N-Acetylcysteine Treatment in Two Patients with Relapsed Thrombotic Thrombocytopenic Purpura Increased ADAMTS13 Activity, Free Thiol Concentration in Plasma, and Inhibited Platelet Activation

Introduction:  Thrombotic thrombocytopenic purpura (TTP) is a catastrophic and potentially fatal disorder caused by systemic microvascular thrombosis due to von Willebrand factor (VWF)–platelet thrombi.  TTP is caused by congenital or acquired deficiency of the plasma metalloprotease ADAMTS13. Based on an earlier study (Chen J et al., J Clin Invest 2011, 121:593-603), we proposed N-acetylcysteine (NAC) as an adjunct treatment for TTP. This study showed that NAC reduced the size and activity of VWF in vitro in human plasma and in vivo in a TTP mouse model.  In 2013 and 2014, two case reports described treatment of refractory TTP patients with NAC, one receiving a low dose of NAC [300 mg/kg (total 15 g) for the 1st 24 hrs, followed by 2.5 g/day for two weeks concurrently with plasma exchange] (Shortt J et al., N Engl J Med 2013, 368: 90-92; Shortt J et al., Transfusion 2014, 54:2362-2363) and the other receiving high-dose NAC [300 mg/kg/day (11 g/day) for 10 days between plasma exchanges] (Li GW et al. Transfusion 2014, 54: 1221-1224). The patient treated with high-dose NAC improved rapidly (the patient woke up from coma 18 hr after NAC treatment was initiated), but the patient treated with lower dose NAC did not appear to respond.  Thus, it is as yet unclear whether NAC is an effective treatment for TTP.  Therefore, more clinical studies and detailed analyses are required to examine the effects of NAC in TTP patients.  Here we report the results of clinical and biochemical studies on two patients with relapsed TTP treated with NAC. Before, during, and after NAC treatment, we determined the concentrations of NAC, cysteine, and glutathione in plasma; VWF concentration, multimer structure, and functions; ADAMTS13 concentration and activity; and platelet counts and activation status (P-selectin expression and phosphatidylserine exposure).

Methods: Two females with a history of prior episodes of TTP presented with acute TTP [ADAMTS13 < 10%, positive for ADAMTS13 inhibitors, platelet count ≤ 10,000/uL, lactate dehydrogenase (LDH) > 600 IU/L] and both were treated with NAC per IRB-approved protocol [150 mg/kg bolus over 1 hr and 150 mg/kg as continuous infusion until the next therapeutic plasma exchange (TPE)]. They received daily TPE until their platelet counts normalized, and intravenous NAC during days 2–5. Blood was collected daily for 8 days for research assays. ADAMTS13 concentrations in patient plasma were measured by ELISA.  ADAMTS13 activity was measured using HRP-conjugated A2 peptide substrate (Wu J-J et al. J Thromb Haemost 2006, 4:129-136). Concentrations of NAC, total cysteine, and total free thiols (free thiol cysteine and free thiol NAC) in plasma were determined by mass spectrometry. Plasma VWF multimer patterns were analyzed by 1.5% agarose gel electrophoresis followed by western blotting with an HRP-conjugated polyclonal VWF antibody. Platelets in whole blood were labeled for platelet markers (CD41a or CD42b) together with one of the activation markers, P-selectin or phosphatidylserine (lactadherin).  The labeled platelets were analyzed by flow cytometry.

Results: Platelet counts in both patients started to increase 1 day after NAC infusion and continued to increase after discontinuation of NAC and TPE.  After NAC infusion, the free thiol concentration (NAC and cysteine) in plasma increased 4 and 59 fold in patients 1 and 2, respectively. This was accompanied by increasing ADAMTS13 specific activity (ADAMTS13 activity/ADAMTS13 antigen). In patient 1, the specific activity increased from 127% (prior to NAC infusion but after TPE) to 270% during NAC infusion; in patient 2, the specific activity increased from 56% to 86%.  In patient 1, the VWF multimer size decreased during NAC treatment and the VWF multimers migrated slightly faster. NAC also appeared to inhibit platelet activation. Before NAC infusion, the platelets in both patients were positive for phosphatidylserine (PS, > 30%) and P-selectin (> 15%), compared to 2% and 5%, respectively, in a normal control. The percentages of PS- and P-selectin–positive platelets decreased to less than 18% and 10% respectively, during NAC treatment.

Summary: NAC treatment of two patients with TTP in conjunction with TPE was well tolerated and associated with recovery of platelet count and LDH, increased ADAMTS13 specific activity and total free thiol concentration in plasma, reduced platelet activation, and decreased VWF multimer size in one patient.