The mechanistic and therapeutic studies of heparin-induced thrombocytopenia (HIT) in this Program Project will rely heavily on standardized, high quality reagents, especially platelet factor 4 (PF4) and relevant antibodies (Abs). Core B will provide the following 3 support services: Service 1: Recombinant PF4 proteins. We will provide highly purified, endotoxin free, recombinant human (h) and mouse (m) wild type (WT) PF4. WT hPF4 will be needed in large quantities for ex vivo studies in Projects 1, 2 and 4. Project 1 will use hPF4 for studies of platelet clearance, thrombosis and endothelial activation in its Specific Aims (SA) 1, 2 and 3, respectively. Project 2 will make major use of WT hPF4 for studies its interaction with KKO and HIT IgGs as well as studies of the structure and binding properties of surface-bound PF4/GAG complexes in its first and second aims, respectively and cell activation in SA 3. In addition, the hPF4'^^?^ mutant and other specific mutants of hPF4 to be developed in SA 1 of Project 2 will also be prepared for the needs of other projects. Large amounts WT m and hPF4 and potentially specific variants will be needed for studying the biophysical properties of PF4/hepann (H) complexes essential for immunogenicity in SA 1 of Project 3 and also for studying the interaction of optimal PF4/H complexes with T cells in SA 2 and B cells in SA 3. Project 4 needs hPF4 and likely mPF4 to identify antagonists to disrupt tetramerization and HIT complex formation. Service 2: Preparation of monoclonal Abs (moAbs). This Core will produce large amounts of endotoxin free, HlT-like moAb KKO, the PF4-specific moAb RTO and an isoimmune moAb control TRA. Projects 1, 2 and 4 will make heavy usage of these Abs for their ex vivo and in vivo studies. Chimeric moAbs KTO designed by Project 2 will also be prepared in quantities sufficient for in vivo tests. F(ab)2 fragments and other modifications of these moAbs will also be prepared for the various Projects. Service 3: Preparation of polyclonal HIT Abs. In conjunction with Core C, polyclonal specific anti PF4/H Abs as well as control human IgG will be affinity purified in relatively large quantities, standardized, and made available for all Projects to validate clinical implications of studies performed with murine Abs and models of HIT. Individual sample preparations will be used by Projects 2 and 4 to validate the structural basis of pathogenic HIT Abs and the therapeutic efficacy of small molecule inhibitors of PF4 tetramerization, respectively. Large scale-batch products will be prepared for proposed studies involving the passive murine model of HIT proposed in Projects 1 and 4. Thus, this Core is expected to provide a ready supply recombinant PF4, PF4 variants, HlT-like and related monoclonal Abs (moAbs), and human HIT and control IgGs that should allow the four Projects to employ products of the highest quality and in sufficient quantities to foster rapid scientific progress and coordination in the pursuit of the main themes of this application: to better understand the molecular mechanisms underlying HIT and to use that knowledge to develop new therapeutic approaches for the treatment of HIT.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL110860-03
Application #
8695465
Study Section
Heart, Lung, and Blood Program Project Review Committee (HLBP)
Project Start
Project End
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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