2-O, 3-O-Desulfated Heparin (ODSH) Mitigates Chemotherapy-Induced Thrombocytopenia (CIT) by Blocking the Negative Paracrine Effect of Platelet Factor 4 (PF4) On Megakaryopoiesis

Abstract 386

Thrombocytopenia is a significant complication of myelosuppressive chemotherapy treatments, which are a mainstay in cancer therapy. We and others have previously shown that PF4 is a negative paracrine affecting megakaryocyte number in culture. In murine studies we showed that platelet PF4 levels are inversely related to steady-state platelet counts and is a major contributor to the duration and severity of CIT. Recently, we have shown that PF4 influences both steady-state platelet count and platelet count recovery in pediatric patients treated for acute lymphoblastic leukemia. Pre-clinical studies in murine models suggest that blocking the effect of PF4 (using polyclonal anti-PF4 antibodies) can mitigate the effect of PF4 on megakaryopoiesis and results in a shortened time to platelet count recovery and higher nadir platelet count. Heparin is known to bind PF4 tightly and can clear PF4 from the vascular endothelium. Pre-clinical studies using heparin to mitigate CIT failed to show an effect, but were limited by dose considerations due to the anti-coagulant effect of unfractionated heparin (UFH) and likely by the inability of this highly negative polysaccharide to reach the intramedullary space in high enough amounts to alter available PF4. ODSH does not exhibit the same effect as unfractionated heparin (UFH) in enhancing antithrombin effects and may allow studies of non-anticoagulant pharmacologic effects of heparin. For example, ODSH retains UFH's anti-inflammatory effects and its ability to bind tightly to PF4. In preliminary results of a clinical trial using ODSH in conjunction with myelosuppressive chemotherapy in patients with metastatic pancreatic cancer, there was a striking lack of clinically significant CIT as will be reported separately at this meeting. This prompted our evaluation of whether ODSH affects CIT and modulates platelet count recovery and whether this is through its interaction with PF4. We first examined the ability of ODSH to prevent the PF4 effect on megakaryopoiesis in vitro. Using a megakaryocyte colony assay, we show that PF4 treatment of PF4^null murine megakaryocytes decreases megakaryocyte colony numbers (54±3% control vs. 23±6% PF4-treated, p<0.001) and treatment with ODSH completely blocks the PF4 effect (55±9% ODSH/PF4-treated, p<0.001 vs. PF4-treated). This suggested that the major mechanism by which ODSH prevents CIT is through inhibition of the PF4 effect. We then examined the effect of ODSH on liquid murine bone marrow culture and showed that ODSH treatment (50 μg/mL) was able to improve cell counts in the presence of added recombinant human PF4 (50 μg/mL) (9.2±3.3 × 10⁴ cells/mL in PF4-treated cells vs. 19.3±4.2 × 10⁴ cells/mL in ODSH+PF4-treated cells, p<0.01). Finally, we examined the in vivo effect of ODSH on CIT in a murine model of chemotherapy in transgenic mice that overexpress human (h) PF4. In these hPF4 mice, endogenous PF4 levels significantly affect enhances the degree and duration of 5FU-induced CIT. We previously reported that treatment of animals with anti-hPF4 antibodies was able to completely abolish the PF4 effect. Using the same model, injecting 180 mg/kg 5-fluourouracil (5-FU) intraperitoneally on day 0, we examined the effect on platelet counts of treatment with 2 clinically relevant doses of ODSH (25 mg/kg/dose) given subcutaneously 30 minutes and 24 hours after injection of 5-FU. hPF4 mice treated with 5-FU and ODSH had a higher platelet count nadir (70±14% versus vs. 44±1% of baseline). The nadir platelet count in the ODSH-treated mice was similar to that in mPF4^null mice (57±20% of baseline). In addition, animals treated with ODSH recovered approximately 2 days earlier. In summary, ODSH mitigates CIT resulting in decreased severity and duration of thrombocytopenia. These studies suggest that this effect is mediated in large part by PF4 as in vitro experiments show that ODSH completely blocks the effect of PF4 on megakaryopoiesis. The in vivo studies support that sufficient ODSH reaches the marrow to block intramedullary-released PF4 and prevents its inhibition of megakaryopoiesis. This drug is already in clinical trials in humans and may be the first clinically relevant inhibitor of CIT. Further studies will examine its effect in other thrombocytopenic settings.