Abstract
Background: In idiopathic thrombocytopenic purpura (ITP), autoantibodies bind to platelets which are then phagocytosed by monocytes/macrophages and removed by the reticuloendothelial system. PRTX-100 (Staphylococcal protein A) is being investigated for the treatment of ITP.
Objective: To assess the effect of PRTX-100 on phagocytosis of platelets in an in vitro assay.
Methods: Human monocytes were isolated from whole blood peripheral blood mononuclear cells (PBMCs) by adherence and cultured for 6 days in RPMI + 5% human serum. 48 hours prior to phagocytosis assay, PRTX-100 was added at 250, 25, and 2.5ng/ml. Human platelets were labeled with a fluorescent (PerCP) lipophilic dye and opsonized with an antibody to MHC Class I (W632). 2×10−5 monocytes were co-cultured with 2×10−7 labeled platelets for 1 hour at 37 ° C. All conditions were performed in triplicate. After an hour, phycoerythrin (PE) labeled anti-CD61 antibody was added to assess surface bound platelets versus ingested platelets. Phagocytosis was determined by flow cytometric analysis. The monocyte population was gated upon by forward and side scatter properties, then verified by staining with CD14-FITC. Percent phagocytosis was calculated as the fraction of ingested platelets (PerCP +/CD61−) to the total PerCP population (PerCP +/CD61−) + ( PerCP+/CD61+) within the gated monocyte population.
Results: PRTX-100 inhibits the phagocytosis of W632 opsonized platelets by human monocytes. Phagocytosis of W632 opsonized platelets was 40%, while phagocytosis in the presence of PRTX-100 at concentrations of 250, 25, and 2.5ng/ml was 18.3%, 23%, and 24.3%, respectively. Phagocytosis at 250ng/ml and 25ng/ml was significantly different from control phagocytosis with p values of 0.014 and 0.001 respectively by Student’s t test.
Conclusions: PRTX-100 inhibits the phagocytosis of platelets by monocytes, the effector limb of ITP. Prevention of platelet phagocytosis is an important treatment goal in ITP. PRTX-100 has been shown to be generally safe and well-tolerated in a phase I study in healthy volunteers (J Clin Pharmacol, in press). PRTX -100 is a promising therapeutic option for ITP and deserves further study.
Effect of PRTX-100 on In Vitro Phagocytosis of Opsonized Human Platelets
Disclosures: The authors are full-time employees of Protalex, Inc.; The authors hold stock and/or stock options in Protalex, Inc.; The research presented was funded entirely by Protalex, Inc.
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