Data presented here confirm previous findings that although all plasma volume expanders tested shortened initial coagulation time, clot functional characteristics varied greatly. As expected, increased haemodilution progressively compromised clot mechanical characteristics, reflecting at least in part, an impact of reduced fibrinogen and platelet concentrations. However, at a given level of haemodilution, hetastarches, particularly high MW hetastarches, produced the greatest impairment compared with albumin and crystalloid solutions. Additionally, blood diluted with hetastarch solutions appeared less responsive to rFVIIa, especially with high MW hetastarches.
To enable us to examine a range of volume expanders and conditions, we focused on a single method, TEG, to assess blood clotting in vitro. Additionally, we performed an in vivo experiment to further investigate a single key finding. We did not specifically address thrombin generation, fibrin assembly, or platelet function. These limitations must temper our interpretation. Because experimental modification of normal blood or animals cannot fully reflect the pathophysiological mechanisms present in patients, caution should be exercised in extrapolating the present findings to the patient setting. These limitations notwithstanding, we believe that we can draw some conclusions regarding potential mechanisms.
Hetastarches exert a number of effects on the haemostatic systems that are distinct from a simple dilutional effect, including reductions in FVIII and vWF levels,6,8,17
impaired fibrin polymerization and cross-linking,18–20
and reduced platelet function.5
These have been related both to MW and to the molar substitution ratio of the hetastarch, with effects generally more pronounced with higher MW and higher molar substitution ratios.5–8
As the lower MW hetastarches used in the present study also had the lowest molar substitutions [HES 130 (130/0.4), HES 200 (200/0.5), HES600 (600/0.7), and HES670 (670/0.75)], it is not possible to differentiate between the potential effects of MW or molar substitution ratio.
In a recent study that examined plasma clotting kinetics after infusion of 20 ml kg−1
high MW hetastarch solution in rabbits (28% estimated blood volume), alpha angle and shear elastic modulus (a measure of clot strength) were reduced by 29 and 73%, respectively.20
These parameters were partially restored by in vitro
addition of either factor XIII or fibrinogen. Addition of thrombin shortened the R
time but had no effect on clot development or strength. It was concluded that hetastarch decreased clot propagation and strength primarily because of decreased FXIIIa-fibrin polymer cross-linking. These results suggest that the decrement in clot development and strength cannot be overcome by the actions of thrombin alone, although rate of clot initiation can be enhanced. Recombinant FVIIa functions by enhancing thrombin production at the site of injury.9
The fact that R
was shortened by rFVIIa at all levels of haemodilution in the present study suggests that thrombin production was enhanced by the addition of rFVIIa. Therefore, it does not appear that hetastarch impaired the ability of rFVIIa to enhance thrombin generation. Hetastarch-induced fibrin structural defects may be resistant to correction by thrombin, which may explain why rFVIIa did not consistently improve alpha angle or MA when hetastarch was used in the present study.
Reducing the pH from 7.4 to 7.0 reduces rFVIIa enzymatic activity 60–90%.16
Therefore, the reduced clinical efficacy of rFVIIa observed in acidosis,12,21–23
may be related to lower enzymatic rates. However, it is not clear at what point reduced enzymatic rate might impact clinical efficacy because rFVIIa administration increases circulating FVIIa levels approximately 100-fold. Our findings suggest that rFVIIa may offset pH-related changes in enzymatic rates. This may, in part, explain clinical reports documenting some individual patients who did and others who did not respond to rFVIIa at a pH <7.2.12,21,22
Further studies are required to determine the relative roles of blood pH and other acidosis-related factors in determining the efficacy of rFVIIa in acidotic patients.
Our inability to detect a statistical impairment of TEG parameters under hypothermic conditions contrasts with previous findings.24,25
Hypothermia reduces coagulation enzymatic rates16
and platetet enzymatic and secretory rates.26
In a recent report, rFVIIa improved TEG parameters at temperatures of 28–31°C.25
Our data provide further evidence for this effect. The ability of rFVIIa to enhance haemostatic parameters in hypothermia is consistent with its retention of enzymatic activity between 37 and 32°C in vitro16
and with clinical reports of efficacy in hypothermic patients.13,21
When the conditions of hypothermia, acidosis, and 20% haemodilution were combined, R was consistently prolonged above normal baseline. This was similar to the pattern observed for undiluted hypothermic blood, but quite different from the effects of haemodilution alone. Alpha angle and MA were reduced below the baseline, a pattern that was also more similar to the responses to hypothermia and acidosis alone than to the responses to 20% haemodilution. These findings suggest that hypothermia and acidosis had the greater impact on TEG parameters than did the type of fluid.
A different pattern was noted when examining the responsiveness to 25 nM rFVIIa. The R was shortened by addition of rFVIIa, regardless of the plasma volume expander used, consistent with the results for acidosis, hypothermia, or haemodilution alone. However, the responses for the alpha angle and MA under combined conditions diverged from the rFVIIa responses for acidosis or hypothermia alone, and more closely followed the patterns observed in the isolated haemodilution experiments. When NaCl or albumin was used in the presence of combined acidosis and hypothermia, the alpha angle was normalized by rFVIIa. However, when hetastarches were used in the presence of acidosis and hypothermia, the alpha angle was improved in only one case (HES670) and the post-rFVIIa values remained below the normal baseline for all hetastarches. These data suggest that the response to rFVIIa was more dependent on the type of plasma expander used than on the effects of hypothermia and acidosis.
The potential differential effect of low vs
high MW hetastarch on the activity of rFVIIa was further addressed in vivo
. The consistent extension of BT after haemodilution with hetastarch solutions in normal rabbits in the present study is consistent with results reported for patients undergoing acute normovolemic haemodilution in preparation for surgery.27
Administration of rFVIIa reduced BT in rabbits that were haemodiluted with low MW hetastarch (HES200) but not in rabbits administered with high MW hetastarch (HES600). In vitro
, we observed that the alpha angle was improved in response to rFVIIa at 40% haemodilution with lower MW hetastarches, HES130, and HES200, but not with higher MW hetastarches, HES600 or HES670. Martinowitz et al
reported improved haemostasis in response to rFVIIa after severe liver injury in pigs that were made coagulopathic by induction of hypothermia and haemodilution with a 200 000 MW hetastarch. However, in a study using a similar animal model, but with a 600 000 MW hetastarch, rFVIIa did not alter blood loss.14
Infusion of high MW hetastarch yields reductions in vWF and FVIII:c well beyond those expected by simple haemodilution.6,8,17
Considering the well-established efficacy of rFVIIa in haemophilia, it does not seem likely that rFVIIa efficacy would be limited by a hetastarch effect on FVIII:c levels. As mentioned earlier, fibrin polymerization is negatively impacted by hetastarches,18–20
and this could have affected the efficacy of rFVIIa. High MW has also been related to a greater impairment of platelet function.5
It appears that hetastarch molecules coat platelets non-specifically, altering the interaction of GPIIb/IIIa receptors with fibrinogen. The present in vitro
finding that the alpha angle, a parameter related to platelet function, was more responsive to rFVIIa when low MW than high MW hetastarches were used, is consistent with such a platelet effect. BT procedures are believed to be more dependent on primary haemostasis than secondary haemostasis, suggesting that the effects observed in the present, in vivo
study might have been related to platelet function.