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.     

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.

     

Ten candidate ADAMTS13 mutations in six French families with congenital thrombotic thrombocytopenic purpura (Upshaw–Schulman syndrome)

Summary.  ADAMTS13, the specific von Willebrand factor (VWF)-cleaving metalloprotease, prevents the spontaneous formation of platelet thrombi in the microcirculation by degrading the highly adhesive ultralarge VWF multimers into smaller forms. ADAMTS13 severe enzymatic deficiency and mutations have been described in the congenital thrombotic thrombocytopenic purpura (TTP or Upshaw–Schulman syndrome), a rare and severe disease related to multivisceral microvascular thrombosis. We investigated six French families with congenital TTP for ADAMTS13 enzymatic activity and gene mutations. Six probands with congenital TTP and their family were tested for ADAMTS13 activity in plasma using a two-site immunoradiometric assay and for ADAMTS13 gene mutations using polymerase chain reaction and sequencing. ADAMTS13 activity was severely deficient (< 5%) in the six probands and one mildly symptomatic sibling but normal (> 50%) in all the parents and the asymptomatic siblings. Ten novel candidate ADAMTS13 mutations were identified in all families, showing either a compound heterozygous or a homozygous status in all probands plus the previous sibling and a heterozygous status in the parents. The mutations were spread all over the gene, involving the metalloprotease domain (I79M, S203P, R268P), the disintegrin domain (29 bp deletion in intron/exon 8), the cystein-rich domain (acceptor splice exon 12, R507Q), the spacer domain (A596V), the 3rd TSP1 repeat (C758R), the 5th TSP1 repeat (C908S) and the 8th TSP1 repeat (R1096stop). This study emphasizes the role of ADAMTS13 mutations in the pathogenesis of congenital TTP and suggests that several structural domains of this metalloprotease are involved in both its biogenesis and its substrate recognition process.     

Acute activation of the endothelium results in increased levels of active von Willebrand factor in hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome

BACKGROUND: HELLP (hemolysis, elevated liver enzymes and low platelets) syndrome is a severe complication of pre-eclampsia in pregnancy, characterized by microvascular platelet thrombi. Activation of the endothelium is thought to play a key role in pre-eclampsia and HELLP syndrome. Activation of endothelial cells may lead to release of von Willebrand factor (VWF) multimers, which are highly reactive with platelets. Normally, newly released multimers are cleaved by ADAMTS13, resulting in less reactive derivatives. OBJECTIVE: We hypothesized that HELLP syndrome is characterized by increased amounts of active VWF compared with healthy pregnancy and pre-eclampsia, due to acute activation of endothelial cells. This might contribute to thrombocytopenia and thrombotic microangiopathy. METHODS: Active VWF and ADAMTS13 activity were measured in healthy pregnant volunteers (n = 9), patients with pre-eclampsia (n = 6) and patients with HELLP syndrome (n = 14) at similar gestational ages. To study the role of endothelial cell activation, the propeptide/mature VWF ratio was determined, and VWF released by cultured endothelial cells was analyzed. RESULTS: Active VWF levels were increased 2.1-fold in HELLP syndrome compared with healthy pregnant volunteers (P < 0.001) and 1.6-fold compared with patients with pre-eclampsia (P = 0.001). ADAMTS13 activity was moderately decreased in patients with HELLP syndrome compared with healthy pregnant volunteers (P < 0.004), but not compared with patients with pre-eclampsia. The propeptide/mature VWF ratio was increased 1.7-fold compared with healthy pregnant volunteers (P < 0.001) and 1.5-fold compared with patients with pre-eclampsia (P < 0.05). A significant correlation was found between this ratio and the activation factor of VWF (r = 0.68, P < 0.001). The amount of active VWF was increased 1.4-fold in medium of stimulated endothelial cells when compared with non-stimulated cells (P < 0.05). CONCLUSION: Acute endothelial cell activation in HELLP syndrome and decreased ADAMTS13 activity result in increased amounts of active VWF. This might explain the consumptive thrombocytopenia and thrombotic microangiopathy associated with HELLP syndrome. Inhibition of circulating active VWF could be a potential new approach in the treatment of patients with HELLP syndrome.     

