Eculizumab is highly effective in controlling complement activation in patients with the atypical hemolytic uremic syndrome (aHUS). However, the course of responses to the treatment is not well understood. We reviewed the responses to eculizumab therapy for aHUS. The results show that, in patients with aHUS, eculizumab therapy, when not accompanied with concurrent plasma exchange therapy, led to steady increase in the platelet count and improvement in extra-renal complications within 3 days. By day 7, the platelet count was normal in 15 of 17 cases. The resolution of hemolytic anemia and improvement in renal function were less predictable and were not apparent for weeks to months in two patients. The swift response in the platelet counts was only observed in one of five cases who received concurrent plasma exchange therapy and was not observed in a case of TMA due to gemcitabine/carboplatin. In summary, eculizumab leads to rapid increase in the platelet counts and resolution of extrarenal symptoms in patients with aHUS. Concurrent plasma exchange greatly impedes the response of aHUS to eculizumab therapy. Eculizumab is ineffective for gemcitabine/carboplatin associated TMA.
Recent studies have demonstrated that thrombotic thrombocytopenic purpura (TTP), a serious thrombotic disorder affecting the arterioles and capillaries of multiple organs, is caused by a profound deficiency in the von Willebrand factor cleaving metalloprotease, ADAMTS13. ADAMTS13, a 190-kD plasma protease originating primarily in hepatic stellate cells, prevents microvascular thrombosis by cleaving von Willebrand factor when the substrate is conformationally unfolded by high levels of shear stress in the circulation. Deficiency of ADAMTS13, due to genetic mutations or inhibitory autoantibodies, leads to accumulation of superactive forms of vWF, resulting in vWF-platelet aggregation and microvascular thrombosis. Analysis of ADAMTS13 has led to the recognition of subclinical TTP and atypical TTP presenting with thrombocytopenia or acute focal neurological deficits without concurrent microangiopathic hemolysis. Infusion of plasma replenishes the missing ADAMTS13 and ameliorates the complications of hereditary TTP. The patients are at risk of both acute and chronic renal failure if they receive inadequate plasma therapy. The more frequent, autoimmune type of TTP requires plasma exchange therapy and perhaps immunomodulatory measures. Current studies focus on the factors affecting the phenotypic severity of TTP and newer approaches to improving the therapies for the patients.
ADAMTS13; shear stress; thrombosis; thrombotic thrombocytopenic purpura; von Willebrand factor
Thrombotic thrombocytopenic purpura (TTP) affects 1 in 1600 to 1 in 5000 patients who receive ticlopidine, but little is known about the pathogenesis of this complication.
To investigate whether von Willebrand factor (vWF), which has been associated with idiopathic TTP, is involved in the pathogenesis of ticlopidine-associated TTP.
Three tertiary care, university-affiliated medical centers.
Seven patients who developed TTP 2 to 7 weeks after initiation of ticlopidine therapy. Controls were 7 consecutive patients without thrombocytopenia who had been receiving ticlopidine for 3 to 5 weeks and 10 randomly selected hospitalized patients.
Platelet-bound vWF in patients’ EDTA-anticoagulated whole blood samples; vWF proteinase activity in patients’ plasma samples; inhibitory activity of IgG isolated from patients’ plasma samples against the proteinase from the controls’ plasma samples; and vWF multimeric patterns in patients’ EDTA-anticoagulated plasma samples.
Binding of vWF to single platelets was increased in the three patients tested during the most thrombocytopenic phase of TTP episodes. Initial plasma samples from all seven patients lacked the largest vWF multimers and were severely deficient in vWF metalloproteinase. IgG molecules, isolated from plasma samples of five patients, inhibited metalloproteinase in plasma samples from the controls. In patients examined, these abnormalities resolved upon the remission that accompanied plasma exchange and discontinuation of ticlopidine therapy.
In the patients who developed ticlopidine-associated TTP, autoantibodies to the vWF metalloproteinase were formed; this led to the same type of vWF abnormalities observed in patients with idiopathic acute TTP. The findings suggest that failure to process large and unusually large vWF multimers in vivo caused binding of vWF to platelets, systemic platelet thrombosis, and TTP.
