BackgroundType 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. Design and MethodsSixty-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. ResultsPlasma 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. ConclusionsThis 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.
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 ϳ8-fold. Conversely, in the presence of a physiological concentration of lyophilized platelets (150 ؋ 10 3 /l), the half-maximal concentration of FVIII required to accelerate VWF proteolysis by ADAMTS13 reduces by ϳ10-fold (to ϳ0.3 nM) compared with that in the absence of platelets (ϳ3.0 nM). Further studies using the FVIII derivative that lacks an acidic region (a3), an antiplatelet glycoprotein 1b␣ IgG, and a purified recombinant VWF-A1 domain or glycoprotein 1b␣-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. ADAMTS13, a member of the A Disintegrin And Metalloprotease with ThromboSpondin type repeats (ADAMTS)2 family (1, 2), controls the sizes of von Willebrand factor (VWF) by cleaving VWF at the Tyr 1605 -Met 1606 bond in the central A2 domain (3,4). This proteolytic cleavage appears to be critical for regulating VWF adhesive function and maintaining normal hemostasis (5). The inability to cleave the ultralarge (UL) VWF into the smaller forms due to a hereditary (1, 6 -8) or acquired deficiency (9 -11) of plasma ADAMTS13 results in thrombotic thrombocytopenic purpura, a potentially fatal thrombotic microangiopathy (5, 12, 13). Conversely, excessive proteolytic cleavage of plasma multimeric VWF by ADAMTS13 leads to a certain subtype of von Willebrand disease (5, 14), the most common bleeding disorder seen in the hematology clinic.Proteolytic processing of UL-VWF by ADAMTS13 appears to occur at least at two different sites: one is on the endothelial cells where UL-VWF is newly released from Weibel-Palade bodies upon stimulation (15,16), and the other may occur in solution (or blood) (3, 10). The cleavage of the cell membraneanchored UL-VWF by ADAMTS13 occurs very rapidly and requires low (15-17) or almost no shear stress (18, 19). However, the cleavage of UL-VWF on the endothelial cell membrane does not appear to be sufficient to reduce the VWF multimer sizes that have been observed in plasma. This is inferred from our study on cultured endothelial cells. The soluble VWF cleaved from the endothelial cell membrane by ADAMTS13 remains ultralarge in size, and the multimer distribution is not different from that released from Weibel-Palade...
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