| Objective:Thrombotic thrombocytopenic purpura(TTP) is a rare but life-threatening disorder characterised by systemic thrombotic microangiopathy. Main clinical manifestations of TTP are as follows: thrombocytopenia, microangiopathic hemolytic anemia, neuropsychiatric symptoms, renal injury and fever. TTP triad syndrome includes thrombocytopenia, microangiopathic hemolytic anemia and neuropsychiatric symptoms. In addition, renal injury and fever accompanying with these triad syndrome is called as pentad syndrome of TTP. Recent studies have showed that the severely reduced activity of ADAMTS13, a plasma metalloprotease which cleaves von Willebrand factor, is the fundamental pathogenesis of TTP. According to its etiology and pathogenesis, TTP is often classified into two general types: congenital(Upshaw-Schulman syndrome) and acquired. The former is presented with severe ADAMTS-13 deficiency caused by mutations in the ADAMTS13 gene, while the latter is caused by autoantibodies or inhibitors against ADAMTS13 metalloprotease. The annual incidence of aquired TTP in the United States is estimated to be 3-10 cases in 1 000 000 and appears to be increasing. According to this incidence, about 5,000 patients with aquired TTP should emerge annually in China. In the initial period of discovery ADAMTS13 some authors consider that:ADAMTS13 deficiency alone could induce the develepment of TTP. However, with some detailed studies, more and more researchers began to doubt about this above viewpoint. The aim of this experiment is to create an acquired ADAMTS13-deficient animal model based on 129×1/Sv J mice through a polyclonal anti-ADAMTS13 antibody(ab71550), and to further explore an acquired ADAMTS13 deficiency alone whether can cause the development of TTP in mice or just produce hypercoagulable state in vivo like ADAMTS13 KO mice.Methods:1 Subjects and Groups: Seven 129×1/Sv J mice were divided into experimental group(three mice) and control group(four mice) at random, another mouse was injected with increased amount of antibody ab71550.2 Testing items: Neuropsychiatric symptom, microscopic examination of ragged red cells in mice peripheral blood smear, platelet count, hemoglobin, plasma ADAMTS13 activity, VWF multimers, and histopathology of heart, kidney and brain, et al.3 Acquisition and processing of specimens: Blood was obtained from mice inner canthus vein and was anticoagulated with sodium citrate. Plasmas obtained by centrifuge were kept it in-70℃ for using. Another blood sample was used for peripheral blood smear and testing of platelet counts and hemoglobin concentration. Finally, all animals were killed after anaesthesia and were taken anatomy immediately to get brain, heart, liver, kidney, and spleen. Then all of the organs were fixed with formalin for macroscopic inspection.4 Statistical analysis: Testing normality of statistics(platelet count, hemoglobin and plasma ADAMTS13 activity) was made with SPSS16.0, and its distribution was expressed by mean±standard( x ± s). Student t- test was used between experimental group and control group, and P < 0.05 was considered to be significant.Results:1 Experimental group and control group(Antibody drug dose was 600ug/kg)①Neuropsychiatric symptom: no neuropsychiatric symptoms happened in experimental group and control group mice.②Microscopic examination of ragged red cells in mouse peripheral blood smear: no schistocytes were found within observation time in two groups.③Platelet count: no statistical significance between experimental group and control group before injection and after 24 h, 48 h, 72 h and 96 h.(The experimental data fit a normal distribution, homogeneity of variance, P>0.05)④Hemoglobin: there was no difference in two groups before injection and after 24 h, 48 h, 72 h and 96 h. Linear graph displayed: the change of hemoglobin with time in experimental group was more steady than control group.(The experimental data fit a normal distribution, homogeneity of variance, P>0.05)⑤Plasma ADAMTS13 activity: no difference between experimental group and control group before injection, while there were statistical differences after 24 h, 48 h, 72 h and 96 h injection. Linear graph displayed: ADAMTS13 activity was almost steady in control group, while experimental group dropped and reached lowest below 10% at 48 h then rebounded.(The experimental data fit a normal distribution, homogeneity of variance, P>0.05).⑥VWF multimers: ULVWF multimers were found in experimental group, while not in control group.⑦Histopathology: No microvascular thrombosis were found in brain, heart, liver, kidney, and spleen.2 One mouse injected with more antibody ab71550.(1000ug/kg)Mouse died when we got blood sample after injection 48 h. During the whole experiment no neuropsychiatric symptom, schistocytes, microvascular thrombosis emerged, platelet count and hemoglobin were steady. But ADAMTS13 activity of this mice was below 10% and VWF Vertical electrophoresis showed the presence of ULVWF.Conclusions:ADAMTS13 inhibitor ab71550 could reduce its activity and form ULVWF in 129×1/Sv J mouse, but could not induce human TTP-like symptoms, such as thrombocytopenia, microangiopathic hemolytic anemia, neuropsychiatric symptoms, renal injury and fever. Therefore, it is inferred that acquired ADAMTS13 deficiency induced by antibody is not sufficient for the development of TTP in mouse, and a second hit is needed, such as additional genetic and/or environmental factors. |
