro towards FRETS‐VWF73 and the C‐terminal T6‐8 (del(T6‐CUB)) and CUB domains (delCUB) are dispensable under these assay conditions. In addition, mADAMTS13 fragments without the spacer domain (MDT and M) had reduced catalytic efficiencies. Our results hence indicate that similar domains in murine and human ADAMTS13 are required for activity in vitro, supporting the use of mouse models to study ADAMTS13 function in vivo. Interestingly, using intravital microscopy we show that removal of the CUB domains abolishes proteolysis of platelet‐decorated VWF strings in vivo. In addition, whereas MDTCS is fully active in vivo, partial (del(T6‐CUB)) or complete (delCUB) addition of the T2‐8 domains gradually attenuates its activity. Conclusions: Our data demonstrate that the ADAMTS13 CUB and T2‐8 domains influence proteolysis of platelet‐decorated VWF strings in vivo.     

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.     

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.     

Emerging roles of adjunct therapies in acquired thrombotic thrombocytopenia purpura

• Acquired thrombotic thrombocytopenia purpura (aTTP) is caused by autoantibody‐mediated severe deficiency of the von Willebrand factor (vWF) cleaving protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13), with subsequent accumulation of ultra‐large vWF‐multimers that spontaneously form platelet‐VWF complexes and microthrombi within the microcirculation.
• Therapeutic plasma exchange (TPE), by removing autoantibodies and excess ultra‐large vWF multimers and replenishing ADAMTS13 activity, remains the urgent primary initial treatment. Although heterogeneity in treatment exists, most centers add upfront immunosuppression with steroids, and many also add upfront rituximab. Refractoriness, exacerbation and relapse are commonly treated with adjunct rituximab.
• Despite adjunct steroids and rituximab, TTP refractoriness, exacerbation, relapse, morbidity, and mortality remain problematic. Newer adjunct therapies include suppression of ADAMTS13 autoantibody production via plasma cell depletion, inhibition of vWF‐platelet interaction, and replenishment of ADAMTS13 function with recombinant ADAMTS13 protein.

     

The membrane‐proximal intermolecular disulfide bonds in glycoprotein Ib influence receptor binding to von Willebrand factor

Summary. Background: In the platelet glycoprotein (GP)Ib–IX complex, the binding site for its ligand von Willebrand factor (VWF) is restricted to the N‐terminal domain of the GPIbα subunit. How the other subunits in the complex, GPIbβ and GPIX, regulate the GPIbα–VWF interaction is not clear. Objectives and methods: As GPIbα connects with two GPIbβ subunits via disulfide bonds, we tested whether these intersubunit covalent links were important to the proper VWF‐binding activity of the GPIb–IX complex by characterizing the structure and VWF‐binding activity of a mutant GPIb–IX complex that lacked the GPIbα–GPIbβ disulfide bonds. Results: Mutating both Cys484 and Cys485 of GPIbα to serine prevents GPIbα from forming covalent disulfide bonds with GPIbβ, while maintaining the integrity of the complex in the membrane. The mutations cause two GPIbβ subunits to form a disulfide bond between themselves. As compared to Chinese hamster ovary (CHO) cells stably expressing the wild‐type GPIb–IX complex at a comparable level, CHO cells stably expressing the mutant GPIb–IX complex bind to significantly less soluble VWF in the presence of ristocetin and roll on the immobilized VWF under flow at a higher velocity. Conclusions: The disulfide bonds between GPIbα and GPIbβ are necessary for optimal GPIbα binding to VWF. The structural plasticity around the disulfide bonds may also help to shed light on the inside‐out mechanism underlying GPIbβ modulation of VWF binding.     

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.

     

Recombinant ADAMTS13 normalizes von Willebrand factor‐cleaving activity in plasma of acquired TTP patients by overriding inhibitory antibodies

Summary. Background: Severe deficiency of the von Willebrand factor (VWF)‐cleaving protease ADAMTS13 as observed in acquired thrombotic thrombocytopenic purpura (TTP) is caused by inhibitory and non‐inhibitory autoantibodies directed against the protease. Current treatment with plasma exchange is considered to remove circulating antibodies and to concurrently replenish the deficient enzyme. Objectives: To explore the use of recombinant ADAMTS13 (rADAMTS13) as a potential therapeutic agent in acquired TTP, we investigated its efficacy in normalizing VWF‐cleaving activity in the presence of ADAMTS13 inhibitors. Methods: Thirty‐six plasma samples from TTP patients were adjusted to predefined inhibitor titers, and recovery of ADAMTS13 activity was analyzed following supplementation with rADAMTS13. Results: We showed a linear relation between the inhibitor titer measured and effective rADAMTS13 concentration necessary for reconstitution of VWF‐cleaving activity in the presence of neutralizing autoantibodies. Conclusions: Our results support the further investigation of the potential therapeutic applicability of rADAMTS13 as an adjunctive therapy in acquired TTP.     