Complement factor I (CFI) mutations are implicated in the pathogenesis of atypical hemolytic uremic syndrome (aHUS). Nevertheless, there is evidence that CFI deficiency is a weak effector of aHUS. Bienaime et al. report that homozygous deletion of CFHR-1 in the RCA gene cluster of chromosome 1q is a major risk factor for poor outcome for patients with CFI mutations. The basic and clinical implications of the findings are further elaborated here.
Functional assays are commonly used to measure the antibodies of ADAMTS13 found in patients of thrombotic thrombocytopenic purpura (TTP). In this study we used an enzyme-linked immunoassay to analyze the ADAMTS13-binding IgG levels in six groups of individuals: normal, random hospitalized patients, acute TTP,TTP after receiving plasma therapy, TTP in remission, and other types of thrombotic microangiopathy (TMA). The results showed thatADAMTS13-binding IgG levels were elevated in 100% of the acute TTP group, 75% of the TTP group after receiving plasma therapy, and 40% of the remission group. Overall, the ADAMTS13-binding IgG levels correlated with the inhibitory activity levels against ADAMTS13 (r=−0.69,P<0.0001). The assay also detected elevated IgG binding levels in 5% – 15% of the normal, random, and other TMA control groups. Addition of purified ADAMTS13 protein to the plasma samples suppressed the IgG binding in each of the acute TTP patients, but in none of the non-TTP groups. Serial measurement in a patient that had two exacerbations of TTP within the first three weeks revealed that the ADAMTS13 activity levels remained < 0.1 U/ml during this period, and the ADAMTS13-binding IgG remained elevated, suggesting thatADAMTS13 analysis may provide valuable insight to the disease status during the course of therapy. Analysis of ADAMTS13-binding IgG is helpful for the diagnosis and management of TTP.
ADAMTS13; thrombotic thrombocytopenic purpura; antibody
In a patient with fatal thrombotic thrombocytopenic purpura, the inhibitory activity of antibodies against ADAMTS13 rapidly escalated to extremely high levels despite daily plasma exchange and corticosteroid therapy. This increase was found to be due to a combination of higher antibody concentration and potency. Furthermore, during her course of the disease, the percentage of IgG1 antibody progressively decreased while that of IgG2 antibody increased, suggesting Th1-type cytokine response. These changes suggest that the course of TTP may be exacerbated by complex immune reactions. Further characterization of the factors contributing to this exacerbation may have important pathogenetic and therapeutic implications.
thrombotic thrombocytopenic purpura; von Willebrand factor; ADAMTS13; somatic hypermutation; antibody class switch
Thrombotic microangiopathy, which includes thrombotic thrombocytopenic purpura (TTP), shiga-toxin associated hemolytic uremic syndrome (Stx-HUS) and atypical HUS, is characterized pathologically by the development of hyaline thrombi in the microvasculature and clinically by the manifestations of thrombocytopenia, microangiopathic hemolysis, and organ dysfunction. Renal failure is a predominant complication of both Stx-HUS and atypical HUS, while neurological complications are more prominent in TTP. Other disorders such as lupus or bone marrow transplantations may occasionally present with features of thrombotic microangiopathy. Recent studies have found autoimmune inhibitors or genetic mutations of a von Willebrand factor cleaving metalloprotease ADAMTS13 in patients with TTP. In approximately 30% – 50% of patients with atypical HUS, mutations have been detected complement factor H, membrane cofactor protein (CD46), or factor I. All three proteins are involved in the regulation of complement activation. Additionally autoantibodies of factor H have been described in patients without genetic mutations. These advances illustrate that dysregulation of VWF homeostasis or complement activation due to genetic or autoimmune mechanisms may lead to syndrome of thrombotic microangiopathy.
