Deficiency Of Plasma ADAMTS13 Activity Research Articles

Characterization of Five Homozygous ADAMTS13 Mutations in Hereditary Thrombotic Thrombocytopenic Purpura – Towards a Phenotype-Genotype Correlation?

Homozygous or compound heterozygous mutations in the ADAMTS13 gene cause hereditary thrombotic thrombocytopenic purpura (TTP), an inherited disorder characterized by severe deficiency of plasma ADAMTS13 activity resulting in occlusive microvascular thrombosis. More than eighty ADAMTS13 mutations have been identified, of which 15 have been found in homozygous state. Homozygous mutations are of special interest to unravel a correlation between the ADAMTS13 genotype and the often variable clinical phenotype in hereditary TTP. We studied 5 ADAMTS13 mutations found in homozygous state in 6 patients with hereditary TTP. Detailed patients' histories were recorded and plasma ADAMTS13 activity and antigen levels were determined. Recombinant ADAMTS13 mutants were transiently expressed in COS-7 cells and stably transfected HEK293 cell lines were generated for all five mutants. The biosynthesis, secretion and specific enzymatic activity under both static and flow conditions were studied. Intracellular localization of the mutants was also determined by immunofluorescent microscopy. Patients homozygous for W688X, R692C or C804R became manifest with acute TTP as neonates and several siblings had died of a TTP-like disorder. Plasma ADAMTS13 activity was < 1% of the normal and ADAMTS13 antigen was undetectable. In patients with homozygous R1060W mutation, milder courses with adult onset of TTP after strong triggering factors were observed. One patient presented with the first TTP attack at the age of 19 years during her first pregnancy while a second patient experienced a first bout at the age of 61 years during a severe pneumonia. Both patients had 3% of residual plasma ADAMTS13 activity and 6% of antigen of normal human plasma. Homozygous mutation G1239V was found in a patient with frequent TTP bouts in childhood starting at 3 years of age and an attenuated course in adulthood with attacks triggered by excessive alcohol consumption. His plasma ADAMTS13 activity was <1% along with undetectable antigen. Recombinant mutants R692C, C804R, R1060W and G1239V migrated at 190 kDa like wild-type ADAMTS13. Secretion of all 4 missense mutants was severely reduced and was highest for R1060W. The truncated mutant W688X was efficiently secreted with a molecular weight of 97 kDa. This mutant could cleave the VWF73 peptide as did wild-type ADAMTS13. Proteolytic activity towards multimeric von Willebrand factor (VWF) under static conditions was moderately impaired and even substantially reduced under fluid shear stress as compared to wild-type. Immunofluorescent staining showed the mutant ADAMTS13 proteases within endoplasmatic reticulum and Golgi apparatus, consistent with the distribution of secretory proteins. We conclude that all 5 investigated ADAMTS13 mutations are causative for hereditary TTP. The homozygous missense mutations in the carboxyl-terminal part of ADAMTS13 severely impair the secretion of the protease while the truncating mutation affects the proteolytic activity towards multimeric VWF under shear stress. Our findings highlight the significance of the middle and distal carboxylterminal domains of ADAMTS13 for secretion and enzymatic activity in vivo. This study of homozygous patients showed an early onset and severe course of TTP for mutations in the 2nd and 3rd TSP-1 repeat while the mutations in the 7th TSP-1 and the first CUB domain led to a later onset and a milder course of TTP with bouts requiring strong triggering factors. The two patients homozygous for R1060W showed low levels of residual plasma ADAMTS13 activity and antigen and the mildest clinical courses which is consistent with the cell culture studies. Together, our findings suggest a phenotype-genotype correlation in hereditary TTP.

Phenotypic Correction of Adamts13-Deficient Mice by Early Intra-Amniotic Gene Transfer of Lentiviral Vector Encoding ADAMTS13 Genes.

