[show abstract][hide abstract] ABSTRACT: 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.
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.
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 CaCl(2) and 1.7 µmol/L ZnCl(2) 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.
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.
[show abstract][hide abstract] ABSTRACT: BACKGROUND: The hemostatic property of “fresh” whole blood (WB) has been observed in military application and cardiac surgery and is associated with reduced blood loss, transfusion requirements, and donor exposures. The time from donation to transfusion defining “fresh” has not been systematically studied. We undertook an in vitro study of coagulation properties of refrigerated WB stored for 31 days.STUDY DESIGN AND METHODS: Twenty-one WB units were obtained from healthy volunteer donors and stored under standard AABB refrigerated conditions. Samples were obtained on the day after donation and again on Days 2, 4, 7, 11, 14, 17, 21, 24, and 31. Tests included complete blood count, pH, pO2, pCO2, glucose, lactate, thromboelastography (TEG), and platelet function by light transmission aggregometry (LTA).RESULTS: There was progressive decline in pH, pO2, glucose, and sodium, but progressive increase in potassium, pCO2, and lactate. TEG variables in all units were normal through Day 11; abnormal values in some variables in some units began on Day 14. Final aggregation levels exhibited no change from Day 1 to Day 21 with adenosine diphosphate and epinephrine, but a decline with collagen (Day 7) and ristocetin (Day 17).CONCLUSION: This in vitro study of coagulation properties demonstrates preservation of normal integrated coagulation function to a minimum of 11 days under standard conditions of refrigerated storage of WB for transfusion. These observations strongly suggest that the hemostatic quality of WB may extend beyond current transfusion practices. If confirmed clinically, this would increase availability and extend benefits of reduced donor exposure and transfusion requirements.
[show abstract][hide abstract] ABSTRACT: Previous studies have demonstrated that factor VIII (FVIII) or platelets alone increase cleavage of von Willebrand factor (VWF) by ADAMTS13 under mechanically induced shear stresses. We show in this study that the combination of FVIII and platelets at the physiological concentrations is more effective than either one alone. In the absence of FVIII, lyophilized platelets increase the formation of cleavage product by 2-3-fold. However, in the presence of physiological concentration of FVIII (1 nm), the formation of VWF cleavage product increases dramatically as a function of increasing platelets with the maximal rate enhancement of approximately 8-fold. Conversely, in the presence of a physiological concentration of lyophilized platelets (150 x 10(3)/microl), the half-maximal concentration of FVIII required to accelerate VWF proteolysis by ADAMTS13 reduces by approximately 10-fold (to approximately 0.3 nm) compared with that in the absence of platelets ( approximately 3.0 nm). Further studies using the FVIII derivative that lacks an acidic region (a3), an antiplatelet glycoprotein 1balpha IgG, and a purified recombinant VWF-A1 domain or glycoprotein 1balpha-stripped platelets demonstrate that the synergistic rate-enhancing effect of FVIII and platelets depends on their specific binding interactions with VWF. Our findings suggest that FVIII and platelets are cofactors that regulate proteolysis of multimeric VWF by ADAMTS13 under physiological conditions.
Journal of Biological Chemistry 09/2010; 285(37):28596-603. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Type G immunoglobulins against ADAMTS13 are the primary cause of acquired (idiopathic) thrombotic thrombocytopenic purpura. However, the domains of ADAMTS13 which the type G anti-ADAMT13 immunoglobulins target have not been investigated in a large cohort of patients with thrombotic thrombocytopenic purpura.
Sixty-seven patients with acquired idiopathic thrombotic thrombocytopenic purpura were prospectively collected from three major U.S. centers. An enzyme-linked immunosorbent assay determined plasma concentrations of anti-ADAMTS13 type G immunoglobulins, whereas immunoprecipitation plus western blotting determined the binding domains of these type G immunoglobulins.
