Essential Domains of A Disintegrin and Metalloprotease With Thrombospondin Type 1 Repeats-13 Metalloprotease Required for Modulation of Arterial Thrombosis (original) (raw)
Related papers
Blood, 2009
ADAMTS13 is a multidomain protease that limits platelet thrombogenesis through the cleavage of von Willebrand factor (VWF). We previously identified 2 types of mouse Adamts13 gene: the 129/Sv-strain Adamts13 gene encodes the long-form ADAMTS13 having the same domains as human ADAMTS13, whereas the C57BL/6-strain Adamts13 gene encodes the short-form ADAMTS13 lacking the distal C-terminal domains. To assess the physiologic significance of the distal C-terminal domains of ADAMTS13, we generated and analyzed 129/Sv-genetic background congenic mice (Adamts13 S/S ) that carry the short-form ADAMTS13. Similar to wild-type 129/Sv mice (Adamts13 L/L ), Adamts13 S/S did not have ultralarge VWF multimers in plasma, in contrast to 129/Svgenetic background ADAMTS13-deficient mice (Adamts13 ؊/؊ ). However, in vitro thrombogenesis under flow at a shear rate of 5000 s ؊1 was accelerated in Adamts13 S/S compared with Adamts13 L/L . Both in vivo thrombus formation in ferric chlorideinjured arterioles and thrombocytopenia induced by collagen plus epinephrine challenge were more dramatic in Adamts13 S/S than in Adamts13 L/L but less than in Adamts13 ؊/؊ . These results suggested that the C-terminally truncated ADAMTS13 exhibited decreased activity in the cleavage of VWF under high shear rate. Role of the C-terminal domains may become increasingly important under prothrombotic conditions. (Blood. 2009;113:5323-5329)
The function of ADAMTS13 in thrombogenesis in vivo: insights from mutant mice
International Journal of Hematology, 2010
Recently, two independent groups have established ADAMTS13-deficient mice using gene-targeting techniques. In humans, genetic or acquired deficiency in ADAMTS13 leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Surprisingly, ADAMTS13-deficient mice are viable with no apparent signs of TTP. However, these mouse models indicate that ADAMTS13 down-regulates platelet adhesion and aggregation in vivo, and ADAMTS13 deficiency can provide enhanced thrombus formation at the site of vascular lesions. In addition, ADAMTS13 by cleaving hyperactive ultra-large von Willebrand factor multimers not only down-regulates thrombosis but also inflammation. ADAMTS13-congenic mice that carry a truncated form of ADAMTS13 lacking the C-terminal domains have also been developed. Phenotypes of the congenic mice indicate the physiological significance of the C-terminal domains of ADAMTS13 in down-regulating thrombus growth. The studies mentioned here in different mouse models uncover the in vivo function of ADAMTS13 and strengthened the understanding of the mechanism of systemic disease TTP.
Arteriosclerosis, Thrombosis, and Vascular Biology, 2013
Objective— ADAMTS13 (A Disintegrin And Metalloprotease with Thrombospondin type 1 repeats, 13) cleaves von Willebrand factor (VWF), thereby inhibiting thrombus formation. Proteolytic cleavage relies on the amino-terminal (MDTCS) domains, but the role of the more distal carboxyl-terminal domains of ADAMTS13 is not fully understood. A previous study demonstrated the presence of multiple surface-exposed free sulfhydryls on ADAMTS13 that seemed to interact with those on VWF under shear. Here, we determined the physiological relevance of such an interaction in antithrombotic responses under flow. Approach and Results— A microfluidic assay demonstrated that a carboxyl-terminal fragment of ADAMTS13, comprising either 2 to 8 thrombospondin type 1 (TSP1) repeats and CUB domains (T2C) or 5 to 8 Thrombospondin type 1 (TSP1) repeats and CUB domains (T5C), directly inhibited platelet adhesion/aggregation on a collagen surface under arterial shear. In addition, an intravital microscopic imaging a...