ADAMTS13; Hemolytic uremic syndrome; Shear stress; Thrombotic microangiopathy; Thrombotic thrombocytopenic purpura; Regulators of complement activation; von Willebrand factor
The kidney is commonly affected in thrombotic thrombocytopenic purpura (TTP), a multi-system disorder with microvascular thrombosis of the capillaries and arterioles. Nevertheless, due to difference in its diagnostic criteria, the frequency and severity of renal dysfunction in TTP remains controversial. With the recent studies indicating that severe deficiency of a VWF cleaving protease, ADAMTS13, is the main cause of platelet thrombosis in TTP, it is now possible to define TTP at the molecular level. Among patients with acquired TTP due to inhibitory antibodies of ADAMTS13, renal dysfunction is usually mild; oliguria, fluid overload, hypertension, and need of dialysis support are infrequent. When any of these complications occur, one must re-examine the diagnosis of TTP and consider the possibility that the patient has another cause for these complications. In a patient with hereditary TTP, acute renal failure may ensue. However, the renal dysfunction is mostly reversible if the patients are promptly treated with plasma to replenish ADAMTS13. Patients with TTP, particularly of the hereditary type, may develop chronic renal failure. This complication may be a consequence of repeated insults by overt or subclinical microvascular thrombosis to the kidney, or it may have a separate cause. Therapy of hereditary TTP should aim not only to prevent acute exacerbations but also to minimize the risk of irreversible renal injury.
Thrombotic thrombocytopenic purpura; ADAMTS13; von Willebrand factor; Thrombosis; Renal failure; Shear stress
Severe deficiency of ADAMTS13, a von Willebrand factor cleaving metalloprotease, causes thrombotic thrombocytopenic purpura. When analyzed with VWF multimers, but not with an abbreviated VWF peptide (VWF73) as the substrate, the plasma ADAMTS13 activity levels of mouse strains segregated in a high and a low group that differed by approximately 10 folds. Low ADAMTS13 activity was detected in mice containing two alleles of IAP-type retrotransposon sequence in the ADAMTS13 gene. Molecular cloning of mouse ADAMTS13 identified two truncated variants (IAP-a and IAP-b) in the low activity mice. Both the IAP variants lacked the two carboxyl terminus thrombospondin type 1 repeat (TSR) and CUB domains of full-length ADAMTS13. The IAP-b variant also had splicing abnormalities affecting the spacer domain sequence and had miniscule enzymatic activity. Compared to full-length ADAMTS13, the IAP-a variant was approximately one ninth as active in cleaving VWF multimers but was only slightly less active in cleaving VWF73 peptide. Recombinant human ADAMTS13 was also less effective in cleaving VWF multimers than VWF73 when the C-terminal TSR sequence was deleted. In summary, the carboxyl terminus TSR sequence is important for cleaving VWF multimers. Assay results should be interpreted with caution when peptide substrates are used for analysis of variant ADAMTS13 proteins.
ADAMTS13; IAP; Mouse; Retrotransposon; von Willebrand factor
ADAMTS13, encoded on chromosome 9q34, is a member of the ADAMTS (a disintegrin and metalloprotease with thrombospondin type 1 motif) metalloprotease family, containing the common domain structure of (from the amino terminus) signal peptide, propeptide, reprolysin type metalloprotease, thrombospondin type 1 motif, cysteine-rich region, and spacer domain. ADAMTS13 cleaves von Willebrand factor (VWF) in a shear stress dependent manner. Deficiency of the enzyme causes the platelet aggregation of thrombotic thrombocytopenic purpura (TTP). Inhibitory antibodies of ADAMTS13 are detected in patients with acquired TTP, while homozygous or double heterozygous mutations of the ADAMTS13 gene cause the hereditary form of the disease 1. Targeting of the ADAMTS13 gene by recombinant technology has reproduced the phenotype of human TTP in ADAMTS13-null mice 2.
Despite these advances, intense controversy and confusion persist regarding the role of ADAMTS13 assays in the diagnosis of TTP. This brief review highlights some of the contentious issues and proposes steps to improve the diagnostic value of ADAMTS13 assays.
Studies with riboflavin in the 1960s showed that it could be effective at inactivating pathogens when exposed to light. The principal mode of action is through electron transfer reactions, most importantly in nucleic acids. This suggested that it could act as a photosensitizer useful in the inactivation of pathogens found in blood products.
To study the influence of photo-inactivation with riboflavin on the coagulation factors of plasma.