Plasma anti-ADAMTS13 type G immunoglobulins from 67 patients all bound full-length ADAMTS13 and a variant truncated after the eighth TSP1 repeat (delCUB). Approximately 97% (65/67) of patients harbored type G immunoglobulins targeted against a variant truncated after the spacer domain (MDTCS). However, only 12% of patients' samples reacted with a variant lacking the Cys-rich and spacer domains (MDT). In addition, approximately 37%, 31%, and 46% of patients' type G immunoglobulins interacted with the ADAMTS13 fragment containing TSP1 2-8 repeats (T2-8), CUB domains, and TSP1 5-8 repeats plus CUB domains (T5-8CUB), respectively. The presence of type G immunoglobulins targeted against the T2-8 and/or CUB domains was inversely correlated with the patients' platelet counts on admission.
This multicenter study further demonstrated that the multiple domains of ADAMTS13, particularly the Cys-rich and spacer domains, are frequently targeted by anti-ADAMTS13 type G immunoglobulins in patients with acquired (idiopathic) thrombotic thrombocytopenic purpura. Our data shed more light on the pathogenesis of acquired thrombotic thrombocytopenic purpura and provide further rationales for adjunctive immunotherapy.
[show abstract][hide abstract] ABSTRACT: Previous studies have shown that ADAMTS13 spacer domain is required for cleavage of von Willebrand factor (VWF). However, the exact amino acid residues within this domain critical for substrate recognition are not known. Epitope mapping of anti-ADAMTS13 immunoglobulin G from patients with thrombotic thrombocytopenic purpura and sequence alignment of the ADAMTS13 spacer domains of human, mouse, and zebrafish with these of human and murine ADAMTS1, a closely related member of ADAMTS family, have provided hints to investigate the role of the amino acid residues between Arg(659) and Glu(664) of the ADAMTS13 spacer domain in substrate recognition. A deletion of all these 6 amino acid residues (ie, Arg(659)-Glu(664)) from the ADAMTS13 spacer domain resulted in dramatically reduced proteolytic activity toward VWF73 peptides, guanidine-HCl denatured VWF, and native VWF under fluid shear stress, as well as ultralarge VWF on endothelial cells. Site-directed mutagenesis, kinetic analyses, and peptide inhibition assays have further identified a role for amino acid residues Arg(659), Arg(660), and Tyr(661) in proteolytic cleavage of various substrates under static and fluid shear stress conditions. These findings may provide novel insight into the structural-function relationship of ADAMTS13 and help us to understand pathogenesis of thrombotic thrombocytopenic purpura and other arterial thromboses associated with compromised VWF proteolysis.
[show abstract][hide abstract] ABSTRACT: Dengue is a mosquito-borne viral disease with an increasing incidence worldwide. Thrombocytopenia is a common finding in dengue virus (DV) infection; however, the underlying mechanisms remain unknown.
Here we provide the first evidence of a case of antibody formation against ADAMTS13 (ADAMTS13 inhibitor) in the course of a severe acute DV infection resulting in thrombotic microangiopathy (TMA). The patient presented with classical dengue symptoms (positive epidemiology, high fever, myalgia, predominantly in the lower limbs and lumbar region for 1 week) and, after 11 days of initial symptoms, developed TMA. Clinical and laboratorial investigation of dengue and TMA was performed.
The patient presented with ADAMTS13 inhibitor (IgG) during the acute phase of the disease, without anti-platelet antibodies detectable. Dengue infection had laboratorial confirmation. There were excellent clinical and laboratory responses to 11 serial plasma exchanges. Anti-ADAMTS13 inhibitor disappeared after remission of TMA and dengue resolution. No recurrence of TMA symptoms was observed after 2-year follow-up.
Although the real incidence of dengue-related TMA is unknown, this case provides the basis for future epidemiologic studies on acquired ADAMTS13 deficiency in DV infection. The prompt clinical recognition of this complication and early installment of specific therapy with plasma exchange are likely to improve the outcome of severe cases of dengue.