ADAMTS13, a member of A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, is mainly synthesized in the hepatic stellate cells, endothelial cells and megakaryocytes or platelets. It controls the sizes of von Willebrand factor (VWF) multimers by cleaving VWF at the Tyr1605-Met1606 bond. Genetic deficiency of plasma ADAMTS13 activity results in hereditary thrombotic thrombocytopenic purpura (TTP), also named Upshaw-Schülman syndrome. To develop a potential gene therapy approach and to determine the domains of ADAMTS13 required for recognition and cleavage of VWF in vivo, a self-inactivating lentiviral vector encoding human wild-type ADAMTS13 or variant truncated after the spacer domain (construct MDTCS) was administrated by intra-amniotic injection on embryonic day 8. Direct stereomicroscopy and immunofluorescent microscopic analysis revealed that the green fluorescent protein (GFP) reporter, ADAMTS13 and MDTCS were predominantly expressed in the heart, kidneys and skin. The synthesized ADAMTS13 and truncated variant were detectable in mouse plasma by immunoprecipitation and Western blot, as well as by proteolytic cleavage of FRETS-VWF73 substrate. The levels of proteolytic activity in plasma of mice expressing ADAMTS13 and MDTCS were 5 ± 7% and 60 ± 70%, respectively using normal human plasma as a standard, and this proteolytic activity persisted for at least 24 weeks in Adamts13−/−mice and 42 weeks in wild-type mice tested (the duration of observation). The mice expressing both recombinant ADAMTS13 and MDTCS showed a significantly decreased ratio of plasma VWF collagen-binding activity to antigen and a reduction in VWF multimer sizes as compared to those in the controls. Moreover, the mice expressing ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time (9.0 ± 0.6 and 25.2 ± 3.2 min, respectively) as compared to the Adamts13−/− mice expressing GFP alone (5.6 ± 0.5 min) (p<0.01). The ferric chloride-induced carotid occlusion time in Adamts13−/− mice expressing ADAMTS13 was almost identical to that in wild type mice with same genetic background (C56BL/6) (8.0 ± 0.2 min) (p>0.05). The data demonstrate the correction of the prothrombotic phenotype in Adamts13−/−mice by gene transfer to the fetus by viral vectors encoding human wild type ADAMTS13 and the carboxyl terminal truncated variant (MDTCS), supporting the feasibility of developing a gene therapy based treatment for hereditary TTP. The discrepancy in the proteolytic activity of MDTCS between in vitro (Zhang P et al. Blood, 2007 in press) and in vivo in the present study suggests the potential cofactors in murine circulation that may rescue the defective proteolytic activity of the carboxyl-terminal truncated ADAMTS13 protease seen in vitro.

Increased Plasma VWF Antigen over Decreased ADAMTS13 Activity in Patients with Liver Cirrhosis: Relationship to Degree of Liver Disturbance and Clinical Features.