The photo-inactivation procedure of riboflavin plus light was applied. Fifty isogroup pools of two plasmas were made from 100 U of plasma that were derived from whole blood products that had previously been held overnight. Pools were split into two bags. One of them was photo-inactivated, and post inactivation samples were obtained. The second bag was not photo-inactivated and samples were taken. Total protein, fibrinogen, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII, antithrombin III, PC, PS, α-2 antiplasmin and vWF:Ag, the multimeric structure of vWF and ADAMTS-13 were analyzed.
In plasma, the proteins most sensitive to photo-inactivation were fibrinogen, FXI, FVIII, FV, and FIX (33%, 32%, 30%, 18% and 18% loss, respectively). Coagulation inhibitors, PS, antithrombin III and PC showed little decrease (all 2%). Retention of vWF and ADAMTS-13 were 99% and 88%, respectively.
As with other pathogen reduction procedures for plasma products, treatment with riboflavin and UV light resulted in reduction in the activity levels of several pro-coagulant factors. Coagulation inhibitors are well preserved.
Plasma; Coagulation factors; Pathogen reduction; Riboflavin
Thrombotic thrombocytopenic purpura (TTP) is a disorder with characteristic von Willebrand factor (VWF)-rich microthrombi affecting the arterioles and capillaries of multiple organs. The disorder frequently leads to early death unless the patients are treated with plasma exchange or infusion. Studies in the last decade have provided ample evidence to support that TTP is caused by deficiency of a plasma metalloprotease, ADAMTS13. When exposed to high shear stress in the microcirculation, VWF and platelets are prone to form aggregates. This propensity of VWF and platelet to form microvascular thrombosis is mitigated by ADAMTS13, which cleaves VWF before it is activated by shear stress to cause platelet aggregation in the circulation. Deficiency of ADAMTS13, due to autoimmune inhibitors in patients with acquired TTP and mutations of the ADAMTS13 gene in hereditary cases, leads to VWF–platelet aggregation and microvascular thrombosis of TTP. In this review, we discuss the current knowledge on the pathogenesis, diagnosis and management of TTP, address the ongoing controversies, and indicate the directions of future investigations.
TTP; von Willebrand factor; ADAMTS13; Shear stress; Microvascular thrombosis
Thrombotic thrombocytopenic purpura is a potentially fatal disease characterized by widespread platelet thrombi in the microcirculation. In the normal circulation, von Willebrand factor is cleaved by a plasma protease. We explored the hypothesis that a deficiency of this protease predisposes patients with thrombotic thrombocytopenic purpura to platelet thrombosis.
We studied the activity of von Willebrand factor–cleaving protease and sought inhibitors of this protease in plasma from patients with acute thrombotic thrombocytopenic purpura, patients with other diseases, and normal control subjects. We also investigated the effect of shear stress on the ristocetin cofactor activity of purified von Willebrand factor in the cryosupernatant fraction of the plasma samples.
Thirty-nine samples of plasma from 37 patients with acute thrombotic thrombocytopenic purpura had severe deficiency of von Willebrand factor–cleaving protease. No deficiency was detected in 16 samples of plasma from patients with thrombotic thrombocytopenic purpura in remission or in 74 plasma samples from normal subjects, randomly selected hospitalized patients or outpatients, or patients with hemolysis, thrombocytopenia, or thrombosis from other causes. Inhibitory activity against the protease was detected in 26 of the 39 plasma samples (67 percent) obtained during the acute phase of the disease. The inhibitors were IgG antibodies. Shear stress increased the ristocetin cofactor activity of von Willebrand factor in the cryosupernatant of plasma samples obtained during the acute phase, but decreased the activity in cryosupernatant of plasma from normal subjects.
Inhibitory antibodies against von Willebrand factor–cleaving protease occur in patients with acute thrombotic thrombocytopenic purpura. A deficiency of this protease is likely to have a critical role in the pathogenesis of platelet thrombosis in this disease.
