The role of platelet factor 4 in radiation-induced thrombocytopenia

Abstract

Purpose: Factors affecting the severity of radiation-induced thrombocytopenia (RIT) are not well described. We address whether platelet factor 4 (PF4; a negative paracrine for megakaryopoiesis) affects platelet recovery postradiation.

Methods and materials: Using conditioned media from irradiated bone marrow (BM) cells from transgenic mice overexpressing human (h) PF4 (hPF4+), megakaryocyte colony formation was assessed in the presence of this conditioned media and PF4 blocking agents. In a model of radiation-induced thrombocytopenia, irradiated mice with varying PF4 expression levels were treated with anti-hPF4 and/or thrombopoietin (TPO), and platelet count recovery and survival were examined.

Results: Conditioned media from irradiated BM from hPF4+ mice inhibited megakaryocyte colony formation, suggesting that PF4 is a negative paracrine released in RIT. Blocking with an anti-hPF4 antibody restored colony formation of BM grown in the presence of hPF4+ irradiated media, as did antibodies that block the megakaryocyte receptor for PF4, low-density lipoprotein receptor-related protein 1 (LRP1). Irradiated PF4 knockout mice had higher nadir platelet counts than irradiated hPF4+/knockout litter mates (651 vs. 328 × 106/mcL, p = 0.02) and recovered earlier (15 days vs. 22 days, respectively, p <0.02). When irradiated hPF4+ mice were treated with anti-hPF4 antibody and/or TPO, they showed less severe thrombocytopenia than untreated mice, with improved survival and time to platelet recovery, but no additive effect was seen.

Conclusions: Our studies show that in RIT, damaged megakaryocytes release PF4 locally, inhibiting platelet recovery. Blocking PF4 enhances recovery while released PF4 from megakaryocytes limits TPO efficacy, potentially because of increased release of PF4 stimulated by TPO. The clinical value of blocking this negative paracrine pathway post-RIT remains to be determined.

Conflict of interest statement

Conflict of Interest statement:

The authors have no conflicts of interest to declare.

Copyright © 2011 Elsevier Inc. All rights reserved.

Figures

Figure 1. In vitro effect of irradiated cell lysates on megakaryocyte colony formation

(A) and (B) Effect on megakaryocyte colony numbers of indicated conditioned media from irradiated BM on megakaryocyte colony formation on KO BM (A) and hPF4+/KO BM (B). WT = wildtype BM cells used as a control. N = 4 separate experiments performed in duplicate. Mean ± 1 standard error (SE) is shown. * = p <0.01 compared to no added conditioned media or KO media, ** = p <0.01 compared to WT BM. (C) and (D) Blocking the effects of irradiated hPF4+/KO conditioned media on megakaryocyte colony formation from KO BM (C) and hPF4+/KO BM (D). N = 3 separate experiments performed in duplicate. Mean ± 1 standard error (SE) is shown. (C) p < 0.05 by ANOVA. (D) * = p <0.01 compared to added conditioned media. In (C) and (D), a-LRP is anti-LRP1 antibody and a-PF4 is anti-hPF4 antibody.

Figure 2. Effect of endogenous PF4 levels on platelet count recovery after irradiation

(A) Platelet counts of irradiated mice after 660cGy irradiation delivered in a single dose. Grey boxes represent mPF4 knockout animals (KO) and black circles represent hPF4 over-expressing animals on the knockout background (hPF4+/KO). Shown is mean ± 1 SE. * = p < 0.05 when corrected for differences in baseline platelet count. (B) Survival of irradiated animals in each group. Survival of KO animals (broken line) was significantly better than hPF4+/KO animals (p<0.05) (solid line). n = 17 KO animals and 10 hPF4+/KO animals.

Figure 3. In vivo studies on the effect of anti-hPF4 antibodies in irradiated mice

(A) Recovery of animals treated with antihPF4 antibodies. hPF4+/KO were treated with either Anti-hPF4 F(ab’)2 fragments (grey circles) or control F(ab’)2 fragments (black circles) on day 0 and 4 after irradiation (250 mg/kg by tail vein injection) shown in comparison with KO animals (white squares). N = 8–12 animals per arm. Shown is mean ± 1 SE. p<0.01 for curves. (B) Survival of the three groups of animals. The grey line represents animals treated with anti-hPF4 antibody. These animals had the best survival although not statistically different from KO animals (solid black line). hPF4+/KO animals treated with control antibody had the worst survival (broken line) p<0.02. N = 8–12 animals per arm.

Figure 4. In vivo effect of TPO and anti-hPF4 antibodies in irradiated mice

(A) Platelet count (shown as % baseline count) recovery in hPF4+/KO mice treated with 660 cGy irradiation on day 0. Animals then received anti-hPF4 F(ab’)2 fragments (125 mg/kg iv on days 0 and 4 post irradiation, grey circle) or TPO (100 mcg/kg iv on days 0 and 4 post irradiation, black circle) or both (diamond) and were compared to control animals (open circle and dashed line). N = 4 animals per arm. Shown is mean ± 1 SE. Black arrows represent days of injections. (B) The same data as (A) shown as survival of hPF4+/KO mice after irradiation compared with treated animals. All of the animals that were treated with agents to improve platelet count survived (superimposed solid line) while none of the control animals survived (p<0.002) (broken line). (C) Same as (A) except for WT animals. Animals then received anti-mPF4 F(ab’)2 fragments (250 mg/kg iv on days 0 and 3 post irradiation, grey circle) or TPO (100 mcg/kg iv on days 0 and 3 post irradiation, black circle) or both (diamond) compared to control animals (open square and dashed line). N = 5 animals per arm. Mean ± 1 SE are shown. (D) Same data as (C) but shown as survival of WT animals after irradiation compared with treated animals. All of the animals which were treated with agents to improve platelet count survived (solid line). Two of the control animals survived (p=0.01) (broken line).