The role of ADAMTS13 in the pathogenesis of thrombotic thrombocytopenic purpura-hemolytic uremic syndrome

The identification, characterization, and clinical observation of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin-1-like domains) have provided important insights into the pathogenesis of thrombotic thrombocytopenic purpura (TTP). ADAMTS13 is a plasma enzyme essential for postsecretion proteolytic processing of von Willebrand factor (VWF). Absence of ADAMTS13 is associated with the occurrence of abnormally large multimers of VWF and is also associated with the occurrence of TTP. Initial assumptions that absent ADAMTS13 was itself the etiology of TTP have been tempered by subsequent observations that ADAMTS13 activity can be severely deficient without clinical abnormalities and that patients can have characteristic clinical features of TTP without severe ADAMTS13 deficiency. A current interpretation of these observations is that ADAMTS13 deficiency is a major risk factor for the development of TTP, but it is neither always necessary nor sufficient to cause TTP. This interpretation is consistent with other vascular and thrombotic disorders in which multiple risk factors and associated conditions contribute to the etiology of acute events.     

Use of n-acetylcysteine therapy in patients with relapsed refractory thrombotic thrombocytopenic purpura

There is limited data on the use of NAC in the literature. We would like to present the satisfactory results we obtained in our resistant and relapsed patients as a case series.Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy caused by ADAMTS13 (a disintegrin with thrombospondin type 1 motif and metalloprotease activity, member13) deficiency. Von Willebrand factor (vWF) initiates platelet aggregation and thus thrombus formation. The multimers of vWF are cleaved by ADAMTS13. Because of the decreased activity of ADAMTS13, ultra-large multimers accumulate and end-organ damage occurs. TTP is characterized by microangiopathic hemolytic anemia (MAHA), severe thrombocytopenia, and organ ischemia resulting from vascular occlusion caused by thrombi. Plasma exchange therapy (PEX) remains the mainstay of TTP therapy. Patients who do not respond to PEX and corticosteroids require additional treatments such as rituximab and caplacizumab. NAC reduces disulfide bonds in mucin polymers through its free sulfhydryl group. Thus, the size and viscosity of the mucins are reduced. VWF is structurally similar to mucin. Based on this similarity, Chen and colleagues showed that NAC can reduce the size and reactivity of ultralarge multimers of vWF, such as ADAMTS13. Currently, there is not much information to suggest that NAC has any clinical value in the treatment of TTP. In this case series of 4 refractory patients, we would like to present the responses we obtained with the addition of NAC therapy. NAC can be added to PEX and glucocorticoid therapy as supportive therapy, especially in unresponsive patients.     

ADAMTS13: origins,applications, and prospects

ADAMTS13 is an enzyme that acts by cleaving prothrombotic von Willebrand factor (VWF) multimers from the vasculature in a highly regulated manner. In pathologic states such as thrombotic thrombocytopenic purpura (TTP) and other thrombotic microangiopathies (TMAs), VWF can bind to the endothelium and form large multimers. As the anchored VWF chains grow, they provide a greater surface area to bind circulating platelets (PLTs), generating unique thrombi that characterize TTP. This results in microvasculature thrombosis, obstruction of blood flow, and ultimately end‐organ damage. Initial presentations of TTP usually occur in an acute manner, typically developing due to an autoimmune response toward, or less commonly a congenital deficiency of, ADAMTS13. Triggers for TMAs that can be associated with ADAMTS13 deficiency, including TTP, have been linked to events that place a burden on hemostatic regulation, such as major trauma and pregnancy. The treatment plan for cases of suspected TTP consists of emergent therapeutic plasma exchange that is continued on a daily basis until normalization of PLT counts. However, a subset of these patients does not respond favorably to standard therapies. These patients necessitate a better understanding of their diseases for the advancement of future therapeutic options. Given ADAMTS13’s key role in the cleavage of VWF and the prevention of PLT‐rich thrombi within the microvasculature, future treatments may include anti‐VWF therapeutics, recombinant ADAMTS13 infusions, and ADAMTS13 expression via gene therapy.     

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.     