Deficiency of von Willebrand factor (VWF) cleaving protease ADAMTS13 has been demonstrated to be the proximate cause of a subset of thrombotic microangiopathic haemolytic anaemias (MAHA) typical for thrombotic thrombocytopenic purpura (TTP). ADAMTS13 gene mutations cause the hereditary form; acquired deficiency has been attributed to presence of an autoantibody, which may represent a specific subset of MAHA best termed ‘autoimmune thrombotic thrombocytopenic purpura’. We describe a patient with relapsing TTP because of ADAMTS13 inhibitors, who failed to achieve sustained remission despite therapies with plasma exchange, steroids, vincristine, staphylococcal protein A and splenectomy. The ADAMTS13 inhibitor titre remained elevated and clinical stability was only maintained by plasma exchange every 2–3 d over a period of 268 d. The patient then received rituximab therapy (eight doses of 375 mg/m2 weekly), during which she required five plasma exchanges in the first 10 d, two exchanges in the next 3 weeks, and none thereafter for 450 d and ongoing. The ADAMTS13 inhibitor titre decreased and enzyme activity increased. We compared this case with that of seven previously reported TTP cases also treated with rituximab; experience suggests that rituximab therapy deserves further investigation for patients with either refractory or relapsing TTP caused by ADAMTS13 inhibitors.
thrombotic thrombocytopenic purpura; rituximab; plasma exchange
von Willebrand factor; ADAMTS13; shear stress; thrombotic microangiopathy; thrombotic thrombocytopenic purpura
ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motifs), a circulating multidomain zinc metalloprotease of the reprolysin subfamily, is critical for preventing von Wille-brand factor-platelet interaction under high shear stress conditions. A deficiency of the protease, due to mutations in the ADAMTS13 gene or the presence of antibodies that inhibit the activity of the protease, causes thrombotic thrombocytopenic purpura (TTP). Plasma therapy, the conventional therapy for TTP, may cause serious adverse reactions and is ineffective in some patients. In order to develop new strategies for improving the diagnosis and treatment of TTP, we produced a series of truncated ADAMTS13 proteins in mammalian cells and analyzed their binding with and suppression by the IgG derived from the TTP patients. The results revealed that truncation of the ADAMTS13 protein at its cysteine-rich region eliminated its recognition by the antibodies without abolishing its von Willebrand factor-cleaving activity. This raises the possibility that resistant ADAMTS13 variants may be exploited to circumvent inhibitory antibodies that cause TTP.
ADAMTS13 is a circulating zinc metalloprotease that cleaves the hemostatic glycoprotein von Willebrand factor (VWF) in a shear-dependent manner. Deficiency in ADAMTS13, owing to genetic mutations or autoimmune inhibitors, causes thrombotic thrombocytopenic purpura (TPP). Northern blot analysis has shown that ADAMTS13 is expressed primarily in the liver. By using real-time RT-PCR, we confirmed that in mice the liver had the highest level of the ADAMTS13 transcript. To identify the liver cell-type-specific origin of ADAMTS13, we used in situ hybridization techniques to investigate the pattern of ADAMTS13 expression in the liver; analyzed the ADAMTS13 proteolytic activity in the culture media of fractionated liver cells; and confirmed ADAMTS13 expression with RT-PCR analysis and cloning of the mouse ADAMTS13 gene. The results revealed that ADAMTS13 was expressed primarily in cell fractions enriched in hepatic stellate cells. The mouse ADAMTS13 cloned from primary hepatic stellate cells was similar to its human counterpart in digesting VWF and was susceptible to suppression by EDTA or the IgG inhibitors of patients with TTP. Since hepatic stellate cells are believed to play a major role in the development of hepatic fibrosis and cirrhosis, the identification of the liver cell-type expressing ADAMTS13 will have important implications for understanding pathophysiological mechanisms regulating ADAMTS13 expression.
ADAMTS13; hepatic stellate cell; von Willebrand factor; metalloprotease
Interaction between platelet and von Willebrand factor (VWF), a circulating adhesive glycoprotein, is essential for hemostasis under the high shear environments of arterioles and capillaries. If unregulated, this interaction may lead to unwarranted platelet thrombosis ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, number 13), a plasma zinc metalloprotease synthesized primarily in the stellate cells of the liver, cleaves shear stress activated VWF, thereby preventing the occurrence of VWF-platelet interaction in the circulation. A profound deficiency of ADAMTS13, due to genetic mutations or autoimmune inhibitors, results in intravascular VWF-platelet aggregation and widespread microvascular thrombosis characteristic of thrombotic thrombocytopenic purpura (TTP). Cloning of ADAMTS13 and structure-function analysis of the enzyme are leading to exciting advances in the diagnosis and therapy of this hitherto mysterious disease.
