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.     

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.     

Comparison and stability of ADAMTS13 activity in therapeutic plasma products

BACKGROUND: The von Willebrand factor (VWF)-cleaving protease, ADAMTS13, is often deficient in cases of thrombotic thrombocytopenic purpura (TTP). The primary treatment of TTP is therapeutic plasma exchange (TPE) utilizing a variety of plasma products that help restore ADAMTS13 activity. However, multiple replacement products are available to choose from. Thawed plasma products have a variable refrigerated shelf life depending on the product type; stability of ADAMTS13 in thawed products stored at 1 to 6 degrees C has not been determined. STUDY DESIGN AND METHODS: ADAMTS13 activity was measured in three types of plasma products and cryoprecipitate. Fresh-frozen plasma (FFP) aliquots and cryoprecipitate-poor plasma (CPP) products were produced from 10 whole-blood (WB) donations. Twenty-four-hour plasma products were manufactured from 10 additional WB donations. ADAMTS13 activity in these products at time of thaw and after 5 days of storage at 1 to 6 degrees C was measured with a modified version of the FRETS-VWF73 fluorogenic assay. ADAMTS13 activity at time of thaw was measured in 10 units of cryoprecipitate and five related CPP products. RESULTS: ADAMTS13 is present in similar amounts in FFP, CPP, and 24-hour plasma products. Storage at 1 to 6 degrees C for up to 5 days did not significantly diminish ADAMTS13 activity. The concentration of ADAMTS13 in cryoprecipitate was significantly higher than that observed in plasma products. CONCLUSION: FFP, CPP, and 24-hour plasma products should be equally effective for ADAMTS13 restoration through TPE and should remain so for the duration of the shelf life of the thawed products.     

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.     

Rituximab maintenance for relapsed refractory thrombotic thrombocytopenic purpura

BACKGROUND: Rituximab, an anti‐CD20 chimeric monoclonal antibody, has been used successfully to treat patients with relapsed or refractory thrombotic thrombocytopenic purpura (TTP); however, the optimal dose and frequency and the role of rituximab maintenance remain uncertain. STUDY DESIGN AND METHODS: We describe a 45‐year‐old woman with chronic relapsing immune thrombocytopenia who responded to rituximab retreatment administered in four doses over the course of 12 months. Previously, she had received four doses of rituximab and sustained a remission for 19 months. During her latest TTP relapse, multiple treatments were administered including rituximab retreatment. After the first dose (375 mg/m²), she developed serum sickness requiring further doses to be deferred. Three subsequent doses were administered at 4‐month intervals over the course of 12 months. ADAMTS13 activity was measured by von Willebrand factor (VWF) digestion. ADAMTS13 inhibition was measured by a modification of the VWF digestion assay and anti‐ADAMTS13 antibodies were measured by enzyme‐linked immunoassay (enzyme‐linked immunosorbent assay, American Diagnostica). RESULTS: Clinical and laboratory remission were achieved after one dose of rituximab, with normalization of ADAMTS13 activity and disappearance of ADAMTS13 inhibitor. Three subsequent doses of rituximab were given without incident and the patient remained in remission after 3.5 years of follow‐up (2.5 years since her last dose of rituximab). CONCLUSION: Maintenance dosing of rituximab should be considered in some patients with relapsing TTP.     

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.     

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.     

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: 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.     

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.     

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.     

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.     