A shear-based assay for assessing plasma ADAMTS13 activity and inhibitors in patients with thrombotic thrombocytopenic purpura

BACKGROUND: Severe deficiency of plasma ADAMTS13 activity is a frequent finding in patients with hereditary and acquired thrombotic thrombocytopenic purpura (TTP). To date, plasma ADAMTS13 activity is determined by cleavage of either predenatured von Willebrand factor (VWF) or small peptides derived from the VWF‐A2 domain. The physiologic relevance of the assay results is uncertain. STUDY DESIGN AND METHODS: We sought to develop a novel shear‐based assay to assess plasma ADAMTS13 activity and inhibitors. We also compared this assay with a fluorogenic peptide assay. RESULTS: We found that an incubation of purified plasma VWF with 0.5 to 1.0 µL of citrated plasma under constant vortexing at 2500 rpm for 60 minutes in the presence of 5 mmol/L CaCl₂ and 1.7 µmol/L ZnCl₂ and low concentration of NaCl resulted in the maximal cleavage of VWF. The cleavage product could be separated by a 2.5% agarose gel and detected by Western blotting. The assay revealed that plasma and recombinant ADAMTS13 are highly sensitive to inhibition by zinc and chloride ions. Under the optimal conditions, the shear‐based assay appeared to be more sensitive than the guanidine‐denaturization assay for determining plasma ADAMTS13 activity. CONCLUSIONS: Our fluid shear‐based assay may be useful for investigating basic biologic function and regulation of ADAMTS13 metalloprotease. It may also be applicable for assessing plasma ADAMTS13 activity and inhibitors in TTP patients.     

Platelet reactive conformation and multimeric pattern of von Willebrand factor in acquired thrombotic thrombocytopenic purpura during acute disease and remission

Summary. Background: Binding of von Willebrand factor (VWF) multimers of ultra‐large size to platelets is considered the triggering mechanism of microvascular thrombosis in thrombotic thrombocytopenic purpura (TTP). Objective: To assess the potential of VWF‐related measurements as markers of disease activity and severity in TTP. Methods: VWF antigen (VWF:Ag), platelet glycoprotein‐Ib‐α binding‐conformation (GPIb‐α/BC) and multimeric pattern were investigated in 74 patients with acquired TTP during acute disease, remission or both and 73 healthy controls. In patients with both acute and remission samples available, VWF ristocetin co‐factor activity (VWF:RCo) and collagen binding (VWF:CB) were also measured. The relationships of study measurements with the presence of acute disease and remission and with markers of disease severity were assessed. Results: VWF:Ag and VWF‐GPIb‐α/BC were higher in TTP patients than controls (P < 0.001 and 0.004). However, there was no statistically significant difference in VWF‐GPIb‐α/BC between samples obtained during acute TTP and remission. Larger VWF multimers were frequently lacking in acute TTP patients, who displayed ultra‐large multimers at remission. The degree of loss of larger VWF multimers correlated with the degree of abnormality of hemoglobin, platelet counts and serum lactate dehydrogenase (LDH) and was associated with low levels of both VWF:RCo/Ag and VWF:CB/Ag ratios. Conclusions: In TTP the platelet‐binding conformation of VWF is not exclusively present in acute disease, nor is it associated with its clinical and laboratory severity. The loss of larger VWF multimers, accompanied by low VWF:RCo/Ag and VWF:CB/Ag ratio values, represents an index of disease activity and severity of acute TTP in patients with severe ADAMTS‐13 deficiency.     

Cleavage of ultra-large von Willebrand factor by ADAMTS-13 under flow conditions

von Willebrand factor (VWF) is a critical ligand for platelet adhesion and aggregation. It is synthesized and released as multimers composed of various numbers of monomers. When first released from the storage granules of endothelial cells, VWF multimers are rich in the ultra-large (UL) forms that spontaneously bind the GP Ib-IX complex and aggregate platelets. These prothrombotic ULVWF multimers are rapidly cleaved by the metalloprotease ADAMTS-13 (A Disintegrin and Metalloprotease with ThromboSpondin motif) to smaller and much less active forms. Recently, several methods have been developed to measure ADAMTS-13 activity in vitro and to link its deficiency to thrombotic thrombocytopenic purpura. Correlations between the structure and functions of the metalloprotease have also been extensively studied using recombinant technologies. However, questions remain regarding the proper substrate for the metalloprotease, the time and location of ULVWF proteolysis, and the role of fluid shear stress. In this brief review, we have discussed a potential model for ULVWF proteolysis by ADAMTS-13 in vivo. In this model, ULVWF is anchored to the surface of endothelial cells to form string-like structures under fluid shear stress. Such an elongated conformation facilitates ULVWF cleavage by exposing either the cleavage or binding sites for the metalloprotease. When ADAMTS-13 is deficient, the uncleaved ULVWF accumulates in plasma and on endothelial cells to capture platelets. This leads to platelet aggregation and thromboembolism. Dissecting the process of ULVWF proteolysis is important for not only understanding the pathophysiology of thrombotic microangiopathies, but also developing more effective means to treat these deadly diseases.     

