Models of thrombotic thrombocytopenic purpura and methods of use thereof

Abstract

The invention relates to the development of an animal model for testing various agents in the treatment of a clotting disorder. More specifically, the invention relates to the use of ultra-large molecular weight multimers of von Willebrand factor (VWF) in various mouse strains to induce thrombotic thrombocytopenic purpura (TTP)-like symptoms for the development of a mouse model of TTP. The invention also provides methods for generating such animal disease models and screening methods for identifying biologically active compounds which are effective in the treatment of TTP.

1 - 7 . (canceled) 
     
     
         8 . A method of testing an agent for its ability to reduce blood clotting in a mammal administered recombinant von Willebrand factor in an amount effective to cause mortality comprising comparing mortality rates in an animal model in the presence and absence of a test agent wherein decreased mortality in the presence of the test agent indicates that the test agent has the ability to reduce blood clotting. 
     
     
         9 . The method of  claim 8 , wherein the animal model is deficient in a disintegrin and metalloprotease with thrombospondin type 1 domains 13 (ADAMTS13) polypeptide. 
     
     
         10 . The method of  claim 9 , wherein the amount of recombinant von Willebrand factor is greater than 1000 RCoU/kg. 
     
     
         11 . The method of  claim 10 , wherein the amount of recombinant von Willebrand factor is greater than 2000 RCoU/kg. 
     
     
         12 . The method of  claim 11 , wherein the amount of recombinant von Willebrand factor is greater than 4000 RCoU/kg. 
     
     
         13 . A method of testing an agent for its ability to reduce blood clotting in a mammal administered recombinant von Willebrand factor in an amount effective to cause a pathology comprising comparing the pathology in an animal model in the presence and absence of a test agent wherein decreased incidence or severity of the pathology in the presence of the test agent indicates that the test agent has the ability to reduce blood clotting. 
     
     
         14 . The method of  claim 8  further comprising administering the test agent over a range of dosages. 
     
     
         15 . The method of  claim 8 , wherein the recombinant von Willebrand factor (VWF) polypeptide is human. 
     
     
         16 . The method of  claim 13 , wherein the animal model is deficient in a disintegrin and metalloprotease with thrombospondin type 1 domains 13 (ADAMTS13) polypeptide. 
     
     
         17 . The method of  claim 16 , wherein the amount of recombinant von Willebrand factor is greater than 250 RCoU/kg. 
     
     
         18 . The method of  claim 17 , wherein the amount of recombinant von Willebrand factor is greater than 500 RCoU/kg. 
     
     
         19 . The method of  claim 18 , wherein the amount of recombinant von Willebrand factor is greater than 1000 RCoU/kg. 
     
     
         20 . The method of  claim 19 , wherein the amount of recombinant von Willebrand factor is greater than 2000 RCoU/kg. 
     
     
         21 . The method of  claim 20 , wherein the amount of recombinant von Willebrand factor is greater than 4000 RCoU/kg. 
     
     
         22 . The method of  claim 13  wherein the animal model is deficient in a von Willebrand factor polypeptide. 
     
     
         23 . The method of  claim 22 , wherein the amount of recombinant von Willebrand factor is greater than 500 RCoU/kg. 
     
     
         24 . The method of  claim 23  wherein the amount of recombinant von Willebrand factor is greater than 1000 RCoU/kg. 
     
     
         25 . The method of  claim 24  wherein the amount of recombinant von Willebrand factor is greater than 2000 RCoU/kg. 
     
     
         26 . The method of  claim 25  wherein the amount of recombinant von Willebrand factor is greater than 4000 RCoU/kg. 
     
     
         27 . The method of  claim 13  wherein the animal model is a mouse of a C57BL/6J strain. 
     
     
         28 . The method of  claim 27 , wherein the amount of recombinant von Willebrand factor is greater than 250 RCoU/kg. 
     
     
         29 . The method of  claim 28  wherein the amount of recombinant von Willebrand factor is greater than 500 RCoU/kg. 
     
     
         30 . The method of  claim 29  wherein the amount of recombinant von Willebrand factor is greater than 1000 RCoU/kg. 
     
     
         31 . The method of  claim 30  wherein the amount of recombinant von Willebrand factor is greater than 2000 RCoU/kg. 
     
     
         32 . The method of  claim 31  wherein the amount of recombinant von Willebrand factor is greater than 4000 RCoU/kg. 
     
     
         33 . The method of  claim 8  wherein the animal model is a mouse. 
     
