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.     

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.     

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.     

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.     

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.     

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.     

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.     

Collagen‐mimetic peptides mediate flow‐dependent thrombus formation by high‐ or low‐affinity binding of integrin α2β1 and glycoprotein VI

Summary. Background: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen‐derived triple‐helical peptides have identified the GXX’GER motif as an adhesive ligand for platelet integrin α2β1, and (GPO)_n as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). Objective: The potency was investigated of triple‐helical peptides, consisting of GXX’GER sequences within (GPO)_n or (GPP)_n motifs, to support flow‐dependent thrombus formation. Results: At a high‐shear rate, immobilized peptides containing both the high‐affinity α2β1‐binding motif GFOGER and the (GPO)_n motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co‐immobilized VWF was needed for thrombus formation. The (GPO)_n but not the (GPP)_n sequence induced GPVI‐dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low‐affinity (GASGER, GAOGER) α2β1‐binding motifs formed procoagulant thrombi only if both (GPO)_n and VWF were present. At a low‐shear rate, immobilized peptides with high‐ or low‐affinity α2β1‐binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)_n. Conclusions: Triple‐helical peptides with specific receptor‐binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high‐shear rate, either GPIb or high‐affinity (but not low‐affinity) GXX’GER mediates GPVI‐dependent formation of procoagulant thrombi. By extension, high‐affinity binding for α2β1 can control the overall platelet‐adhesive activity of native collagens.     

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.     

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.     

Functional association of phosphoinositide‐3‐kinase with platelet glycoprotein Ibα, the major ligand‐binding subunit of the glycoprotein Ib–IX–V complex

Summary. Background: The adhesion receptor glycoprotein (GP)Ib–IX–V, which binds von Willebrand factor (VWF) and other ligands, initiates platelet activation and thrombus formation at arterial shear rates, and may control other vascular processes, such as coagulation, inflammation, and platelet‐mediated tumor metastasis. The cytoplasmic C‐terminal domain of the ligand‐binding GPIbα subunit contains binding sites for filamin (residues 561–572, critically Phe568/Trp570), 14‐3‐3ζ (involving phosphorylation sites Ser587/590 and Ser609), and the phosphoinositide‐3‐kinase (PI3‐kinase) regulatory subunit, p85. Objectives: We previously showed that, as compared with wild‐type receptor, deleting the contiguous sequence 580–590 or 591–610, but not upstream sequences, of GPIbα expressed as a GPIb–IX complex in Chinese hamster ovary cells inhibited VWF‐dependent Akt phosphorylation, which is used as a read‐out for PI3‐kinase activity. Pulldown experiments using glutathione‐S‐transferase (GST)–p85 or GST–14‐3‐3ζ constructs, and competitive inhibitors of 14‐3‐3ζ binding, suggested an independent association of 14‐3‐3ζ and PI3‐kinase with GPIbα. The objective of this study was to analyze a further panel of GPIbα deletion mutations within residues 580–610. Results: We identified a novel deletion mutant, Δ591–595, that uniquely disrupts 14‐3‐3ζ binding but retains the functional p85/PI3‐kinase association. Deletion of other sequences within the 580–610 region were less discriminatory, and either partially affected p85/PI3‐kinase and 14‐3‐3ζ binding (Δ580–585, Δ586–590, Δ596–600, Δ601–605), or strongly inhibited binding of both proteins (Δ606–610). Conclusions: Together, these findings have significant implications for interpreting the functional role of p85 and/or 14‐3‐3ζ in GPIb‐dependent signaling or platelet functional studies involving truncation of the C‐terminal residues in cell‐based assays and mouse models. The Δ591–595 mutation provides another strategy for determining the function of GPIbα‐associated 14‐3‐3ζ by selective disruption of 14‐3‐3ζ but not p85/PI3‐kinase binding.     