Variations among normal individuals in the cleavage of endothelial-derived ultra-large von Willebrand factor under flow.

von Willebrand factor (VWF) freshly released from endothelial cells is normally cleaved by the ADAMTS-13 metalloprotease to prevent the direct release of these ultra-large (UL) and hyper-reactive multimers into plasma. The balance of ULVWF proteolysis may be regulated by the amount of ULVWF released and the processing capacity of ADAMTS-13. The former associates with the size of ULVWF storage pool, sensitivity of vascular endothelial cells to stimulation, and the type of agonists, whereas the latter associates with the activity of ADAMTS-13. These parameters may vary significantly among individuals. We have determined the variations of ADAMTS-13 activity in 68 normal individuals by a flow-based assay and a static assay using ULVWF strings and recombinant VWF A2 domain as substrates, respectively. We found that the levels of ADAMTS-13 activity required to cleave the platelet-decorated ULVWF strings under flow is significantly higher than that of static assays. Normal plasma diluted to 25% significantly reduced its ability to cleave ULVWF strings under flow, whereas 2% plasma retained 48% enzyme activity in static assay. ADAMTS-13 activity varied from 33 to 100% among individuals and the variations were greater at shorter incubations of plasma with the substrate. Furthermore, the production of ULVWF from endothelial cells also varied among individuals. These results suggest that the commonly used static assays may underestimate the ADAMTS-13 activity required to cleave newly released ULVWF. They also demonstrated that the proteolysis of ULVWF may vary significantly among individuals, potentially contributing to the individual's vulnerability to thrombosis so that measurement of ADAMTS-13 may serve as a marker for TTP and other thrombotic diseases.     

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.     

Novel monoclonal antibody-based enzyme immunoassay for determining plasma levels of ADAMTS13 activity

BACKGROUND: ADAMTS13 specifically cleaves unusually large von Willebrand factor (VWF) multimers, which induce platelet thrombi formation under high shear stress. ADAMTS13 activity is deficient in patients with thrombotic thrombocytopenic purpura (TTP). The determination of plasma levels of ADAMTS13 activity is a prerequisite for a differential diagnosis of thrombotic microangiopathies. Here, a unique and highly sensitive enzyme immunoassay (EIA) of ADAMTS13 activity is described. STUDY DESIGN AND METHODS: ADAMTS13 hydrolyzes the peptide bond between Y1605 and M1606 of VWF. In this assay, a recombinant fusion protein (GST-VWF73-His) is used as a substrate. A panel of mouse monoclonal antibodies (MoAbs) that specifically recognizes Y1605, which is the C-terminal edge residue of the VWF-A2 domain and is generated by the enzymatic cleavage, has been produced. These antibodies were prepared with a synthetic decapeptide, termed N-10 (1596-DREQAPNLVY-1605), as the immunogen. Twenty-six clones specific to N10 were obtained, and one anti-N10 MoAb was used in this study. RESULTS: With horseradish peroxidase-conjugated anti-N10 MoAb, a standard enzyme assay was established. This assay was highly sensitive, and the detection limit was 0.5 percent of the normal. Further, an inhibitor of ADAMTS13 was measured to a level of 0.1 Bethesda units per mL. ADAMTS13 activity was measured in 20 patients with Upshaw-Schulman syndrome, a congenital TTP, and 61 acquired TTP patients. The activity measured by this assay and by the classic VWF multimer assay showed high correlation. CONCLUSION: A convenient and highly sensitive EIA for ADAMTS13 activity has been established. This assay can be introduced for routine laboratory work in transfusion medicine.     

Redox control of β2‐glycoprotein I–von Willebrand factor interaction by thioredoxin‐1

Summary. Background: β₂‐Glycoprotein I (β₂GPI) is an abundant plasma protein that is closely linked to blood clotting, as it interacts with various protein and cellular components of the coagulation system. However, the role of β₂GPI in thrombus formation is unknown. We have recently shown that β₂GPI is susceptible to reduction by the thiol oxidoreductases thioredoxin‐1 and protein disulfide isomerase, and that reduction of β₂GPI can take place on the platelet surface. Methods: β₂GPI, reduced by thioredoxin‐1, was labeled with the selective sulfhydryl probe N^a‐(3‐maleimidylpropionyl)biocytin and subjected to mass spectrometry to identify the specific cysteines involved in the thiol exchange reaction. Binding assays were used to examine the affinity of reduced β₂GPI for von Willebrand factor (VWF) and the effect of reduced β2GPI on glycoprotein (GP)Ibα binding to VWF. Platelet adhesion to ristocetin‐activated VWF was studied in the presence of reduced β₂GPI. Results: We demonstrate that the Cys288–Cys326 disulfide in domain V of β₂GPI is the predominant disulfide reduced by thioredoxin‐1. Reduced β₂GPI in vitro displays increased binding to VWF that is dependent on disulfide bond formation. β₂GPI reduced by thioredoxin‐1, in comparison with non‐reduced β₂GPI, leads to increased binding of GPIbα to VWF and increased platelet adhesion to activated VWF. Conclusions: Given the importance of thiol oxidoreductases in thrombus formation, we provide preliminary evidence that the thiol‐dependent interaction of β₂GPI with VWF may contribute to the redox regulation of platelet adhesion.