Murine In Vivo Studies Support Platelet Factor 4 as a Negative Autocrine of Megakaryopoiesis with Clinical and Therapeutic Implications.

Platelet Factor 4 (PF4) is a member of the CXC family of chemokines that appears to act as an autocrine regulator of megakaryopoiesis in vitro. To demonstrate that PF4 plays a physiological role in vivo, we examined megakaryopoiesis in animals that over- or under-expressed PF4. We generated PF4^−/− knockout animals as well as transgenic animals that overexpress human (h) PF4 to a level that is ~6-fold greater than control human platelet samples (hPF4⁺). Relative to wildtype (WT) littermate controls (n=26), platelet counts were elevated by ~15% in knockout animals (n=18, p=0.001) and reduced by ~33% in hPF4⁺ animals (n=13, p=0.0004). Platelet survival studies insured that the observed changes were not due to a consumptive process. The hypothesis that PF4 results in a decrease in megakaryocyte and/or platelet production was supported by in vitro evidence that growth of megakaryocyte-containing colonies was inversely related to the PF4 phenotype. The hPF4⁺ mice formed 52%±15% fewer megakaryocyte colonies compared to WT (n=8 each arm, p<0.00002). This inhibition was blocked by the inclusion of a rabbit polyclonal anti-hPF4 antibody. Based on these observations, we hypothesized that lysis of megakaryocytes induced by cytolytic chemotherapy with resultant release of PF4 may contribute to post-cytolytic-therapy thrombocytopenia. Exposing mice to intraperitoneal 5-fluorouracil (5-FU) decreased WT platelet counts below normal for 10.8±1.3 days (n=23). PF4^−/− mice had a significantly more rapid recovery of 8.2±1.4 days (n=14, p<0.0001), while hPF4⁺ mice had significantly delayed recovery of 15.3±1.7 days (n=15, p<0.000006). Importantly, the duration of thrombocytopenia in WT animals could by shortened to that seen in PF4^−/− mice by administration of 50 mg/kg of F(ab)₂ fragments of a polyclonal rabbit anti-murine PF4 antibody, but not by rabbit non-specific polyclonal IgG. Specific anti-PF4 therapy improved recovery of platelet count in WT mice to 8.8±1.5 days vs. 10.7±1.0 days in non-treated WT mice (n=10 and 8, respectively, p<0.008). Forty percent of the specific-treated animals recovered by day 7 post injection of 5-FU, while none of the control animals recovered before day 10. Similar interventions in hPF4⁺ animals resulted in a marked improvement in platelet count recovery compared to controls with the anti-hPF4 antibody-treated group recovering by 9.0±1 vs. 13.6±1.2 for the control mice (n=8 and 5, respectively, p<0.002). Our data suggest that efforts to block the effect of PF4 during cytotoxic therapy may limit the duration of thrombocytopenia. These observations have clinical implications, particularly for patients who express high endogenous levels of PF4. To that end, we studied 250 well adults and found that 10% of these individuals have 2–4.5 times the average platelet PF4 content. We propose that these individuals with high platelet PF4 levels may be particularly sensitive to developing thrombocytopenia after cytolytic therapy and may specifically benefit from therapy that blocks the effects of released PF4.