[show abstract][hide abstract] ABSTRACT: The function of von Willebrand factor (VWF) is regulated by proteolysis, which limits its multimeric size and ability to tether platelets. The importance of ADAMTS13 metalloprotease in VWF regulation is demonstrated by the association between severe deficiency of ADAMTS13 and thrombotic thrombocytopenic purpura (TTP). However, ADAMTS13 activity levels do not always correlate with the clinical course of TTP, suggesting that other proteases could be important in regulating VWF. We identified 4 leukocyte proteases that cleave the synthetic VWF substrate FRETS-VWF73 and multimeric VWF. Elastase and proteinase 3 (PR3) cleave multimeric VWF and FRETS-VWF73 at the V(1607)-T(1608) peptide bond; cathepsin G and matrix metalloprotease 9 cleave VWF substrates at the Y(1605)-M(1606) and M(1606)-V(1607) bonds, respectively. Isolated intact human neutrophils cleave FRETS-VWF73 at the V(1607)-T(1608) peptide bond, suggesting that elastase or PR3 expressed on leukocyte surfaces might cleave VWF. In the presence of normal or ADAMTS13-deficient plasma, cleavage of FRETS-VWF73 by resting neutrophils is abolished. However, activated neutrophils retain proteolytic activity toward FRETS-VWF73 in the presence of plasma. Although the in vivo relevance remains to be established, these studies suggest the existence of a "hot spot" of VWF proteolysis in the VWF A2 domain, and support the possibility that activated leukocytes may participate in the proteolytic regulation of VWF.
[show abstract][hide abstract] ABSTRACT: Enteropeptidase, a type II transmembrane serine protease, is localized to the brush border of the duodenal and jejunal mucosa. It is synthesized as a zymogen (proenteropeptidase) that requires activation by another protease, either trypsin or possibly duodenase. Active enteropeptidase then converts the pancreatic precursor, trypsinogen, to trypsin by cleavage of the specific trypsinogen activation peptide, Asp-Asp-Asp-Asp-Lys- Ile that is highly conserved in vertebrates. Trypsin, in turn, activates other digestive zymogens such as chymotrypsinogen, proelastase, procarboxypeptidase and prolipase in the lumen of the gut. The important biological function of enteropeptidase is highlighted by the manifestation of severe diarrhea, failure to thrive, hypoproteinemia and edema as a result of congenital deficiency of enteropeptidase activity in the gut. Conversely, duodenopancreatic reflux of proteolytically active enteropeptidase may cause acute and chronic pancreatitis.
Frontiers in bioscience (Elite edition) 06/2009; 1:242-9.
[show abstract][hide abstract] ABSTRACT: Patients who have undergone a Fontan procedure face an increased risk for thromboembolic complications. This study aimed to evaluate whether thromboelastography, a global whole-blood assay of coagulation, can be used to detect hypercoagulability in pediatric Fontan patients compared with healthy children. This prospective, cross-sectional study investigated 25 Fontan patients and 51 healthy children in three age groups: 1-5 years, 6-10 years, and 11-16 years. Kaolin-activated thromboelastography was performed on citrated samples. No statistically significant differences in thromboelastography parameters were found among the different age groups of the 51 healthy children. None of the 25 Fontan patients demonstrated evidence of hypercoagulability on thromboelastography (95% confidence interval, 0-7%), as defined by two standard deviations above or below the normal mean. The findings suggest that the percentage of Fontan patients demonstrating hypercoagulability on thromboelastography is substantially lower than the reported incidence of thromboembolic complications. Whether thromboelastography could be helpful in predicting patients at increased risk for thromboembolic complications or not still is not known. Further studies comparing the thromboelastography of Fontan patients with the thromboembolic complications of those without Fontan are needed to delineate these issues.
[show abstract][hide abstract] ABSTRACT: Thrombotic thrombocytopenic purpura (TTP) is a fulminant disease characterized by platelet aggregates, thrombocytopenia, renal insufficiency, neurologic changes, and mechanical injury to erythrocytes. Most idiopathic cases of TTP are characterized by a deficiency of ADAMTS13 (a disintegrin and metalloprotease, with thrombospondin-1-like domains) metalloprotease activity. Ironically, use of anti-platelet agents, the thienopyridine derivates clopidogrel and ticlopidine, is associated with drug induced TTP. Data were abstracted from a systematic review of English-language literature for thienopyridine-associated TTP identified in MEDLINE, EMBASE, the public website of the Food and Drug Administration, and abstracts from national scientific conferences from 1991 to April 2008. Ticlopidine and clopidogrel are the two most common drugs associated with TTP in FDA safety databases. Epidemiological studies identify recent initiation of anti-platelet agents as the most common risk factor associated with risks of developing TTP. Laboratory studies indicate that most cases of thienopyridine-associated TTP involve an antibody to ADAMTS13 metalloprotease, present with severe thrombocytopenia, and respond to therapeutic plasma exchange (TPE); a minority of thienopyridine-associated TTP presents with severe renal insufficiency, involves direct endothelial cell damage, and is less responsive to TPE. The evaluation of this potentially fatal drug toxicity can serve as a template for future efforts to comprehensively characterize other severe adverse drug reactions.