     
         34 . The method of  claim 33  wherein the mouse is of a C57BL/6J strain. 
     
     
         35 . The method of  claim 13  wherein the pathology is a clinical pathology. 
     
     
         36 . The method of  claim 35  wherein the clinical pathology is a change in lactate dehydrogenase level, creatinine kinase level, hematocrit, hemoglobulin concentration, erythrocyte count, reticulocyte count, total leukocyte count, differential leukocyte count, blood morphology abnormality, platelet count, mean cell volume, mean cell hemoglobulin concentration, or blood cell level in the urine. 
     
     
         37 . The method of  claim 13  wherein the pathology is a histological pathology. 
     
     
         38 . The method of  claim 37  wherein the histological pathology is microthrombi, myocardial necrosis, increased coronary perivasculitis, myocardial degeneration, myocardial infarction, myocardial reparation, glia cell foci, cortical necrosis, hemorrhage, increased incidence or mean severity of a microthrombi, a disseminated intravascular coagulopathy (DIC), thrombotic thrombocytopenic purpura (TTP), ischemic heart disease, a thromboembolic change, reactive coronary perivasculitis, inflammation, fibrosis, necrosis, hemosiderin deposition, calcification, renal infarction, or a reduction in body mass. 
     
     
         39 . The method of  claim 13  wherein the pathology is a behavioral pathology. 
     
     
         40 . The method of  claim 39  wherein the behavioral pathology is behavioral depression, a prone body position, a side body position, an abnormal body position, dyspnea, ataxia, immobility, convulsions, cramps, or piloerection. 
     
     
         41 . A method of inducing symptoms of Thrombotic Thrombocytopenic Purpura (TTP) in a mouse, said method comprising the step of administering to the mouse a composition comprising recombinant human von Willebrand factor (rVWF), wherein the rVWF composition forms high molecular weight multimers, and wherein the administration results in at least one symptom of TTP in the mouse, selected from the group consisting of: reduced platelet levels, anemia, histopathological effects, increased blood creatinine kinase levels, increased lactate dehydrogenase levels, and increased microthrombi. 
     
     
         42 . The method of  claim 41 , wherein the mouse has normal levels of endogenous von Willebrand factor (VWF). 
     
     
         43 . The method of  claim 41 , wherein the mouse has deficient levels of endogenous VWF. 
     
     
         44 . The method of  claim 41 , wherein the mouse has deficient levels of endogenous ADAMTS13. 
     
     
         45 . The method of  claim 41 , wherein the mouse has deficient levels of both endogenous VWF and endogenous ADAMTS13. 
     
     
         46 . The method of  claim 41 , further comprising administering to the mouse recombinant Factor VIII. 
     
     
         47 . The method of  claim 41 , wherein the rVWF is administered in a dose of at least 1000 RCoU/kg body weight. 
     
     
         48 . The method of  claim 41 , wherein the rVWF is administered in a dose of at least 2000 RCoU/kg body weight. 
     
     
         49 . The method of  claim 41 , wherein the rVWF is administered in a dose of at least 4000 RCoU/kg body weight. 
     
     
         50 . The method of  claim 41 , wherein the rVWF is administered intravenously. 
     
     
         51 . The method of  claim 41 , wherein the rVWF is administered more than once. 
     
     
         52 . The method of  claim 50 , wherein the rVWF is administered periodically. 
     
     
         53 . The method of  claim 50 , wherein the rVWF is administered at a dose of between 250 and 1000 RCoU/kg body weight. 
     
     
         54 - 63 . (canceled) 
     
     
         64 . A method for assessing the effect of a test composition on symptoms of Thrombotic Thrombocytopenic Purpura (TTP), the method comprising the steps of:
 (a) administering to a mouse a composition comprising recombinant von Willebrand Factor (rVWF), wherein the rVWF composition forms high molecular weight multimers, and wherein the administration results in formation of at least one symptom of TTP selected from the group consisting of: reduced platelet levels, anemia, histopathological effects, increased blood creatinine kinase levels, increased lactate dehydrogenase levels, and increased microthrombi;   (b) administering the test composition to the mouse; and   (c) determining the effects of the test composition on the symptoms of TTP.   
     
     
         65 . The method of  claim 64 , wherein the test composition and the rVWF composition are administered at the same time. 
     
     
         66 . The method of  claim 64 , wherein the test composition is administered before the rVWF composition. 
     
     
         67 . The method of  claim 64 , wherein the test composition is administered after the rVWF composition. 
     
     
         68 . The method of  claim 64 , wherein the test composition is administered more than once.