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.     

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.

     

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.     

Effect of von Willebrand disease type 2B and type 2M mutations on the susceptibility of von Willebrand factor to ADAMTS-13

BACKGROUND: von Willebrand disease (VWD) type 2 is associated with mutations in von Willebrand factor (VWF) that affect its secretion, multimeric pattern, affinity for platelet receptors and clearance of the protein. While increased proteolysis by a disintegrin-like and metalloprotease with thrombospondin type 1 motifs-13 (ADAMTS-13) has been clearly established for VWF type 2A, only little is known about VWF types 2B and 2M in this regard. OBJECTIVES: Sensitivity of wild-type (WT) and mutated recombinant (r) VWF to proteolysis by ADAMTS-13 was investigated to better understand the role of this process in the pathophysiology of VWD. METHODS: We used human rADAMTS-13-WT to digest 11 full-length recombinant forms of VWF carrying molecular abnormalities identified in patients with VWD type 2A (E1638K and P1648S), type 2B (InsM1303, R1306W, R1308P and V1314F) and type 2M (G1324A, E1359K, K1362T, R1374H and I1425F). RESULTS: Using low ionic strength conditions, all mutations induced increased proteolysis of rVWF by rADAMTS-13 as compared with rVWF-WT. The susceptibility of mutants decreased in the following order: type 2A > type 2B > type 2M > rVWF-WT. At physiological salt concentration (150 mm NaCl) the sensitivity of all rVWF to rADAMTS-13 was significantly decreased. However, type 2A and type 2B mutants still exhibited a significantly higher susceptibility to rADAMTS-13 than rVWF-WT, whereas type 2M mutants normalized. CONCLUSIONS: Type 2M mutants and rVWF-WT exhibit a similar sensitivity to rADAMTS-13-mediated proteolysis, in agreement with the normal multimeric pattern in vivo. In VWD type 2B, the spontaneous binding to platelets and excessive degradation by ADAMTS-13 of VWF high-molecular-weight multimers may account for their clearance from plasma.     

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.     

Role of von Willebrand factor and ADAMTS‐13 in early brain injury after experimental subarachnoid hemorrhage

Essentials

• von Willebrand Factor (VWF) and ADAMTS13 may affect early injury after subarachnoid hemorrhage (SAH).
• Early brain injury was assessed in VWF^?/?, ADAMTS13^?/? and recombinant (r) ADAMTS13 treated mice.
• VWF^?/? and rADAMTS13 treated mice had less brain injury than ADAMTS13^?/? and wild‐type mice.
• Early administration of rADAMTS13 may improve outcome after SAH by reducing early brain injury.

Summary

Background

Early brain injury is an important determinant of poor functional outcome and case fatality after aneurysmal subarachnoid hemorrhage (SAH), and is associated with early platelet aggregation. No treatment exists for early brain injury after SAH. We investigated whether von Willebrand factor (VWF) is involved in the pathogenesis of early brain injury, and whether ultra‐early treatment with recombinant ADAMTS‐13 (rADAMTS‐13) reduces early brain injury after experimental SAH.

Methods

Experimental SAH in mice was induced by prechiasmatic injection of non‐anticoagulated blood from a littermate. The following experimental SAH groups were investigated: C57BL/6J control (n = 21), VWF^?/? (n = 25), ADAMTS‐13^?/? (n = 23), and C57BL/6J treated with rADAMTS‐13 (n = 26). Mice were killed at 2 h after SAH. Primary outcome measures were microglial activation (IBA‐1 surface area) and neuronal injury (number of cleaved caspase‐3‐positive neurons).

Results

As compared with controls, microglial activation was decreased in VWF^?/? mice (mean difference of ? 20.0%, 95% confidence interval [CI] ? 4.0% to ? 38.6%), increased in ADAMTS‐13^?/? mice (mean difference of + 34.0%, 95% CI 16.2–51.7%), and decreased in rADAMTS‐13‐treated mice (mean difference of ? 22.1%, 95% CI ? 3.4% to ? 39.1%). As compared with controls (185 neurons, interquartile range [IQR] 133–353), neuronal injury in the cerebral cortex was decreased in VWF^?/? mice (63 neurons, IQR 25–78), not changed in ADAMTS‐13^?/? mice (53 neurons, IQR 26–221), and reduced in rADAMTS‐13‐treated mice (45 neurons, IQR 9–115).

Conclusions

Our findings suggest that VWF is involved in the pathogenesis of early brain injury, and support the further study of rADAMTS‐13 as a treatment option for early brain injury after SAH.

     

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.     

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.     

房颤患者vWF与ADAMTS-13水平的变化及意义

目的 研究房颤患者vWF、ADAMTS-13的变化及意义.方法 分别测定27例持续性房颤、20例阵发性房颤患者和25例正常对照组血浆血管性血友病因子（vWF）、血管性血友病因子裂解酶（ADAMTS-13）水平、D-二聚体（D-D）水平,测vWF/ADAMTS-13比值.结果 与对照组比较,阵发性房颤组、持续性房颤组的vWF、D-D均显著增高、ADAMTS-13明显下降,vWF/ADAMTS-13比值明显增高;vWF/ADAMTS-13比值在阵发性房颤组和持续性房颤组间差异有统计学意义,且持续性房颤组比值与D-D水平呈正相关.结论 房颤患者存在有明显vWF、ADAMTS-13水平变化,且与其可能出现的血栓栓塞并发症有密切关系.