Kidney International 02/2009; 112:S20-4. · 7.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: ADAMTS13, a metalloprotease primarily synthesized in liver and endothelial cells, cleaves von Willebrand factor (VWF) at the central A2 domain, thereby reducing the sizes of circulating VWF multimers. Genetic or acquired deficiency of plasma ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). To date, plasma infusion or exchange is the only proven effective therapy for TTP. In search for a better therapy, an autologous transplantation of hematopoietic progenitor cells transduced ex vivo with a self-inactivating lentiviral vector encoding a full-length murine Adamts13 and an enhanced green fluorescent protein (GFP) reporter gene was performed in Adamts13(-/-) mice after irradiation. All recipient mice showed detectable ADAMTS13 antigen and proteolytic activity in plasma despite only low levels of bone marrow chimerism. The levels of plasma ADAMTS13 were sufficient to eliminate the ultralarge VWF multimers and offered systemic protection against ferric chloride-induced arterial thrombosis. The data suggest that hematopoietic progenitor cells can be genetically modified ex vivo and transplanted in an autologous model to provide adequate levels of functional ADAMTS13 metalloprotease. This success may provide the basis for development of a novel therapeutic strategy to cure hereditary TTP in humans.
[show abstract][hide abstract] ABSTRACT: In an effort to improve interlaboratory agreement in the monitoring of unfractionated heparin (UFH), the College of American Pathologists (CAP) recommends that the therapeutic range of the activated partial thromboplastin time (APTT) be defined in each laboratory through correlation with a direct measure of heparin activity such as the factor Xa inhibition assay. Whether and to what extent this approach enhances the interlaboratory agreement of UFH monitoring has not been reported.
We conducted a cross-validation study among four CAP-accredited coagulation laboratories to compare the interlaboratory agreement of the anti-FXa-correlated APTT with that of the traditional 1.5-2.5 times the midpoint of normal (1.5-2.5:control) method for defining the therapeutic APTT range.
APTT and FXa inhibition assays were performed in each laboratory on plasma samples from 44 inpatients receiving UFH.
Using the anti-FXa-correlation method, there was agreement among all four laboratories as to whether a sample was subtherapeutic, therapeutic or supratherapeutic in seven (16%) patient samples. In contrast, consensus was achieved in 23 (52%) samples when the 1.5-2.5:control method was employed.
The anti-FXa-correlation method does not appear to enhance interlaboratory agreement in UFH monitoring as compared with the traditional 1.5-2.5:control method. Adoption of the anti-FXa-correlation method produces considerable disparity in UFH dosing decisions among different centers, although the clinical impact of this disparity is not known.
Journal of Thrombosis and Haemostasis 12/2008; 7(1):80-6. · 6.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Deficiency of A Disintegrin And Metalloprotease with ThromboSpondin (ADAMTS13) results in thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective treatment to date. We show in this study that an administration of a self-inactivating lentiviral vector encoding human full-length ADAMTS13 and a variant truncated after the spacer domain (MDTCS) in mice by in utero injection at embryonic days 8 and 14 resulted in detectable plasma proteolytic activity (approximately 5-70%), which persisted for the length of the study (up to 24 weeks). Intravascular injection via a vitelline vein at E14 was associated with significantly lower rate of fetal loss than intra-amniotic injection, suggesting that the administration of vector at E14 may be a preferred gestational age for vector delivery. The mice expressing ADAMTS13 and MDTCS exhibited reduced sizes of von Willebrand factor (vWF) compared to the Adamts13(-/-) mice expressing enhanced green fluorescent protein (eGFP). Moreover, the mice expressing both ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time as compared to the Adamts13(-/-) expressing eGFP. The data demonstrate the successful correction of the prothrombotic phenotypes in Adamts13(-/-) mice by a single in utero injection of lentiviral vectors encoding human ADAMTS13 genes, providing the basis for developing a gene therapy for hereditary TTP in humans.
