The results of these studies indicate that radiation exposure has varying effects on both arms of coagulation, increasing the blood clotting times in irradiated ferrets. The time interval between 3–7 h post-radiation exposure appears to be a crucial period for observing an increase in PT values in ferrets, suggesting that radiation exposure may have a temporal effect on the extrinsic cascade. Unlike the extrinsic pathway, the intrinsic pathway (measured by aPTT values) is solely affected by LDR exposure at 3–7 h post-irradiation. These results indicate that there appears to be a time frame after radiation exposure in which the clotting effects involving the extrinsic pathway can be measured. Additionally, a dose rate effect is observed, depending on the pathway studied, which can result in coagulopathies in the irradiated animals.
The doses used in these experiments ranged from 25–200 cGy. These doses are within the expected range of previously estimated SPE radiation doses to BFO. Radiation effects on PT values have been reported for radiation doses of 300 up to 3000 cGy (Weisbach et al. 2007
, Rithidech et al. 2009
). There are no data published using low doses (< 100 cGy) for such studies. The estimated dose from the 1972 SPE to BFO inside a spacecraft is (approximately) 46 cGy, nearly twice the lowest dose used in these experiments; during an EVA, the dose to BFO is expected to be approximately 138 cGy (Hu et al. 2009
The INR data presented here suggest that SPE radiation exposure could pose a serious risk to astronauts. Recent publications report bleeding risks associated with trauma victims with an INR > 1.5 or 1.8 (Thomas and DeLoughery 2004
, Yuan et al. 2007
). As the INR levels reported here resulting from ferret exposure to SPE-radiation exceed the current limits in place for trauma patients in the clinic, our results suggest that trauma or injuries could pose a threat to astronauts. Complications of coagulopathies include, but are not limited to, bleeding into joints, muscle, and other tissues. Limited medical care while on an EVA increases the risk of hypocoagulability and in the situation of massive trauma, potential exsanguination. Some 70% of ferrets exposed to a 100 cGy (LDR) dose had an INR that would be deemed a risk for trauma victims (>1.5), indicating an INR in the clinically significant range. Not all the ferrets exhibited coagulopathies, presumably because ferrets represent an outbred model system, and are thus not expected to have a uniform response.
The acute aPTT measurements in this study were solely affected by the LDR exposures (). Increased aPTT values were observed 48 h post-irradiation when compared to pre-irradiation controls for both HDR and LDR exposures, suggesting that there could be long-term radiation-induced changes in hemostasis. While HDR exposures resulted in a delayed aPTT response, their effects were minor (representing approximately a 10%-fold increase) when compared to the changes observed for LDR exposures. Limited sample size for this endpoint could have contributed to the borderline statistical significance at the 100 cGy (LDR) 48 h time-point. One out of the five ferrets analyzed at 48 h post-irradiation did not have increased aPTT values. It is noteworthy that this one ferret was not affected by LDR exposure at the 3 h time-point either; the inclusion of this animal resulted in a higher p-value (p = 0.08). This variability in the ferret response is not surprising due to the outbred nature of the ferrets.
Differences between the dose rates are evident from the clotting studies, and become even more noteworthy when the factor deficiencies were evaluated in the mixing studies. A summary of all the radiation affected factors, with corresponding pathways, are reported in . When evaluating the integrity of the extrinsic pathway, the HDR exposure causes a Factor VII deficiency (), while LDR exposure affected Factors II, V, and VII (). It is difficult to separate which factor deficiency (II, V, or VII) specifically causes the observed clinically significant INR, as the combination of all three contribute to the hypocoagulable state. Factor VII deficiency may be critical to the temporal effect of radiation on PT values which was observed in the 200 cGy doses when plasma was isolated at 3 h or 7 h post-irradiation. Factor VII has a half-life of 3–6 h (Pehlivanov et al. 2004
), which may contribute to the increase in PT values observed at 3 h post-irradiation, but not at 7 h post-irradiation.
A summary of radiation-induced factor deficiencies. Factors are classified by assay (pathway) they are found in, and whether the pathway is PT (extrinsic) and/or aPTT (intrinsic).
The mixing studies performed to evaluate the integrity of the intrinsic pathway suggested that there were coagulation factor deficiencies in Factor VIII, IX, X, XI, and XII, as shown by the increased aPTT values in plasma samples isolated from the 100 cGy dose LDR group (). Factor VIII, XI, and XII are all part of the intrinsic cascade. While Factor IX is referenced as part of the common pathway upon activation from the complex of TF and Factor VII, the predominant form of activation is through the intrinsic cascade. Therefore, in mixing studies, Factor IX signaling is associated with the aPTT cascade. Factor X is a member of the common pathway, where the intrinsic and extrinsic pathways converge. We report that Factor X deficiency is observed in plasma samples obtained from ferrets exposed to LDR radiation in which the PT values were increased, but Factor X is not observed to be deficient when irradiated ferret plasma samples were evaluated for alterations in PT values. This discrepancy may be attributed to mutations caused in Factor X that could exclusively impact aPTT and not PT, which has been reported previously (Denson 1969
, Denson et al. 1970
, Mannucci et al. 2004
Plasma isolated from the animals exposed to the 200 cGy dose (LDR) were Factor IX deficient (). The half-life of Factor IX is 18–24 h (White et al. 1997
); therefore, deficiencies in Factor IX could lead to an increase in aPTT values beyond 3 h after SPE radiation. This question will be assessed in future experiments.
In conclusion, SPE-like proton radiation causes a disruption in secondary hemostasis, as is evidenced by increases in PT and aPTT values measured post-irradiation and compared to pre-irradiation values. The intrinsic pathway was solely affected by LDR exposure at 3–7 h post-irradiation, as evidenced by the radiation-induced increase in aPTT values. Mixing studies were used to assess the integrity of the coagulation factors involved in each of the pathways. The LDR exposed ferrets resulted in plasma samples which were deficient in a number of factors affecting all pathways (). The only observation from the plasma isolated from ferrets exposed to HDR radiation was Factor VII deficiency, affecting the extrinsic pathway. At the highest dose tested, 200 cGy at the LDR, only Factor IX deficiency was observed to cause an increase in aPTT values. Future studies will be focused on the determination of the concentration of various affected factors, long-term effects of radiation exposure on Factor IX deficiency, and estimating the risk of coagulopathy.