Thrombotic thrombocytopenic purpura; Thrombotic microangiopathy; Proteolysis; Metalloprotease; Von Willebrand factor; Rituximab; ADAMTS13; Shear stress
Recent advances have demonstrated that thrombotic thrombocytopenic purpura (TTP), characterized by widespread thrombosis in the arterioles and capillaries, is caused by deficiency of a circulating zinc metalloprotease ADAMTS13. Two types of TTP are recognized: Autoimmune TTP caused by inhibitory antibodies of ADAMTS13 and hereditary TTP in association with genetic mutations of the ADAMTS13 gene. This article reviews the characteristic and function of ADAMTS13, the mechanism by which ADAMTS13 deficiency may cause thrombosis, and the causes of ADAMTS13 deficiency. It also discusses how the new knowledge may improve the diagnosis and treatment of this previously mysterious disorder.
Thrombotic thrombocytopenic purpura; von Willebrand factor; ADAMTS13; Shear stress; Hemolytic uremic syndrome
A serious disorder with characteristic microvascular thrombosis involving the brain and other organs, TTP typically presents with thrombocytopenia, hemolysis with schistocytes on blood smears and mental changes or seizures and may rapidly progresses to a fatal end if the patients are not immediately treated with plasma. Recent advances have shown that TTP is caused by deficiency of a circulating, von Willebrand factor cleaving metalloprotease, ADAMTS13. This new knowledge will provide clues to improve the diagnosis and management of this intriguing disease.
Thrombotic thrombocytopenic purpura; Von Willebrand factor; ADAMTS13; Metalloprotease; Microangiopathic hemolysis; Thrombocytopenia
The activity of ADAMTS13, the von Willebrand factor cleaving protease, is deficient in patients with thrombotic thrombocytopenic purpura (TTP). In the present study, the phenotype of ADAMTS13 in TTP and in normal plasma was demonstrated by immunoblotting. Normal plasma (n = 20) revealed a single band at 190 kD under reducing conditions using a polyclonal antibody, and a single band at 150 kD under non-reducing conditions using a monoclonal antibody. ADAMTS13 was not detected in the plasma from patients with congenital TTP (n = 5) by either antibody, whereas patients with acquired TTP (n = 2) presented the normal phenotype. Following immunoadsorption of immunoglobulins, the ADAMTS13 band was removed from the plasma of the patients with acquired TTP, but not from that of normal individuals. This indicates that ADAMTS13 is complexed with immunoglobulin in these patients. The lack of ADAMTS13 expression in the plasma from patients with hereditary TTP may indicate defective synthesis, impaired cellular secretion, or enhanced degradation in the circulation. This study differentiated between normal and TTP plasma, as well as between congenital and acquired TTP. This method may, therefore, be used as a complement in the diagnosis of TTP.
ADAMTS13; von Willebrand factor; Thrombotic thrombocytopenic purpura; Immunoblotting; Plasma; von Willebrand factor cleaving protease
Thrombotic thrombocytopenic purpura (TTP) is a life-threatening illness caused by deficiency of the vWF-cleaving protease ADAMTS13. Here we show that ADAMTS13-deficient mice are viable and exhibit normal survival, although vWF-mediated platelet-endothelial interactions are significantly prolonged. Introduction of the genetic background CASA/Rk (a mouse strain with elevated plasma vWF) resulted in the appearance of spontaneous thrombocytopenia in a subset of ADAMTS13-deficient mice and significantly decreased survival. Challenge of these mice with shigatoxin (derived from bacterial pathogens associated with the related human disease hemolytic uremic syndrome) resulted in a striking syndrome closely resembling human TTP. Surprisingly, no correlation was observed between plasma vWF level and severity of TTP, implying the existence of TTP-modifying genes distinct from vWF. These data suggest that microbe-derived toxins (or possibly other sources of endothelial injury), together with additional genetic susceptibility factors, are required to trigger TTP in the setting of ADAMTS13 deficiency.