VH1-69 germline encoded antibodies directed towards ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura

Summary.  Background: Autoantibodies directed towards ADAMTS13 are present in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). Analysis of a set of antibodies derived from two patients with acquired TTP revealed frequent use of the VH1-69 heavy chain gene segment for the assembly of anti-ADAMTS13 antibodies. Objective: We explored the ability of two VH1-69 germline gene-encoded antibodies to inhibit the von Willebrand factor (VWF)-processing activity of ADAMTS13 under different experimental conditions. Furthermore, the presence of VH1-69 encoded anti-ADAMTS13 antibodies in 40 patients with acquired TTP was monitored using monoclonal antibody G8, which specifically reacts with an idiotype expressed on VH1-69 encoded antibodies. Methods and Results: Binding of the two VH1-69 encoded monoclonal antibodies was dependent on the presence of the spacer domain. Both antibodies inhibited ADAMTS13 activity under static conditions, as measured by cleavage of FRETS-VWF73 substrate and cleavage of VWF multimers. The recombinant antibodies were also capable of inhibiting the processing of UL-VWF strings on the surface of endothelial cells. G8-reactive antibodies directed towards ADAMTS13 were present in plasma of all patients containing anti ADAMTS13 antibodies. Conclusions: These results suggest that VH1-69 derived antibodies directed towards ADAMTS13 develop in the majority of patients with acquired TTP.     

The activity of the von Willebrand factor cleaving protease ADAMTS-13 in newborn infants

Summary.  Background : Unusually large von Willebrand factor (VWF) multimers have been observed in patients with thrombotic microangiopathies (TMA), and absence of the VWF cleaving protease ADAMTS-13 activity is considered to be involved in the etiology of TMA. Increased amounts of large multimers of VWF have also been identified in neonates. Objective : We assessed ADAMTS-13 activity in healthy neonates, children and adults to establish baseline levels. Patients and methods : Cord blood was collected from 38 full-term newborns; venous samples were taken from 15 neonates on day 2–3 of life. Seventeen children, 24 healthy adults and seven patients with TMA were studied as well. ADAMTS-13 activity was quantified by the binding of the subjects' plasma VWF to collagen before and after enzyme activation. The multimer distribution of VWF was also determined. Results : Neonates and children had percentage ADAMTS-13 activity similar to adults. However, two groups were apparent in the cord blood samples: while 28/38 newborns had percentage activity within the normal range of healthy adults (102 ± 3.0%), 10 had significantly lower percentage activity (53 ± 1.1%; P  < 0.0001) that normalized by day 2–3. The VWF multimer distribution was the same in all cord blood samples and was not different compared with children and adults. High-molecular-weight VWF multimers were significantly increased in the 2–3-day-old neonates and in TMA patients. Conclusions : Although ADAMTS-13 activity was similar in neonates compared with adults, 26% of neonates had mildly reduced activity. Further studies are needed to investigate the complex interaction of VWF production and secretion with its size control by ADAMTS-13 in different age groups.     

Safety and efficacy of cryoprecipitate‐poor plasma as a replacement fluid for therapeutic plasma exchange in thrombotic thrombocytopenic purpura: A single center retrospective evaluation

Introduction: Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy caused by decreased activity of ADAMTS13, resulting in reduced clearance of ultralarge von Willebrand factor (VWF) multimers. Treatment of TTP is therapeutic plasma exchange (TPE) with replacement with fresh frozen plasma (FFP). Cryoprecipitate‐poor plasma (CPP) is a plasma product with lower concentrations of large VWF multimers, and similar amounts of ADAMTS13. CPP is regarded as at least as efficacious as FFP in TTP but evidence of additional benefits has not been demonstrated. Furthermore, there are limited data on the frequency of adverse events associated with CPP. Material and methods: In our center, the choice between CPP and FFP is performed before the 1st TPE session at the physicians' discretion. Here, we retrospectively evaluated the efficacy and safety of CPP based on the number of sessions, volume of plasma exposure, frequency of exacerbations/relapses, and adverse events. Results: Fourteen patients with newly diagnosed TTP were included in this analysis. The proportion of CPP:FFP use was 5:9. There were no significant differences in age, gender, initial hemoglobin, platelet count, LDH, or etiology of TTP between groups. We observed a trend toward a higher number of TPE sessions and higher plasma exposure in CPP, compared to FFP‐treated patients. Acute exacerbations were more frequent among patients treated with CPP (OR 26.6; 95%CI 1.01–703.51; P = 0.03). Mild allergic reactions were the most common treatment‐related adverse event in both groups. Discussion: Our data suggest that CPP should not be used as 1st line treatment for newly diagnosed TTP patients. J. Clin. Apheresis 29:311–315 2014. © 2014 Wiley Periodicals, Inc.     

