Von Willebrand Factor-Cleaving Protease Activity in Thrombotic Microangiopathy: First Report From Iran

Thrombotic microangiopathic (TMA) is a rare but devastating disorder of small vessels that is characterized by intravascular platelet thrombi, thrombocytopenia, and various degrees of organ ischemia and anemia, which is due to erythrocyte fragmentation in microcirculation (1). In thrombotic thrombocytopenic purpura (TTP), systemic microvascular aggregation of platelets mainly causes ischemia in the brain. In the hemolytic-uremic syndrome (HUS), platelet-fibrin thrombi predominantly occlude the renal circulation (2). Adults with major central neurological involvement are labeled mainly as TTP (1). TTP and HUS are not distinct syndromes and their essential diagnostic criteria are the same. Although neurologic abnormalities such as confusion, focal deficits, seizures, or coma are commonly considered characteristics of TTP and renal failure is more common in HUS, some patients with TMA have both neurologic and renal involvement. Prompt recognition of TTP is important because the disease responds well to plasma-exchange while high mortality ensues when it remain untreated. Nevertheless, recognition of TTP can be difficult because of its different features and lack of specific diagnostic criteria. Consistent abnormalities are red cell fragmentation and thrombocytopenia (3). Endothelial damage due to toxins and inhibitory antibodies to von Willebrand factor (vWF)-cleaving protease (ADAMTS13), which impair endothelial defense against complement activation, has a central role in pathogenesis (4). Discovery of ADAMTS13 has offered a new insight into the pathogenesis of TMA (4). ADAMTS13 is a metalloprotease that cleaves vWF at the Tyr1605-Met1606 bond in the central A2 domain. This cleavage progressively converts the vWF polymer to smaller multimers that are less adhesive. When ADAMTS13 activity is deficient, vWF polymers are not cleaved, which results in accumulation of hyperactive intact forms of vWF that causes platelet aggregation and microvascular thrombosis (5). During the course of plasma exchange therapy, increasing ADAMTS13 activity level is associated with clinical and hematologic improvement (6). Severe deficiency of ADAMTS13 (< 6%) is observed in genetic mutations or in the presence of inhibiting autoantibodies. Occasionally, low levels of ADAMTS13 might be observed in disseminated intravascular coagulopathy, liver disease, or sepsis (7).

We aimed to measured ADAMTS13 activity and status of their ADAMTS13-inhibiting antibody during acute phase of TMA.

All patients with the diagnosis of TMA were registered by Chronic Kidney Disease Research Center of Tabriz University of Medical Sciences since 2003 to 2011. The diagnosis of acute TMA was made based on at least three of the following criteria: (a) thrombocytopenia with no other apparent cause; (b) Coombs-negative hemolytic anemia with schistocytes; (c) high serum levels of lactate dehydrogenase (LDH); and (d) signs or symptoms of target organ involvement including’s kidney or central nervous system involvement (8). Demographic, clinical, and laboratory parameters of all studied patients were entered in the standard forms.

Serum samples were obtained from all individuals during the acute phase before the first plasma exchange or at least two weeks after the last therapeutic plasma infusion or plasma exchange, and were stored at -80℃ in tubes containing trisodium citrate as anticoagulant. Patients received detailed information on purposes of the study and signed a written informed consent. According to an agreement and approval by Mario Negri Institute of Pharmacological Research in Bergamo, Italy, all collected samples were sent to that institute while they were kept at < 0℃ during the transfer and their quality were checked by the institute at the time of reception. ADAMTS13 activity was measured as described by Gerritsen et al. (9, 10). The samples with activity levels of < 30% and < 6% were classified as deficient and complete deficient ADAMTS13 activity, respectively. The activity levels of > 30% were considered normal or near normal results. Inhibiting antibodies to ADAMTS13 activity were searched in all patients by measuring residual activity in a 50:50 mixture of heat-inactivated patient’s plasma and normal pooled human plasma. Heat-inactivated plasma was obtained after incubation at 56℃ for 60 minutes to completely neutralize endogenous ADAMTS13 activity. A patient would be considered to have ADAMTS13 inhibitor (1 Bethesda unit) if the residual activity of the protease in the plasma mixture were less than 50% of the control mixture (a 50:50 mixture of heat-inactivated and unheated normal plasma) (9, 11).

We used a simple descriptive analysis of our data.

From total number of 40 studied patients, 14 (35%) were male and 26 (65%) were female. The mean age of patients was 46.12 ± 17.26 years (range, 18-87). The demographic characteristics of the patients are summarized in Table 1.

In terms of underlying disease, infectious diseases were the most prevalent presumptive cause of TMA (50%). Among the infections, urinary tract infection (UTI) was the most common underlying cause. The second cause was gastroenteritis and diarrhea (16.7%). Infections sinusitis, bacterial pneumonia, and in one case, hydatid cyst were the concomitant infections. Pregnancy was the second presumptive causes of TMA. Systemic lupus erythematous (SLE) was the most common autoimmune diseases (85%) and the mean activity of ADAMTS13 activity in this group was 26%. TMA was also detected in a patient with Goodpasture disease. Another patient developed TMA immediately after myocardial infarction while he was taking Clopidogrel (Plavix) for a year; ADAMTS13 activity level in this patient was 25%. We also detected TMA in a 38-year-old woman who was HBsAg positive and had started taking oral contraceptive recently. Acute myeloid leukemia, multiple myeloma, and esophageal squamous cell carcinoma were the concomitant malignancies in three patients. Malignant hypertension was the precipitating cause in two patients with ADAMTS13 activity of 23% and 50%. We also detected TMA in a patient who was hospitalized because of subarachnoid hemorrhage and intracerebral hemorrhage without any episode of malignant hypertension. In general, the mean activity level of ADAMTS13 was 34.58% ± 21.83%. Two patients (5%) had complete deficiency of ADAMTS13 activity because of inhibiting antibodies. In one of them, pregnancy was the probable precipitating factor and she died because of severity of her disease. Another patient had no apparent condition.