A multi‐stage multi‐design strategy provides strong evidence that the BAI3 locus is associated with early‐onset venous thromboembolism

Summary. Background: Factor VIII (FVIII) and von Willebrand factor (VWF) are two known quantitative risk factors for venous thromboembolism (VTE). Objectives: To identify new loci that could contribute to VTE susceptibility and to modulating FVIII and/or VWF levels. Patients/Methods: A pedigree linkage analysis was first performed in five extended French‐Canadian families, including 253 individuals, to identify genomic regions linked to FVIII or VWF levels. Identified regions were further explored using ‘in silico’ genome‐wide association studies (GWAS) data on VTE (419 patients and 1228 controls), and two independent case‐control studies (MARTHA and FARIVE) for VTE, gathering 1166 early‐onset patients and 1408 healthy individuals. Single nucleotide polymorphisms (SNPs) associated with VTE risk were further investigated in relation to plasma levels of FVIII and VWF in a cohort of 108 healthy nuclear families. Results: Four main linkage regions were identified, among which the well‐characterized ABO locus, the recently identified STAB 2 gene, and a third one, on chromosome 6q13‐14, harbouring four non‐redundant SNPs, associated with VTE at P < 10^?4 in the GWAS dataset. The association of one of these SNPs, rs9363864, with VTE was further replicated in the MARTHA and FARIVE studies. The rs9363864‐AA genotype was associated with a lower risk for VTE (OR = 0.58 [0.42–0.80], P = 0.0005) but mainly in non‐carriers of the FV Leiden mutation. This genotype was further found to be associated with the lowest levels of FVIII (P = 0.006) and VWF (P = 0.001). Conclusions: The BAI3 locus where the rs9363864 maps is a new candidate for VTE risk.     

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.

     

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.     

Von Willebrand factor and ADAMTS13 balancing primary haemostasis

Von Willebrand factor (VWF) is an adhesive, multi-functional huge multimerized protein with multiple domains harboring binding sites for collagen, platelet glycoprotein receptors and coagulation factor VIII (FVIII). The functional domains enable VWF to bind to the injured vessel wall, to recruit platelets to the site of injury by adhesion and aggregation and to bind and protect FVIII, an important cofactor of the coagulation cascade. VWF function in primary haemostasis is located in particular in the arterial and micro-circulation. This environment is exposed to high shear forces with hydrodynamic shear rates ranging over several orders of magnitude from 10?1 to 10? s-1 and requires particular mechanisms to enable platelet adhesion and aggregation under these variable conditions. The respective VWF function is strictly correlating with its multimer size. Lack or reduction of large VWF multimers is seen in patients with von Willebrand disease (VWD) type 2A which correlates with reduction of both VWF:platelet GPIb-binding and VWF:collagen binding and a bleeding phenotype. To prevent unlimited platelet adhesion and aggregation which is the cause of the microangiopathic disorder thrombotic thrombocytopenic purpura (TTP), VWF function is regulated by its specific protease ADAMTS13. Whereas a particular susceptibility of VWF to ADAMTS13 proteolysis is the cause of a frequent VWD type 2A phenotype, lack or dysfunction of ADAMTS13, either acquired by ADAMTS13 antibodies or by inherited ADAMTS13 deficiency (Upshaw-Schulman Syndrome), causes TTP. Therefore VWD and TTP represent the opposite manifestations of VWF related disorders, tightly linked to each other.     

AT1 blockade abolishes left ventricular hypertrophy in heterozygous cMyBP‐C null mice: role of FHL1

