Autoimmune thrombocytopenia is often due to antibodies reactive with platelet glycoprotein (GP) complexes, particularly GPIIb/IIIa. Autoepitopes are often divalent cation-dependent. The investigators will focus on two research areas: autoantibody characterization and antigenic localization. Autoantibody characterization. They will: 1) Demonstrate the presence of platelet-associated and plasma antibodies and focus on antibodies against GPIIb/IIIa, since these are most common; 2) Evaluate antibody clonality by examining light chain and IgG subtype characteristics and 3) Study autoantibody binding to megakaryocytes in vitro and determine if bound antibody effects megakaryocyte maturation, activates complement or induces phagocytosis. Antigen localization. They will: 1) Determine if rabbit antibody to the GPIIb/IIIa calcium-bindings sites will inhibit autoantibody binding; 2) Test autoantibody binding to a series of large recombinant peptides, spanning glycoproteins Iib and IIIa. They will insert specific PCR-generated cDNA fragments into a vector which results in their expression by E. Coli as fusion proteins with a 6X-histidine tag which can then be affinity-purified using a nickel nitrilo-tricacetic acid resin column. Antibodies which bind to one of the large peptides will be studied further by determining binding patterns to smaller peptides within this region; 3) Evaluate epitopes, not identified by this technique, with random peptide libraries and GPIIb/IIIa chimeras. Antibodies are incubated with fUSE5 filamentous phage expressing several million random peptide sequences. Phage expressing sequences which bind to antibodies are separated from non-specific phage by serial 'planning' with staphylococcal-A protein agarose followed by acid elution. The sequence of the specific peptide(s) is determined. In addition, they will develop human/xenopus and human/avian GPIIb/IIIa chimeras which they will use to examine the binding patterns of human antiplatelet autoantibodies. Results of these studies will be correlated with the patients' clinical course and thrombopoietin levels. In addition, the effect of immune thrombocytopenia on the patients' quality of life will be studied.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL061809-05
Application #
6527511
Study Section
Special Emphasis Panel (ZHL1-CSR-K (S1))
Program Officer
Ganguly, Pankaj
Project Start
1998-09-30
Project End
2003-08-31
Budget Start
2002-09-01
Budget End
2003-08-31
Support Year
5
Fiscal Year
2002
Total Cost
$370,400
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
McMillan, Robert; Durette, Carol (2004) Long-term outcomes in adults with chronic ITP after splenectomy failure. Blood 104:956-60
McMillan, Robert; Wang, Lei; Tomer, Aaron et al. (2004) Suppression of in vitro megakaryocyte production by antiplatelet autoantibodies from adult patients with chronic ITP. Blood 103:1364-9
McMillan, Robert (2003) Antiplatelet antibodies in chronic adult immune thrombocytopenic purpura: assays and epitopes. J Pediatr Hematol Oncol 25 Suppl 1:S57-61
McMillan, R; Wang, L; Tani, P (2003) Prospective evaluation of the immunobead assay for the diagnosis of adult chronic immune thrombocytopenic purpura (ITP). J Thromb Haemost 1:485-91
McMillan, R (2002) Classical management of refractory adult immune (idiopathic) thrombocytopenic purpura. Blood Rev 16:51-5
McMillan, Robert; Wang, Lei; Lopez-Dee, Jennifer et al. (2002) Many alphaIIbbeta3 autoepitopes in chronic immune thrombocytopenic purpura are localized to alphaIIb between amino acids L1 and Q459. Br J Haematol 118:1132-6
Pockros, Paul J; Duchini, Andrea; McMillan, Robert et al. (2002) Immune thrombocytopenic purpura in patients with chronic hepatitis C virus infection. Am J Gastroenterol 97:2040-5
McMillan, R; Lopez-Dee, J; Loftus, J C (2001) Autoantibodies to alpha(IIb)beta(3) in patients with chronic immune thrombocytopenic purpura bind primarily to epitopes on alpha(IIb). Blood 97:2171-2
McMillan, R; Lopez-Dee, J; Bowditch, R (2001) Clonal restriction of platelet-associated anti-GPIIb/IIIa autoantibodies in patients with chronic ITP. Thromb Haemost 85:821-3
McMillan, R (2000) Autoantibodies and autoantigens in chronic immune thrombocytopenic purpura. Semin Hematol 37:239-48