Apart from decreasing the activity levels of factors II, VII, and X,17
the present study shows that intravenous infusion of NAC to healthy subjects also depresses plasma levels of other vitamin K dependent proteins participating in the coagulation cascade (factor IX activity, protein C activity, and free protein S reactivity). The maximal effect of NAC on vitamin K dependent proteins occurred within one hour in our study and after several hours in a previously reported study,17
probably because we used a threefold higher bolus dose of NAC. Data on the effect of NAC on vitamin K dependent proteins in healthy subjects can most likely be extended to include patients treated with NAC, as a comparable decrease in factor II+VII+X activity as observed in the present study after initiation of NAC infusion was seen in patients with paracetamol poisoning but without any sign of hepatocellular damage.18
In agreement with the just cited study,17
we found that NAC infusion did not affect APTT, which is in agreement with the notion that APTT is rather insensitive to changes in activities of coagulation factors, contributing to the outcome of the test.20
Depressed levels of vitamin K dependent proteins are most likely caused by modification of their structure/function by NAC. This is supported by the following evidence. (A) Concentrations of NAC affecting levels of the respective proteins in vivo and in vitro were comparable. The dose regimen of NAC used in the present study has been reported to give rise to a total plasma mean concentration of the drug of 3.4 mmol/l after the initial loading dose.21
Then, the mean concentration fell to a steady state level of 0.21 mmol/l after approximately 12 hours. After discontinuation of NAC it disappeared from plasma with a half life of approximately six hours. In vitro, we observed that NAC in the range 0.2–10 mmol/l induced a progressive decrease in levels of vitamin K dependent proteins. (B) In all instances the maximal fall in vitamin K dependent protein levels occurred within one hour after the initial loading dose of NAC, irrespective of their plasma half lives, which range from about six hours (factor VII) to approximately 70 hours (factor II).22–24
(C) Factor II and IX activities reached their baseline values by six hours after the start of the NAC infusion, despite the fact that their half lives are approximately 24 and 70 hours, respectively. As these two factors are among the least sensitive to the effect of NAC in vitro, the relatively rapid normalisation of activity levels could be explained by a decline in the concentration of NAC after the initial loading dose, leading to dissociation of NAC from the two proteins and restoration of their normal function. (D) Depression of factor X activity and free protein S reactivity was sustained several hours after cessation of the NAC infusion. This sustained effect is in agreement with the observation that the activities of these two proteins are the most sensitive to the effect of NAC in vitro and therefore are also affected by the drug at the low concentrations achieved after the initial loading dose. The sustained effect of NAC on factor X activity is most likely responsible for the sustained effect of the drug on factor II+VII+X activity.
The fall in free protein S reactivity in response to NAC could be due to a modifying effect of the drug, leading to a decrease in the affinity of one or both of the targets for the protein S detecting reagents in the assay. The targets are the sex hormone binding globulin-like domain and the N terminal Gla domain, which are recognised by C4 binding protein and monoclonal antibody coated latex particles, respectively.25
Vitamin K dependent proteins are homologous multidomain proteins that share a unique Gla domain of pivotal significance for their function,26
indicating that NAC affects the structure/function of these proteins by a common mechanism. One mechanism could be reduction of exposed sensitive intramolecular disulphide bonds required for maintenance of their structure/function. By using thiol reactive agents, evidence has been provided that NAC can reversibly reduce disulphide bonds in the insulin receptor, matrix proteins, and intracellular signal transducers at concentrations similar to those impairing the vitamin K dependent proteins in the present study.27–29
Another mechanism of impairment could be that the vitamin K dependent proteins analogous to other plasma proteins (primarily albumin) are nitrosylated,30
that this nitrosylation enhances their activity, and that NAC induces denitrosylation resulting in a decrease in activity. This mechanism seems less likely as preliminary experiments in vitro showed that the nitrosylating agent DETA NONOate at 1–10 mmol/l had no effect on factor II or X activity or on free protein S reactivity in plasma incubated for one or two hours at 37°C (unpublished).
VWf serves as a stabilising carrier of factor VIII and any change in the plasma level of vWf is associated with a concordant change in the level of factor VIII.31,32
In the present work, four of five subjects with moderate adverse reactions in response to NAC showed concordant and rapid marked increases in factor VIII activity and vWf antigen followed by progressive slow decreases in their levels. This is compatible with the immediate release of vWf from storage sites (Weibel-Palade bodies) in endothelial cells and the subsequent elimination of the factor VIII-vWf complex from plasma with the reported half life of approximately 14 hours.23,32
Histamine may be the responsible agonist for the release of vWf as NAC liberates histamine in cell cultures33,34
and histamine liberates vWf from its storage pools in vivo.35
The proposed mechanism agrees with the observation that a controlled insect sting challenge resulted in a rapid and marked parallel increase in plasma levels of histamine and vWf in subjects who developed anaphylactic reactions and hypotension.36
Interestingly, the one subject with systemic adverse reactions, but without an increase in levels of factor VIII activity and vWf antigen, was among the three subjects who received antihistamines. Both factor VIII and vWf are acute phase reactants.37
It is unlikely that the NAC induced increases in the levels of these two factors in the four subjects with systemic adverse reactions were mediated by an acute phase reaction, as the level of the sensitive acute phase reactant CRP was unaffected. It is also unlikely that a gain in function mechanism was involved, as NAC did not affect plasma levels of factor VIII activity and vWf antigen in subjects without adverse reactions or in vitro.
The frequency of systemic adverse reactions in response to intravenous NAC infusion in patients with paracetamol intoxication varies from 3% to 48%.38–40
This great variation in frequency could be caused by underestimation of adverse reactions in some studies as paracetamol can produce nausea and vomiting, making it difficult to distinguish these symptoms from those caused by NAC itself. Another explanation could be that adverse reactions may be underreported. In the present study, the frequency of systemic adverse reactions was 50%. The nature of these reactions and their early manifestation in relation to bolus infusion of NAC are in agreement with results obtained by others.38–40
In a similar study to ours in healthy subjects, no systemic reactions occurred.17
This may be explained by the three times lower bolus dose used in this study.
There is no general consensus on when to stop NAC administration in patients being treated for paracetamol intoxication but the activity of coagulation factor II+VII+X remains essential in the monitoring of these patients.15,41–46
It remains to be determined whether derangements of the haemostatic system observed here will be of future clinical relevance for decision making in the treatment of paracetamol intoxication. It is notable that NAC infusion has little effect on factor V activity and may increase factor VIII activity, as levels of both of these factors have been reported to be of prognostic value in fulminant hepatic failure.47,48
The bioavailability of NAC after oral administration is much lower (approximately 9%) than after intravenous administration.49
This means that the conclusions of the present study cannot be directly transferred to patients receiving NAC orally.
In conclusion, we have demonstrated that NAC infusion in doses used to treat paracetamol intoxication causes small to moderate derangements of the coagulation system, which affect both vitamin K dependent coagulant and anticoagulant proteins, some of which are used as early predictors of hepatic damage. We also found that adverse reactions can be associated with increases in the levels of factor VIII and its carrier protein vWf. Further studies are needed to elucidate the biochemical and pharmacokinetic bases for the observed changes in haemostatic parameters.