Background/aims: Deficiency of plasma ADAMTS13 activity (:act) accumulates unusually large von Willebrand factor multimer (UL-VWFM) in circulation that might induce platelet thrombi formation. We demonstrated that hepatic stellate cells are major ADAMTS13-producing cells in human liver using in situ hybridization and immunohistochemistry (Blood , 2005, 106:922), and a decreased plasma ADAMTS13:act in patients with cirrhotic biliary atresia can be fully restored after living-related liver transplantation (Blood 2000, 96:636a). Taken these findings together, ADAMTS13 may play a role on the regulation of sinusoidal microcirculation in the liver. We, therefore, investigated the relationship of ADAMTS13 and its substrate (UL-VWFM) to clinical features in patients with chronic liver diseases.Methods: Plasma levels of ADAMTS13:act, ADAMTS13 antigen (:ag) and VWF antigen (VWF:ag) were determined in 33 patients with chronic hepatitis (CH) and 109 liver cirrhosis (LC). ADAMTS13:act was measured by both the classic VWFM assay and the novel monoclonal antibody-based ELISA (Transfusion 2006, 46:1444). The ADAMTS13:ag was quantified by a sandwich ELISA using two anti-ADAMTS13 murine monoclonal Abs (A10 and C7), and the UL-VWFM was analyzed by a SDS−0.9% agarose gel electrophoresis.Results: ADAMTS13:act in LC progressively decreased from the highest in Child A (mean 79%), to Child B (63%), to the lowest in Child C (30%), compared with CH (87%) and normal healthy subjects (N, 102%). The activity measured by VWFM assay highly correlated with that assayed by ELISA (r=0.75, p<0.001), which was also correlated with ADAMTS13:ag (r=0.79, p<0.001). Markedly decreased ADAMTS13 :act was noted in cirrhotics with hepatic encephalopathy (28%), refractory ascites (25%), and hepatorenal syndrome (13%). The multivariate analysis showed Child-Pugh score and spleen volume as independent factors contributing to the activity (r=0.69, p<0.001). VWF:ag was remarkably high with the progression of liver disturbance (N 100%, CH 245%, Child A LC 320%, Child B LC 436%, Child C LC 486%, respectively), resulting in extremely increased ratio of VWF:ag to ADAMTS13 activity in advanced cirrhotics (N 1.0, CH 2.9, Child A LC 4.3, Child B LC 13.3, Child C LC 43.2, respectively). The cirrhotics with UL-VWFM showed higher Child-Pugh score (10.8 vs. 7.7, p<0.001), lower hemoglobin levels (9.6 g/dl vs. 11.3 g/dl, p<0.005), severe thrombocytopenia (6.2 x104/mm3 vs. 9.2 x104/mm3, p<0.05), higher C-reactive protein (3.8 mg/dl vs. 0.9 mg/dl, p<0.001), and higher serum creatinine (1.5 mg/dl vs. 0,9 mg/dl, p<0.005) than those without.Conclusions: Both the activity and antigen of plasma ADAMTS13 decrease with increasing severity of cirrhotics. The imbalance of a larger amount of UL-VWFM over the decreased plasma level of ADAMTS13 may reflect the predisposing state for the development of microcirculatory disturbance in advanced cirrhotics.

Biosynthesis and Apical Secretion of ADAMTS13 Metalloprotease in Vascular Endothelial Cells and in Madin-Darby Canine Kidney Cells.