ADAMTS13 exerts a thrombolytic effect in microcirculation
Thrombosis and Haemostasis, 2012
Recombinant tissue plasminogen activator (r-tPA) is the drug of choice for thrombolysis, but it is associated with a significant risk of bleeding and is not always successful. By cleaving von Willebrand factor (VWF), the metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats-13) down-regulates thrombus formation in injured vessels. We investigated whether recombinant ADAMTS13 (r-ADAMTS13) induces thrombolysis in vivo in mice. Thrombosis was produced by ferric chloride-induced (FeCl 3 ) injury in the venules of a dorsal skinfold chamber. Phosphate-buffered saline (PBS, vehicle), r-tPA or r-ADAMTS13, supplemented with hirudin (to stop ongoing thrombin generation), was directly applied onto the occluded vessel, and thrombus dissolution was evaluated by intravital microscopy. The incidence of blood flow restoration significantly increased 30 minutes (min) after r-ADAMTS13 vs. PBS treatment (60% vs. 0%, p<0.05) and 60 min after r-tPA treatment (75% vs. 17%, p<0.05). Both r-tPA and r-ADAMTS13 significantly reduced thrombus size 60 min after their superfusion (53.2% and 62.3% of the initial thrombus size, p<0.05 and p<0.01, respectively). Bleeding occurred in all r-tPA-treated chambers, while it was absent in mice treated with r-ADAMTS13 or PBS. We observed that, similar to r-tPA, r-ADAMTS13 can dissolve occlusive thrombi induced by FeCl 3 injury in venules. In contrast to r-tPA, the in vivo thrombolytic effect of ADAMTS13 was not associated with any signs of haemorrhage. ADAMTS13 could represent a new therapeutic option for thrombolysis.
Systemic antithrombotic effects of ADAMTS13
Journal of Experimental Medicine, 2006
Abbreviations used: ADAMTS, a disintegrin-like and metalloprotease with thrombospondin type I repeats; r-hu, recombinant human; TTP, thrombotic thrombocytopenic purpura; UL-VWF, ultra-large VWF; VWF, von Willebrand factor.
Blood, 2015
ADAMTS13 metalloprotease cleaves von Willebrand factor (VWF), thereby inhibiting platelet aggregation and arterial thrombosis. An inability to cleave ultra large VWF resulting from hereditary or acquired deficiency of plasma ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Plasma exchange is the most effective initial therapy for TTP to date. Here, we report characterization of transgenic mice expressing recombinant human ADAMTS13 (rADAMTS13) in platelets and its efficacy in inhibiting arterial thrombosis and preventing hereditary and acquired antibody-mediated TTP in murine models. Western blotting and fluorescent resonance energy transfer (FRETS) assay detect full-length rADAMTS13 protein and its proteolytic activity, respectively, in transgenic (Adamts13(-/-)Plt(A13)), but not in wild type (WT) and Adamts13(-/-) (KO) platelets. The expressed rADAMTS13 is released upon stimulation with thrombin and collagen, but less with 2MesADP....
Journal of thrombosis and haemostasis : JTH, 2014
Congenital thrombotic thrombocytopenic purpura (TTP) is characterized by mutations in the ADAMTS13 gene, which either impair protein secretion or influence ADAMTS13 (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type-1 motif, member 13) activity. Phenotypic consequences of these mutations have not yet been evaluated in animal models for TTP. To identify the in vitro effect of a novel ADAMTS13 mutation and to investigate whether this mutation induces TTP in vivo. All 29 ADAMTS13 exons with exon-intron boundaries of a patient with pregnancy-onset TTP were sequenced. Wild-type and mutant ADAMTS13 proteins were both transiently and stably expressed in human embryonic kidney cells, and their activity was evaluated in vitro using fluorescence resonance energy transfer and flow assays. Molecular dynamics simulations were performed to study Ca(2+) stability. Adamts13(-/-) mice were hydrodynamically injected with wild-type and mutant expression plasmids and triggered with...
Thrombospondin1 and ADAMTS13 competitively bind to VWF A2 and A3 domains in vitro
Thrombosis Research, 2010
Introduction: ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 repeat motif. 13) is the major metalloprotease for VWF degradation. ADAMTS13 deficiency causes the accumulation of uncleaved VWF and might lead to a lethal thrombotic thrombocytopenic purpura (TTP). Thrombospondin-1 (TSP1) is considered as a reductase of VWF (von Willebrand factor) which can mildly downregulate the size of VWF by targeting on disulfide bond between VWF dimers. It was reported that TSP1 might protected VWF from cleaving by ADAMTS13, yet the underlying mechanism of this VWF protection has remained unknown. Materials and Methods: Full-length ADAMTS13 and different domains (A1,A2,A3) of human VWF were constructed and expressed respectively. The binding ability of TSP1 or ADAMTS13 with each VWF domain or full-length VWF was investigated by using enzyme linked immunosorbent assay. The inhibition of ADAMTS13 activities by the different concentrations of TSP1 were observed by western blot and residualcollagen binding assay (R-CBA) under the denaturing condition. Results: We found that ADAMTS13 interacted with the rVWF A1, A2, A3 domains and full-length VWF, while TSP1 also bound to three A domains, especially to A2 and A3 domains. We observed that TSP1 partially blocked ADAMTS13 binding to A2 domain, A3 domain and full length VWF. The results of our assays showed that TSP1 could restrain ADAMTS13 activity up to 70%. Conclusions: Our study suggested that TSP1 played competitively inhibitory role in ADAMTS13 binding and cleaving of VWF, and the potential competition might happen within A2 and A3 domains.
Structure–function and regulation of ADAMTS‐13 protease
Journal of Thrombosis and Haemostasis, 2013
ADAMTS13, a plasma reprolysin-like metalloprotease, cleaves von Willebrand factor (VWF). Severe deficiency of plasma ADAMTS13 activity results in thrombotic thrombocytopenic purpura (TTP), while mild to moderate deficiencies of plasma ADAMTS13 activity are emerging risk factors for developing myocardial and cerebral infarction, preeclampsia, and malignant malaria. Moreover, Adamts13 −/− mice develop more severe inflammatory responses, leading to increased ischaemia/perfusion injury and formation of atherosclerosis. Structure-function studies demonstrate that the N-terminal portion of ADAMTS13 (MDTCS) is necessary and sufficient for proteolytic cleavage of VWF under various conditions and attenuation of arterial/venous thrombosis after oxidative injury. The more distal portion of ADAMTS13 (TSP1 2-8 repeats and CUB domains) may function as a disulphide bond reductase to prevent an elongation of ultra large VWF strings on activated endothelial cells and inhibit platelet adhesion/aggregation on collagen surface under flow. Remarkably, the proteolytic cleavage of VWF by ADAMTS13 is accelerated by FVIII and platelets under fluid shear stress. A disruption of the interactions between FVIII (or platelet glycoprotein 1bα) and VWF dramatically impairs ADAMTS13-dependent proteolysis of VWF in vitro and in vivo. These results suggest that FVIII and platelets may be physiological cofactors regulating VWF proteolysis. Finally, the structure-function and autoantibody mapping studies allow us to identify an ADAMTS13 variant with increased specific activity but reduced inhibition by autoantibodies in patients with acquired TTP. Together, these findings provide novel insight into the mechanism of VWF proteolysis and tools for the therapy of acquired TTP and perhaps other arterial thrombotic disorders. ADAMTS13 and potential human diseases ADAMTS13, first identified and cloned in 2001, is a member of the ADAMTS (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats) family (1,2). It cleaves a large polymeric adhesion protein von Willebrand factor (VWF). VWF is synthesised in vascular endothelial cells and megakaryocytes (3,4). The newly synthesised VWF is stored in intracellular organelles: Weibel-Palade bodies in endothelial cells and αgranules in megakaryocytes and platelets (3,4). VWF is released upon physiological or pathological stimulation and forms an ultra-long or ultra-large (UL) "string-like" structure on the endothelial surface (5-7). These UL-VWF "string-like" structures are hyperactive in recruiting circulating platelets to the site of endothelial activation and/or injury. Plasma ADAMTS13, which is primarily synthesised and released from hepatic stellate cells (8-10) and endothelial cells (11,12), binds and cleaves cell bound UL-VWF strings at the Tyr 1605-Met 1606 bond, thereby eliminating the UL-VWF from the endothelial surface and resulting
ADAMTS13 binds to CD36: a potential mechanism for platelet and endothelial localization of ADAMTS13
Transfusion, 2009
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. A DAMTS13 (a disintegrin and metalloprotease with thrombospondin-1-like domains) was initially named von Willebrand factor (VWF)cleaving protease. Its discovery was triggered when four patients with chronic relapsing thrombotic thrombocytopenic purpura (TTP) were noted to have "unusually large" multimers of VWF in their plasma. 1 This led to the search and discovery of an enzyme responsible for cleavage of these multimers. 2,3 It was subsequently shown that ADAMTS13 cleaves ultralarge VWF (ULVWF) on the endothelial surface under flow conditions. 4 It is now generally recognized that ADAMTS13 plays an important physiologic role in regulation of VWF. 5,6 ADAMTS13 circulates in plasma at a concentration of 0.5 to 1.0 mg per mL 7 and is synthesized principally in the liver. 8 In addition, recent studies have shown that ADAMTS13 is also produced in endothelial cells 9 and platelets (PLTs). 10,11 It has been speculated that the thrombospondin type 1 ABBREVIATIONS: FRET = fluorescence resonance energy transfer; PWB = platelet wash buffer; TTP = thrombotic thrombocytopenic purpura; ULVWF = ultralarge von Willebrand factor.