血栓性血小板减少性紫癜患者vWF裂解蛋白酶抗原和活性的检测及意义

目的研究血管性血友病因子裂解蛋白酶(ADAMTS13)抗原含量和活性在血栓性血小板减少性紫癜(TTP)患者及遗传性 TTP 家族突变携带者中变化的情况。方法用残余胶原结合实验(RCBA)检测13例 TTP 患者共28份血浆标本[含血浆置换(PE)前后]及10例携带者的 ADAMTS13活性;用新近建立的三抗体夹心酶联免疫反应法检测标本的 ADAMTS13抗原含量。结果正常对照组 ADAMTS13含量为(600.93±145.36)mU/ml(设白种人混合血浆的 ADAMTS13抗原含量为1000mU/ml),活性为(74.79±11.81)%。遗传性 TTP 患者 ADAMTS13抗原含量和活性治疗前和发病间期均明显减低,PE 后恢复;其家族中携带者 ADAMTS13抗原含量为(331.40±109.85)mU/ml,活性为(66.79±12.82)%(与对照组比较,P 值分别<0.01和>0.05);原发性 TTP 患者 PE 前 ADAMTS13抗原含量为(98.7±82.08)mU/ml,活性为(22.23±19.07)%(与对照组比较,P 值均<0.01);PE 后ADAMTS13 抗原含量为(449.4±232.33)mU/ml,活性为(60.92±22.33)%(与对照组比较,P 值分别<0.01和>0.05);1例继发性 TTP 患者 PE 后 ADAMTS13抗原含量远高于正常,活性仅为6.00%结论治疗前的 TTP 患者 ADAMTS13抗原含量和活性均明显减低。大多数患者两指标变化趋势一致,也有个别患者两指标变化趋势相反,前者可能因为遗传因素或体内免疫系统的廓清作用,后者可能因为抗 ADAMTS13抗体仅抑制了 ADAMTS13的活性而未影响其抗原的含量或其他未知原因所致。     

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

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

Systemic antithrombotic effects of ADAMTS13

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

Comparison of von Willebrand factor antigen, von Willebrand factor-cleaving protease and protein S in blood components used for treatment of thrombotic thrombocytopenic purpura

Replacement of normal levels of von Willebrand factor-cleaving protease (VWF:CP, ADAMTS13) activity from infused plasma is important in plasma exchange (PEX) for the treatment of thrombotic thrombocytopenic purpura (TTP) patients. We have studied the VWF:CP activity, VWF multimer distribution, VWF:Ag, protein S (PS) activity and free PS antigen levels in fresh frozen plasma (FFP), cryosupernatant (CSP) and virally inactivated components treated with methylene blue/light (MB) or solvent detergent (SD) processes. VWF:CP activity was normal in all components tested and was retained following overnight storage at room temperature. CSP and SD plasma contained reduced levels of the highest molecular weight VWF multimers. Protein S activity was reduced below the normal range in SD plasma, but within the normal range for the other components tested. Virally inactivated SD- and MB-treated plasma may be an effective alternative to FFP and CSP in PEX for TTP. Reduced PS activity in SD plasma may predispose to venous thromboembolism, especially if infused in large volumes.     

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.     

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.     

ADAMTS13 binds to CD36: a potential mechanism for platelet and endothelial localization of ADAMTS13

BACKGROUND: ADAMTS13 cleaves ultralarge von Willebrand factor (VWF) and plays a significant role in vascular biology and thrombotic thrombocytopenic purpura. CD36, a transmembrane protein present on endothelial cells and platelets (PLTs), binds to thrombospondin via three thrombospondin type 1 repeats. ADAMTS13 contains eight thrombospondin type 1 repeats.
STUDY DESIGN AND METHODS: An enzyme-linked immunoassay was used to explore the binding of recombinant human CD36 (rHuCD36) to recombinant human ADAMTS13 (rHuADAMTS13). A competition assay between rHuADAMTS13 and recombinant human (rHu)-thrombospondin-2 for binding to CD36 was then performed. Subsequently, binding of rHuADAMTS13 to PLT membrane fragments expressing CD36 (PLT glycoprotein IV) and glycoprotein Ib/IX was assessed. To examine the functional significance of an ADAMTS13-CD36 interaction, ADAMTS13 activity measured by a fluorescence resonance energy transfer assay was investigated in the presence of either rHuCD36 or concentrated PLTs.
RESULTS: rHuCD36 bound to rHuADAMTS13 in a dose-dependent fashion. rHu-thrombospondin-2 competed with ADAMTS13 for CD36 occupancy, but even high concentrations of rHu-thrombospondin-2 failed to completely block binding of rHuADAMTS13 to rHuCD36. rHuADAMTS13 bound to PLT membrane fragments expressing CD36 (PLT glycoprotein IV) in preference to PLT membrane fragments expressing glycoprotein Ib/IX. ADAMTS13 activity was not inhibited by the presence of either rHuCD36 or concentrated PLTs.
CONCLUSION: rHuADAMTS13 binds to both rHuCD36 and PLT membrane CD36 in vitro. The binding of CD36 to rHuADAMTS13 with retention of its enzymatic activity is consistent with a proposed role for CD36 in localizing ADAMTS13 on the endothelial cell surface where it regulates the cleavage of VWF.