ADAMTS13, a reprolysin-like metalloprotease, limits platelet aggregation by cleaving von Willebrand factor. Deficiency of plasma ADAMTS13 activity can lead to a life-threatening complication, thrombotic thrombocytopenic purpura (TTP). It has been shown that ADAMTS13 is synthesized in human and mouse hepatic stellate cells. But detection of ADAMTS13 mRNA by a reverse transcriptase polymerase chain reaction (RT-PCR) in many other tissues (brain, heart, lungs, kidneys, pancreas, adrenal gland, prostate, uterus and placenta) suggests that the vascular endothelial cells may also produce low level of ADAMTS13 protease. We showed that full-length ADAMTS13 mRNA and protein were detected in human umbilical cord vein endothelial cells (HUVEC), human aortic endothelial cells (HAEC), and microvascular endothelial cells (ECV304) by RT-PCR, SDS-polyacrylamide gel electrophoresis and Western blot. The ADAMTS13 in the cell lysates and the serum-free conditioned media of HUVEC, HAEC and ECV304 cleaved a peptidyl substrate, FRETS-VWF73, specifically, and such a proteolytic cleavage could be completely abolished by 10 mM EDTA. Immunohistochemistry showed that both endogenous and recombinant ADAMTS13 in HUVEC and HAEC appeared to be co-localized with von Willebrand factor in the endoplasmic reticulum and/or Golgi apparatus, but not in the Weibel-Parade bodies, suggesting that ADAMTS13 is readily secreted rather than stored inside the cells. To determine whether ADAMTS13 is secreted into the lumen of the vessels, we assessed the polarity of ADAMTS13 secretion in ECV304 and Madin-Darby canine kidney (MDCK) cells, two well-characterized polarized endothelial and epithelial cell lines for studying protein sorting. Both endogenous ADAMTS13 and recombinant ADAMTS13-V5-His expressed in ECV304 was predominantly secreted into the apical domain of the cells. Similarly, the recombinant ADAMTS13-V5-His in MDCK cells was also secreted predominantly into the apical domain. Apical secretion of ADAMTS13-V5-His appeared to depend on at least a transient association of ADAMTS13-V5-His with Triton X-100 insoluble glycosphingolipid and cholesterol-enriched microdomains, namely rafts. Deletion of the distal carboxyl terminal CUB (Complement C1r/C1s, Urinary EGF, and Bone morphogenic protein) domains or removal of membrane cholesterol by treatment of MDCK cells expressing recombinant ADAMTS13-V5-His with lovastatin and methyl-β-cyclodextrin completely abolished the raft association and apical secretion of ADAMTS13-V5-His in MDCK cells. Moreover, an ADAMTS13 mutation (4143insA), which naturally occurs in a patient with TTP that deletes the second CUB domain not only significantly impaired the amount of total ADAMTS13 protein secreted into the conditioned medium, but also reversed the polarity of ADAMTS13 secretion in MDCK cells. Our data demonstrate that: 1) active full-length ADAMTS13 protease is produced in human vascular endothelial cells; 2) the CUB domains are critical for apical secretion via their interaction with lipid rafts; 3) mutations in ADAMTS13 gene that disrupt sorting signals may result in congenital ADAMTS13 deficiency, leading to TTP in some cases. Considering a large surface area of the vascular endothelial cells, apically secreted ADAMTS13 from these cells may contribute significantly to plasma pool of ADAMTS13 protease.

Molecular Biology of ADAMTS13 and Diagnostic Utility of ADAMTS13 Proteolytic Activity and Inhibitor Assays

ADAMTS13, a reprolysin-like metalloprotease, limits platelet-rich thrombus formation in the small arteries by cleaving von Willebrand factor (vWF) at the Tyr1605-Met1606 peptide bond. Deficiency of plasma ADAMTS13 activity, due to either an inherited or an acquired etiology, may lead to a potentially lethal syndrome, thrombotic thrombocytopenic purpura (TTP). Molecular cloning and characterization of the ADAMTS13 gene have provided further insight into the structure-function relationships, biosynthesis, and regulation of the ADAMTS13 protease, in addition to understanding the pathogenesis of TTP and perhaps other thrombotic disorders. ADAMTS13 consists of a short propeptide, a typical reprolysin-like metalloprotease domain, followed by a disintegrin-like domain, first thrombospondin type 1 (TSP1) repeat, Cys-rich domain, and spacer domain. The carboxyl terminus of ADAMTS13 has seven more TSP1 repeats and two CUB domains. ADAMTS13 is synthesized mainly in hepatic stellate cells, but also in vascular endothelial cells. Recognition and cleavage of vWF require the proximal carboxyl terminal domains, but not the middle and distal carboxyl terminal domains. Cleavage of vWF appears to be modulated by shear force, binding to platelet or platelet glycoprotein-1balpha, heparin, inflammatory cytokine (interleukin-6), and chloride ion. At the site of thrombus formation, the ADAMTS13 may be inactivated by thrombin, plasmin, and factor Xa. Having a sensitive and specific assay for ADAMTS13 activity is not only critical to understand the basic biology of ADAMTS13 protease, but also to facilitate a more timely and accurate clinical diagnosis of TTP, and to initiate potentially life-saving plasma exchange therapy. Although many assays have been developed and tested for clinical applications, the fluorescent resonance energy transfer-vWF73 assay appears to be the simplest and most promising assay to date.