Species-dependent variability of ADAMTS13-mediated proteolysis of human recombinant von Willebrand factor

Summary.  Background: von Willebrand factor (VWF) is composed of a series of multimers, the sizes of which are regulated by the plasma metalloprotease ADAMTS13. Objective: Proteolysis of human recombinant VWF (rVWF) by ADAMTS13 present in the plasma of different species typically used as preclinical animal models was investigated to evaluate the efficacy and safety of rVWF. Methods: Degradation of rVWF was studied in vitro under moderate denaturing conditions and was monitored by multimer analysis, residual collagen binding, and immunoblot analysis. In vivo cleavage was determined by administration of rVWF to cynomolgus monkeys, rabbits and VWF-deficient mice and subsequent analysis of plasma samples by immunoblot. Plasma ADAMTS13 levels were determined with a synthetic human VWF peptide (FRETS-VWF73). Results: From the animals tested, only rabbit plasma was as efficient as human plasma in proteolysing rVWF in vitro . Mouse plasma virtually failed to cleave rVWF. Administration of human rVWF resulted in ADAMTS13-specific cleavage products in rabbits and, to a lesser extent, in cynomolgus monkeys at various doses of rVWF. Virtually no cleavage occurred in mice. ADAMTS13 activity levels in rabbit and monkey plasma were similar to those in human plasma and were not significantly altered upon infusion of rVWF up to very high doses, indicating that rVWF did not lead to an exhaustion of endogenous ADAMTS13 in both species. Conclusions: The differences in susceptibility to cleavage of rVWF by different species need to be considered when interpreting the physiology of human rVWF from results of tests in animal models.     

Complement activation in thrombotic thrombocytopenic purpura

Summary. Background: Ultra‐large von Willebrand factor and deficiency of its cleaving protease are important factors in the events leading to thrombotic microangiopathy; however, the mechanisms involved are only partly understood. Whereas pathological activation of the alternative complement pathway is linked to atypical hemolytic uremic syndrome, the role of complement activation in thrombotic thrombocytopenic purpura (TTP) is unknown. The aim of this study was to investigate whether signs of complement activation are characteristic of TTP. Patients and methods: Twenty‐three patients with TTP (18 women, median age 38 years) and 17 healthy controls (13 women, median age 38 years) were included. Complement parameters (C3, Factors H, I, B and total alternative pathway activity) together with complement activation fragments (C3a) or complexes (C1rs‐INH, C3bBbP, sC5b9) were measured by ELISA or RID. ADAMTS13 activity and anti‐ADAMTS13 inhibitory antibodies were measured by the VWF‐FRET73 assay. Results: Increased levels of C3a, and SC5b9 were observed in TTP during acute episodes, as compared with healthy controls. Decreased complement C3 levels indicative of complement consumption occurred in 15% of acute TTP patients. Significant decrease of complement activation products C3a and SC5b9 was observed during plasma exchange (PEX). The sustained presence of anti‐ADAMTS13 inhibitory antibodies in complete remission was associated with increased complement activation. Conclusion: These data document in an observational study the presence of complement activation in TTP. Further investigation is needed to determine its potential pathogenetic significance.     

ADAMTS13 activity and the presence of acquired inhibitors in human immunodeficiency virus-related thrombotic thrombocytopenic purpura

BACKGROUND: Little is known about the pathophysiology of human immunodeficiency virus (HIV)-related thrombotic thrombocytopenic purpura (TTP). It is generally assumed that acquired ADAMTS13 deficiency is due to the presence of autoantibody inhibitors, but limited data are available regarding ADAMTS13 activity and inhibitors in such patients. STUDY DESIGN AND METHODS: By use of a collagen-binding assay, ADAMTS13 activity was analyzed at presentation in 20 patients with HIV-related TTP. The presence of inhibitors in patients with reduced ADAMTS13 activity was assessed with mixing studies. The correlation between ADAMTS13 activity and inhibitors and other laboratory and clinical parameters was assessed. RESULTS: The patients fell clearly into two groups with regard to ADAMTS13 activity. Six patients (30%) had activity within the normal range, whereas the remaining 14 patients had severely reduced levels. Of the patients with reduced activity, only 5 patients had a detectable inhibitor whereas 8 showed no evidence of an inhibitor. There was significant correlation between normal ADAMTS13 activity and lower CD4 counts (p = 0.049). von Willebrand factor (VWF) antigen levels were significantly higher in patients with reduced ADAMTS13 activity (p = 0.03). Low activity in the absence of a detectable inhibitor was associated with significantly higher D-dimer levels (p = 0.01) and worse clinical outcome. CONCLUSION: The heterogeneity with regard to ADAMTS13 activity and the absence of inhibitors in the majority of patients indicate that other factors are important in the pathogenesis of HIV-related TTP. VWF release and localized coagulation activation due to direct viral or cytokine-mediated endothelial cell injury is likely to be playing a major role.     

Deficient activity of von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a dramatic intravascular platelet-clumping disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, neurologic abnormalities, renal insufficiency and fever. TTP is a rare disease but is almost always fatal if untreated. More than 80% of patients survive with plasma therapy. In healthy individuals, the proteolytic cleavage of ultralarge von Willebrand factor (vWF) multimers prevents spontaneous clumping of platelets in the microcirculation. Patients with TTP have either severe congenital deficiency of von Willebrand factor-cleaving protease (vWF-cp), or have autoantibodies that inhibit the protease. Determination of vWF-cp levels in patient plasma helps to distinguish between TTP and other thrombotic microangiopathies with similar clinical signs and symptoms. vWF-cp is a member of the ADAMTS family of metalloproteases and has been designated ADAMTS13.     

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.     

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.     

von Willebrand factor activation,granzyme‐B and thrombocytopenia in meningococcal disease

Summary. Background: During invasive meningococcal disease, severe thrombocytopenia is strongly associated with a poor outcome. Objectives: In order to elucidate the pathophysiological mechanism behind the development of thrombocytopenia, we studied the role of von Willebrand factor (VWF) in meningococcal disease. Patients/methods: Thirty‐two children with severe meningococcal disease admitted to our university hospital were included in this study. VWF and related parameters were measured and results were correlated with the development of shock and thrombocytopenia. Results: At admission, all patients had increased levels of (active) VWF and VWF propeptide. The highest VWF propeptide levels were observed in patients with shock, indicating acute endothelial activation. Although VWF propeptide levels in patients with shock, with or without thrombocytopenia, were similar, increased active VWF was significantly lower in patients with thrombocytopenia as compared with patients without thrombocytopenia. ADAMTS13 was moderately decreased. However, the VWF multimeric pattern was minimally increased. We assume that these findings are explained by VWF consumption and perhaps by granzyme B (GrB). In vitro experiments showed that GrB is able to cleave VWF multimers in plasma, whereas GrB was high in patients with shock, who developed thrombocytopenia. Conclusions: Our results demonstrate that consumption of VWF, derived from endothelial cells, could be a key feature of meningococcal disease and primary to the development of thrombocytopenia during shock.     

Deficient activity of von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a dramatic intravascular platelet-clumping disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, neurologic abnormalities, renal insufficiency and fever. TTP is a rare disease but is almost always fatal if untreated. More than 80% of patients survive with plasma therapy. In healthy individuals, the proteolytic cleavage of ultralarge von Willebrand factor (vWF) multimers prevents spontaneous clumping of platelets in the microcirculation. Patients with TIP have either severe congenital deficiency of von Willebrand factor-cleaving protease (vWF-cp), or have autoantibodies that inhibit the protease. Determination of vWF-cp levels in patient plasma helps to distinguish between TTP and other thrombotic microangiopathies with similar clinical signs and symptoms. vWF-cp is a member of the ADAMTS family of metalloproteases and has been designated ADAMTS13.     

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.