This research investigated the impact of angiotensin AT1 receptor (Agtr1) blockade on left ventricular (LV) hypertrophy in a mouse model of human hypertrophic cardiomyopathy (HCM), which carries one functional allele of Mybpc3 gene coding cardiac myosin‐binding protein C (cMyBP‐C). Five‐month‐old heterozygous cMyBP‐C knockout (Het‐KO) and wild‐type mice were treated with irbesartan (50 mg/kg/day) or vehicle for 8 weeks. Arterial blood pressure was measured by tail cuff plethysmography. LV dimension and function were accessed by echocardiography. Myocardial gene expression was evaluated using RT‐qPCR. Compared with wild‐type littermates, Het‐KO mice had greater LV/body weight ratio (4.0 ± 0.1 vs. 3.3 ± 0.1 mg/g, P < 0.001), thicker interventricular septal wall (0.70 ± 0.02 vs. 0.65 ± 0.01 mm, P < 0.02), lower Mybpc3 mRNA level (?43%, P < 0.02), higher four‐and‐a‐half LIM domains 1 (Fhl1, +110%, P < 0.01), and angiotensin‐converting enzyme 1 (Ace1, +67%, P < 0.05), but unchanged Agtr1 mRNA levels in the septum. Treatment with irbesartan had no effect in wild‐type mice but abolished septum‐predominant LV hypertrophy and Fhl1 upregulation without changes in Ace1 but with an increased Agtr1 (+42%) in Het‐KO mice. Thus, septum‐predominant LV hypertrophy in Het‐KO mice is combined with higher Fhl1 expression, which can be abolished by AT1 receptor blockade, indicating a role of the renin‐angiotensin system and Fhl1 in cMyBP‐C‐related HCM.     

Functional near‐infrared spectroscopy to probe sensorimotor region activation during electrical stimulation‐evoked movement

This study used non‐invasive functional near‐infrared spectroscopy (fNIRS) neuroimaging to monitor bilateral sensorimotor region activation during unilateral voluntary (VOL) and neuromuscular electrical stimulation (NMES)‐evoked movements. Methods. In eight healthy male volunteers, fNIRS was used to measure relative changes in oxyhaemoglobin (O₂Hb) and deoxyhaemoglobin (HHb) concentrations from a cortical sensorimotor region of interest in the left (LH) and right (RH) hemispheres during NMES‐evoked and VOL wrist extension movements of the right arm. Results. NMES‐evoked movements induced significantly greater activation (increase in O₂Hb and concomitant decrease in HHb) in the contralateral LH than in the ipsilateral RH (O₂Hb: 0·44 ± 0·16 μM and 0·25 ± 0·22 μM, P = 0·017; HHb: ?0·19 ± 0·10 μM and ?0·12 ± 0·09 μM, P = 0·036, respectively) as did VOL movements (0·51 ± 0·24 μΜ and 0·34 ± 0·21 μM, P = 0·031; HHb: ?0·18 ± 0·07 μΜ and ?0·12 ± 0·04 μΜ, P = 0·05, respectively). There was no significant difference between conditions for O₂Hb (P = 0·144) and HHb (P = 0·958). Conclusion. fNIRS neuroimaging enables quantification of bilateral sensorimotor regional activation profiles during voluntary and NMES‐evoked wrist extension movements.     

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.

     

The glycoprotein Ibα–von Willebrand factor interaction induces platelet apoptosis

Summary. Background: The interaction of glycoprotein (GP) Ibα with von Willebrand factor (VWF) initiates platelet adhesion, and simultaneously triggers intracellular signaling cascades leading to platelet aggregation and thrombus formation. Some of the signaling events are similar to those occurring during apoptosis, however, it is still unclear whether platelet apoptosis is induced by the GPIbα–VWF interaction. Objectives: To investigate whether the GPIbα–VWF interaction induces platelet apoptosis and the role of 14‐3‐3ζ in apoptotic signaling. Methods: Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing wild‐type (1b9) or mutant GPIb–IX interacting with VWF by flow cytometry or western blotting. Results: Ristocetin‐induced GPIbα–VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF; however, the shear‐induced apoptosis was eliminated by the anti‐GPIbα antibody AK2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb–IX with GPIbα truncated at residue 551 or a serine‐to‐alanine mutation at the 14‐3‐3ζ‐binding site in GPIbα. Conclusions: This study demonstrates that the GPIbα–VWF interaction induces apoptotic events in platelets, and that the association of 14‐3‐3ζ with the cytoplasmic domain of GPIbα is essential for apoptotic signaling. This finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases.