The function of ADAMTS13 in thrombogenesis in vivo: Insights from mutant mice
National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka, 565-8565, Japan.International journal of hematology (Impact Factor: 1.92). 01/2010; 91(1):30-5. DOI: 10.1007/s12185-009-0477-0
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.
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ABSTRACT: To elucidate how unusually large von Willebrand factor (UL-VWF) multimers facilitate thrombus formation, their behavior was analyzed together with that of platelets in living mice deficient in the gene encoding the protease that cleaves UL-VWF, a disintegrin-like and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13-/-). By crossing ADAMTS13-/- mice with green fluorescent protein-expressing transgenic mice (GFP mice), GFP-ADAMTS13-/- mice were obtained. The dynamics of GFP-expressing platelets were monitored employing intravital confocal fluorescent microscopy. Administration of a vasopressin derivative, DDAVP, a secretagogue of VWF increased the number of platelets adhered to vascular endothelial cells (VECs) on mesentery at sites recognized by an anti-VWF antibody. Some of these platelets were interconnected and aligned as beads on a string. They reached their maximum length at 5 min and were longer in GFP-ADAMTS13-/- mice than in GFP mice (5.3 ± 4.3, N = 6 vs 2.9 ± 2.1 μm, N = 4) (mean±SE). Focal injury of VECs by topical application of FeCl(3) developed longer (25, 3-50 vs 10, 2-25 μm, P < 0.01) (mean, 10th-90th percentile) and more stable (1.3, 0.3-6.3 vs 0.3, 0.2-1.3 s, P < 0.01) connected platelets in GFP-ADAMTS13-/- mice than in GFP mice. This study revealed that ADAMTS13 cleaves platelet-bound UL-VWF multimers, both during their secretion from VECs and after their adherence to injured vascular walls in veins. UL-VWF multimers either being secreted from VECs or circulating in plasma of ADAMTS13-/- mice appeared to facilitate the accumulation of longer and more stable VWF strings with more associated platelets on injured vascular walls.
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ABSTRACT: Plasma ADAMTS13 deficiency results in the clinical disorder thrombotic thrombocytopenic purpura. However, other potential pathophysiological roles of ADAMTS13 in endothelial cell biology remain unexplored. To assess the possible role of ADAMTS13 and its interactions with VEGF-mediated angiogenesis, the effects of ADAMTS13 on human umbilical vein endothelial cell (HUVEC) were studied in Matrigel tube formation, proliferation, cell migration, and scratch wound assays. Treatment of endothelial cells with exogenous recombinant full-length ADAMTS13 alone promoted angiogenesis in a dose-dependent manner. HUVEC incubated with 200 ng/mL ADAMTS13 (1.4 nM) resulted in a 65% increase in cell tube formation when compared to the EBM-2 control. HUVEC treated with 30 ng/mL ADAMTS13 (204.1 pM) resulted in an 83% increase in proliferation in a visual counting assay, whereas HUVEC treated with 10 ng/mL ADAMTS13 (68.0 pM) yielded a 295% increase in EC migration in a Boyden chamber assay. In contrast, ADAMTS13 inhibited VEGF-induced angiogenesis in a dose-dependent manner, with 200ng/mL inhibiting tube formation by 35%. HUVEC co-incubated with ADAMTS13 and an antibody to the ADAMTS13 thrombospondin domains 5-7 reversed the inhibition of tube formation. HUVEC treated with 30 ng/mL ADAMTS13 and 6.2 ng/mL (323.0 pM) VEGF proliferated 40% slower than the VEGF control after 24 h of incubation as measured by visual counting assay. Treatment of HUVEC with 6.2 ng/mL VEGF and 10 ng/mL ADAMTS13 inhibited cell migration by 48%, compared to the VEGF control. Substitution of ADAMTS13 with truncated ADAMTS13 (deletion of C-terminal TSP1 domain) did not significantly increase angiogenesis or suppress VEGF-induced angiogenesis, suggesting that the TSP1 domain is involved in ADAMTS13 angiogenic activities. Co-immunoprecipitation experiments provided further evidence that ADAMTS13 binds to VEGF via its TSP1 domain.
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ABSTRACT: Normal hemostasis requires von Willebrand factor (VWF) to support platelet adhesion and aggregation at sites of vascular injury. VWF is a multimeric glycoprotein built from identical subunits that contain binding sites for both platelet glycoprotein receptors and collagen. The adhesive activity of VWF depends on the size of its multimers, which range from 500 to over 10 000 kDa. There is good evidence that the high-molecular-weight multimers (HMWM), which are 5000-10 000 kDa, are the most effective in supporting interaction with collagen and platelet receptors and in facilitating wound healing under conditions of shear stress. Thus, these HMWM of VWF are of particular clinical interest. The unusually large multimers of VWF are, under normal conditions, cleaved by the plasma metalloproteinase ADAMTS13 to smaller, less adhesive multimers. A reduction or lack of HMWM, owing to a multimerization defect of VWF or to an increased susceptibility of VWF for ADAMTS13, leads to a functionally impaired VWF and the particular type 2A of von Willebrand disease. This review considers the biology and function of VWF multimers with a particular focus on the characterization of HMWM - their production, storage, release, degradation, and role in normal physiology. Evidence from basic research and the study of clinical diseases and their management highlight a pivotal role for the HMWM of VWF in hemostasis.