In our study, majority of patients had an underlying condition during the clinical diagnosis of TMA. Infectious disease, pregnancy, and autoimmune disease were the most common precipitating factors. ADAMTS13 had variable activity levels. We found that even in those patients who had a relevant condition such as malignant hypertension, the low level of ADAMTS13 could be the precipitating cause for TMA. The presence of inhibiting antibodies is an indicator of severity. It is often difficult to differentiate between the two clinical syndromes of TTP and HUS. Early in 1998, Furlan et al. reported 23 patients who were diagnosed with HUS that had some subnormal ADAMTS13 activity (12). Choi et al. in 2011 studied ADAMTS13 activity and ADAMTS13 gene mutation in children with hemolytic uremic syndrome. They found five ADAMTS13 gene mutations without deficiency of ADAMTS13 activity in three patients with HUS and they could not answer whether these mutations without reduced ADAMTS13 activity were a predisposing factors (13). Bianchi et al. determined ADAMTS13 activity level in various conditions that were accompanied by thrombocytopenia such as severe sepsis or septic shock, heparin-induced thrombocytopenia, and idiopathic thrombocytopenic purpura. They noticed that mild and even moderate decrease in ADAMTS13 activity was common in these conditions (7). Severe deficiency of ADAMTS13 activity (< 5%-10%) is a specific finding in clinically diagnosed TTP (2, 14). Tsai and Lian (15) observed severe ADAMTS13 deficiency in all of 37 patients with acute TTP and Furlan et al. observed this situation in 26 out of 30 patients (12). Another study reported a severe deficiency of ADAMTS13 in 47 of 66 patients (71%) with idiopathic or secondary TTP (7).

In 1997, a complete deficiency of ADAMTS13 activity was reported by Furlan et al. in four patients with chronic relapsing TTP (16). In a literature review on ADAMTS13, Mannucci and Peyvandi stated that it is unnecessary to assay ADAMTS13 for the diagnosis of acute TTP and activity measurement is an index to predict the relapse (17). The Oklahoma TTP-HUS registry believes that relapses occur mainly in patients with severe autoantibody-mediated ADAMTS13 deficiency and it most often occurs during the first year of the TTP attack (10, 18). Furlan et al. and Tsai et al. reported complete deficiency of ADMATS13 in patients who had a circulating IgG inhibitor against the ADAMTS13 (12, 15), which was compatible with the results of the present study regarding two patients with severe deficiency of ADAMTS13 activity level and presence of inhibiting autoantibodies. Infections are reported as underlying conditions that induce TTP (19).; some of these infections are pulmonary tuberculosis, cytomegalovirus pneumonitis, mucocutaneous herpes simplex infection, ventilator-associated pneumonia, UTI, gram-negative sepsis, central line infections with staphylococcal bacteremia, and cellulites (4, 20, 21). Ardalan et al. reported cases of TMA in renal transplantation as a result of Parvovirus B19 and aspergillosis (21, 22) Crimean-Congo hemorrhagic fever has been reported as a cause of TMA and acute renal failure (23). In our study, UTI (with and without renal stones), gastroenteritis and diarrhea, bacterial sinusitis, pneumonia, and hydatid cyst were presumptive underlying causes. TTP has been reported in association with SLE, might precede or follow SLE, has similar clinical symptoms, and the levels of ADAMTS13 activity are decreased in both conditions (24). Yang et al. reported a patient with typical TTP who finally ended with the diagnosis of SLE with a good response to the initial plasmapheresis (25). In our cohort, we had six patients with SLE as the underlying cause of TMA and all of them improved with plasmapheresis. TTP might be detected during pregnancy or peripartum period. Characteristic signs of TTP might occur in patients with severe preeclampsia, which is named HELLP syndrome (hemolysis, elevated liver-enzyme levels, and a low platelet count) and make it difficult to distinguish from TTP (26). Molvarec et al. found that although plasma level of vWF were significantly higher in patients with preeclampsia in comparison with healthy pregnant and non-pregnant women, ADAMTS13 activity was normal in majority of these patients (27). We reached the same findings in our study. TTP affects one in 1600 to one in 5000 patients who receive ticlopidine; however, little is known about the pathogenesis of this complication. In a study by Tsai et al. seven patients with ticlopidine-associated TTP had severe decrease in ADAMTS13 levels (5). In our study, we had one case of ticlopidine-associated TMA in whom ADAMTS13 activity was slightly decreased. van den Born et al. reported that ADAMTS13 was deficient in malignant hypertension-induced TMA, probably because of the release of vWF from stimulated endothelium (28). Finally, although kidney is the main target organ, other organs might be affected too. Cardiac complications occur in 3% to 10% of patients with complement-mediated HUS and as a consequence of microangiopathic injury in the coronary microvasculature, sudden cardiac death could happens (29).