[show abstract][hide abstract] ABSTRACT: Proteolytic processing of von Willebrand factor (VWF) by ADAMTS13 metalloproteinase is crucial for normal hemostasis. In vitro, cleavage of VWF by ADAMTS13 is slow even at high shear stress and is typically studied in the presence of denaturants. We now show that, under shear stress and at physiological pH and ionic strength, coagulation factor VIII (FVIII) accelerates, by a factor of approximately 10, the rate of specific cleavage at the Tyr(1605)-Met(1606) bond in VWF. Multimer analysis reveals that FVIII preferentially accelerates the cleavage of high-molecular-weight multimers. This rate enhancement is not observed with VWF predenatured with 1.5 M guanidine. The ability of FVIII to enhance VWF cleavage by ADAMTS13 is rapidly lost after pretreatment of FVIII with thrombin. A FVIII derivative lacking most of the B domain behaves equivalently to full-length FVIII. In contrast, a derivative lacking both the B domain and the acidic region a3 that contributes to the high-affinity interaction of FVIII with VWF exhibits a greatly reduced ability to enhance VWF cleavage. Our data suggest that FVIII plays a role in regulating proteolytic processing of VWF by ADAMTS13 under shear stress, which depends on the high-affinity interaction between FVIII and its carrier protein, VWF.
Proceedings of the National Academy of Sciences 06/2008; 105(21):7416-21. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Profound thrombocytopenia and microangiopathic hemolytic anemia characterize thrombotic microangiopathy, which includes two major disorders: thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). TTP has at least three types: congenital or familial, idiopathic, and nonidiopathic. The congenital and idiopathic TTP syndromes are caused primarily by deficiency of ADAMTS13, owing to mutations in the ADAMTS13 gene or autoantibodies that inhibit ADAMTS13 activity. HUS is similar to TTP, but is associated with acute renal failure. Diarrhea-associated HUS accounts for more than 90% of cases and is usually caused by infection with Shiga-toxin-producing Escherichia coli (O157:H7). Diarrhea-negative HUS is associated with complement dysregulation in up to 50% of cases, caused by mutations in complement factor H, membrane cofactor protein, factor I or factor B, or by autoantibodies against factor H. The incomplete penetrance of mutations in either ADAMTS13 or complement regulatory genes suggests that precipitating events or triggers may be required to cause thrombotic microangiopathy in many patients.
Annual Review of Pathology Mechanisms of Disease 02/2008; 3:249-77. · 25.79 Impact Factor
[show abstract][hide abstract] ABSTRACT: ADAMTS13 cleaves von Willebrand factor (VWF) between Tyr(1605) and Met(1606) residues at the central A2 subunit. The amino-terminus of ADAMTS13 protease appears to be sufficient to bind and cleave VWF under static and denatured condition. However, the role of the carboxyl-terminus of ADAMTS13 in substrate recognition remains controversial. Present study demonstrates that ADAMTS13 cleaves VWF in a rotation speed- and protease concentration-dependent manner on a mini vortexer. Removal of the CUB domains (delCUB) or truncation after the spacer domain (MDTCS) significantly impairs its ability to cleave VWF under the same condition. ADAMTS13 and delCUB (but not MDTCS) bind VWF under flow with dissociation constants (K(D)) of about 50 nM and about 274 nM, respectively. The isolated CUB domains are neither sufficient to bind VWF detectably nor capable of inhibiting proteolytic cleavage of VWF by ADAMTS13 under flow. Addition of the TSP1 5-8 (T5-8CUB) or TSP1 2-8 repeats (T2-8CUB) to the CUB domains restores the binding affinity toward VWF and the inhibitory effect on cleavage of VWF by ADAMTS13 under flow. These data demonstrate directly and quantitatively that the cooperative activity between the middle carboxyl-terminal TSP1 repeats and the distal carboxyl-terminal CUB domains may be crucial for recognition and cleavage of VWF under flow.