Our experiments demonstrate that NAC has many effects that could be of benefit in TTP and other thrombotic disorders involving hyperreactive forms of vWF. These effects included reduction of vWF multimer size, removal of ULVWF strings from the endothelial surface, and marked shortening of the time to reestablish normal blood flow in the microvasculature of ADAMTS13-deficient mice challenged to secrete vWF. Even at concentrations of NAC with no discernible effect on vWF multimer composition, NAC reduced the disulfide bond encompassing the vWF A1 domain, which is important for vWF’s ability to bind platelet GPIb (15
). The effect of NAC on vWF’s hemostatic potency was demonstrated by its inhibition of platelet agglutination to ristocetin and prolongation of the aperture closure time measured by PFA.
The effects of NAC on vWF in mice were obtained at weight-adjusted doses similar to those used to treat acetaminophen overdose in humans (16
). For example, the Danish protocol for treatment of acetaminophen poisoning recommends a total dose of 400 mg/kg delivered as a 150 mg/kg bolus followed by continuous infusion over 36 hours. This dose is identical to the lowest effective dose that was administered to mice as a single bolus in our studies (17
NAC has other hemostatic effects that could be of benefit in treating thrombosis, including a concentration-dependent inhibition of in vitro platelet aggregation in response to both ADP and collagen. Several studies of the effect of NAC on platelet aggregation have been previously reported (18
). For example, NAC prevents NO scavenging by reactive oxygen species in platelets. Some NAC effects may be thiol dependent, as the actions of both ADP and collagen involve thiols. The ADP receptor P2
contains a free thiol that is the target of the platelet function inhibitor clopidogrel (20
). Furthermore, optimal collagen-induced platelet aggregation requires the presence of vWF (22
In reducing vWF disulfide bonds, NAC could have both direct and indirect effects. NAC contains a free thiol, which may reduce the disulfide bonds of vWF directly, as in mucin polymers (14
). This is the likely mechanism in our experiments when NAC was added directly to blood or plasma, as the action was rapid and the only cells available to metabolize NAC to other substances were blood cells. In vivo, NAC may also act indirectly after conversion to l
-cysteine or by acting as a substrate for augmented synthesis of reduced glutathione (GSH) (23
), another bioactive free thiol–containing molecule. It is through this latter mechanism that NAC is effective in treating acetaminophen overdose. Overdose of acetaminophen leads to accumulation of a toxic metabolite, N-acetyl-p-benzo-quinone imine (NAPQI), which is normally only produced in minute amounts (24
). NAPQI is detoxified by conjugation with GSH (26
), which becomes rapidly depleted in the presence of high acetaminophen concentrations. Unconjugated NAPQI is toxic to the liver (27
). NAC works in this case by generating more reduced GSH (28
). Thus, in addition to acting directly to reduce ULVWF multimers to smaller and less reactive forms, NAC would also serve to generate reducing equivalents to allow reduction of the vWF in the plasma by other reducing agents, perhaps GSH itself. The existence of such mechanisms to inactivate ULVWF by means other than proteolysis is suggested by the fact that patients with severe congenital ADAMTS13
deficiency sometimes do not experience acute TTP until the third decade of life (29
). Such a reductase would require the continuous provision of reducing equivalents, an action that would be facilitated by NAC. Recent evidence indicates that oxidation of free thiols facilitates vWF self association on surfaces (30
); provision of reducing equivalents would tend to prevent this phenomenon.
Our data indicate that NAC may prove useful for treating TTP, as a temporary agent or as supplementary therapy in patients refractory to plasma infusion or exchange. The documented safety profile of NAC makes it a particularly attractive drug for this purpose. NAC has been used in humans for over half a century with few reported adverse effects. Doses as high as 500 mg/kg have been employed to treat acute acetaminophen toxicity (16
). NAC has also been evaluated extensively in laboratory animals and found to have an LD50
of more than 10 g/kg in both rats and mice when given orally (32
), and doses of 1 g/kg have been given daily for up to 2 years in these animals without apparent detrimental effects (32
). Caution is warranted, however, because of the recent demonstration that prolonged high-dose NAC therapy or SNO-NAC (the nitric oxide adduct) (10 mg/ml) in the drinking water of mice resulted in pulmonary hypertension (33
Three studies have addressed the potential adverse hemostatic effects of NAC. Jepsen et al. (34
) found that NAC administered intravenously rapidly prolonged the prothrombin time and depressed the activities of clotting factors II, VII, and X. This same group later examined the effect on other hemostatic parameters and concluded that the acute effect was on the vitamin K–dependent factors, with no change noted in vWF antigen levels (17
). In a recent report, patients treated with NAC during cardiovascular surgery had increased blood loss compared with untreated patients (35
). However, no deaths were reported in the NAC-treated group as compared with 7 deaths in the untreated group (P
= 0.007), suggesting another beneficial effect of NAC. Our own studies of the effect of NAC administration on the tail-bleeding times in mice surprisingly showed no difference between NAC-treated mice and control mice at either 30 minutes or 8 hours after administration. This result should be interpreted cautiously, particularly given the many antihemostatic effects of NAC we demonstrated using other tests.
This safety profile makes NAC a potentially useful first-line therapy for TTP when the diagnosis is in doubt or when plasmapheresis is unavailable. For a physician faced with a patient with potential TTP, the diagnosis is rarely clear cut. None of the cardinal signs of TTP are specific for the disease, so they are often ascribed to other syndromes, particularly in the early stages of the clinical course or in patients with concomitant illnesses. Tests to rapidly detect ADAMTS13
deficiency are not at present routinely available in hospitals, and TTP patients may display near-normal ADAMTS13
). The risk of missing the diagnosis of TTP could have disastrous consequences for the patient; yet receiving the presumptive diagnosis of TTP commits that patient to a long and potentially dangerous course of therapy with plasmapheresis. NAC might thus be used to treat TTP while awaiting a definitive diagnosis or availability of plasmapheresis.
Another attractive feature of NAC is its low cost and wide availability. The drug could thus be employed as a temporizing therapy for TTP where plasmapheresis is not readily available. It might also be used in long-term therapy for TTP, especially in those patients with the acquired form of the disorder whose course is self limited when given the appropriate therapy. Clinical trials on healthy individuals and TTP patients will be the next step for determining NAC’s utility as an adjunct or first-line therapy of TTP.
If NAC proves useful in TTP, its use could be extended to other syndromes in which hyperactive vWF might also have a role, such as sickle cell anemia (6
), myocardial infarction (38
), cerebral malaria (39
), and stroke (41
). We therefore expect that the result of this study may stimulate investigation of NAC as a therapy for several other disorders in which excessive vWF-mediated cell adhesion plays a causative role.