Han-Mou Tsai

Antibodies to von Willebrand Factor–Cleaving Protease in Acute Thrombotic Thrombocytopenic Purpura

BACKGROUND Thrombotic thrombocytopenic purpura is a potentially fatal disease characterized by widespread platelet thrombi in the microcirculation. In the normal circulation, von Willebrand factor is cleaved by a plasma protease. We explored the hypothesis that a deficiency of this protease predisposes patients with thrombotic thrombocytopenic purpura to platelet thrombosis. METHODS We studied the activity of von Willebrand factor-cleaving protease and sought inhibitors of this protease in plasma from patients with acute thrombotic thrombocytopenic purpura, patients with other diseases, and normal control subjects. We also investigated the effect of shear stress on the ristocetin cofactor activity of purified von Willebrand factor in the cryosupernatant fraction of the plasma samples. RESULTS Thirty-nine samples of plasma from 37 patients with acute thrombotic thrombocytopenic purpura had severe deficiency of von Willebrand factor-cleaving protease. No deficiency was detected in 16 samples of plasma from patients with thrombotic thrombocytopenic purpura in remission or in 74 plasma samples from normal subjects, randomly selected hospitalized patients or outpatients, or patients with hemolysis, thrombocytopenia, or thrombosis from other causes. Inhibitory activity against the protease was detected in 26 of the 39 plasma samples (67 percent) obtained during the acute phase of the disease. The inhibitors were IgG antibodies. Shear stress increased the ristocetin cofactor activity of von Willebrand factor in the cryosupernatant of plasma samples obtained during the acute phase, but decreased the activity in cryosupernatant of plasma from normal subjects. CONCLUSIONS Inhibitory antibodies against von Willebrand factor-cleaving protease occur in patients with acute thrombotic thrombocytopenic purpura. A deficiency of this protease is likely to have a critical role in the pathogenesis of platelet thrombosis in this disease.

Thrombotic Thrombocytopenic Purpura Associated with Clopidogrel

BACKGROUND The antiplatelet drug clopidogrel is a new thienopyridine derivative whose mechanism of action and chemical structure are similar to those of ticlopidine. The estimated incidence of ticlopidine-associated thrombotic thrombocytopenic purpura is 1 per 1600 to 5000 patients treated, whereas no clopidogrel-associated cases were observed among 20,000 closely monitored patients treated in phase 3 clinical trials and cohort studies. Because of the association between ticlopidine use and thrombotic thrombocytopenic purpura and other adverse effects, clopidogrel has largely replaced ticlopidine in clinical practice. More than 3 million patients have received clopidogrel. We report the clinical and laboratory findings in 11 patients in whom thrombotic thrombocytopenic purpura developed during or soon after treatment with clopidogrel. METHODS The 11 patients were identified by active surveillance by the medical directors of blood banks (3 patients), hematologists (6), and the manufacturer of clopidogrel (2). RESULTS Ten of the 11 patients received clopidogrel for 14 days or less before the onset of thrombotic thrombocytopenic purpura. Although 10 of the 11 patients had a response to plasma exchange, 2 required 20 or more exchanges before clinical improvement occurred, and 2 had relapses while not receiving clopidogrel. One patient died despite undergoing plasma exchange soon after diagnosis. CONCLUSIONS Thrombotic thrombocytopenic purpura can occur after the initiation of clopidogrel therapy, often within the first two weeks of treatment. Physicians should be aware of the possibility of this syndrome when initiating clopidogrel treatment.

Shigatoxin triggers thrombotic thrombocytopenic purpura in genetically susceptible ADAMTS13-deficient mice

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening illness caused by deficiency of the vWF-cleaving protease ADAMTS13. Here we show that ADAMTS13-deficient mice are viable and exhibit normal survival, although vWF-mediated platelet-endothelial interactions are significantly prolonged. Introduction of the genetic background CASA/Rk (a mouse strain with elevated plasma vWF) resulted in the appearance of spontaneous thrombocytopenia in a subset of ADAMTS13-deficient mice and significantly decreased survival. Challenge of these mice with shigatoxin (derived from bacterial pathogens associated with the related human disease hemolytic uremic syndrome) resulted in a striking syndrome closely resembling human TTP. Surprisingly, no correlation was observed between plasma vWF level and severity of TTP, implying the existence of TTP-modifying genes distinct from vWF. These data suggest that microbe-derived toxins (or possibly other sources of endothelial injury), together with additional genetic susceptibility factors, are required to trigger TTP in the setting of ADAMTS13 deficiency.

Antibody inhibitors to von Willebrand factor metalloproteinase and increased binding of von Willebrand factor to platelets in ticlopidine-associated thrombotic thrombocytopenic purpura.

Ticlopidine, a potent antiplatelet agent used to maintain patency after coronary artery stenting and to prevent strokes in high-risk persons (1), has been associated with thrombotic thrombocytopenic purpura (TTP) (2-5). Thrombotic thrombocytopenic purpura, first described by Moschcowitz (6), is characterized by extensive platelet thrombi in the arterioles and capillaries. Abnormalities in von Willebrand factor multimers, including the presence of unusually large multimers and disappearance of the large multimers found in normal plasma, have been detected in many cases of the disease (7, 8). Furthermore, von Willebrand factor is abundant in the thrombi of patients with TTP (9), and flow cytometric studies have demonstrated that the factor is bound to platelets in the circulation of these patients during the most thrombocytopenic phase of the disease (10). The von Willebrand factor, a glycoprotein critical in mediating platelet deposition at sites of vessel injury, is synthesized and secreted by endothelial cells as a disulfide-linked polymer composed of a 2050amino acid monomer (11). Upon release into the circulation, it is cleaved by a plasma metalloproteinase in a shear-dependent manner (11) at the peptide bond between tyrosine-842 and methionine-843 (12). This cleavage decreases the size of the von Willebrand factor polymer, generates a series of multimers found in normal plasma, and produces dimers of 176-kD and 140-kD fragments (11). In the absence of the proteinase, large and unusually large von Willebrand factor multimers accumulate in the plasma. When unfolded by shear stress (13), these multimers exhibit an increased capacity to support platelet aggregation (14). Indeed, a deficiency of the proteinase has been reported in idiopathic TTP (15, 16). We investigated whether von Willebrand factor is involved in ticlopidine-associated TTP. Methods Patients Seven consecutive patients who developed TTP after initiation of ticlopidine therapy and were treated at the participating institutions from 1 January 1996 to 31 December 1998 were investigated. The criteria for the diagnosis of TTP were those described elsewhere (10, 16). We also determined proteinase activity in 17 controls: 7 consecutive, unselected patients without thrombocytopenia (age range, 62 to 81 years; 5 men and 2 women) who donated blood samples at routine follow-up examinations after 3 to 5 weeks of ticlopidine therapy prescribed for cardiac stents, and 10 randomly selected hospitalized patients not taking ticlopidine. Blood samples were obtained by venipuncture or at the time of plasmapheresis. The investigational protocol was approved by the institutional review boards of the participating centers. von Willebrand Factor Studies Platelet-bound von Willebrand factor, von Willebrand factor multimers, von Willebrand factorcleaving metalloproteinase activity, and the inhibitory activity of IgG to the von Willebrand factorcleaving metalloproteinase were measured as described elsewhere (10, 16). The von Willebrand factor bound to single platelets in EDTA-anticoagulated whole-blood samples was quantified by flow cytometry. Proteinase activity was expressed as a percentage of that in the pooled normal plasma control. Results The initial clinical and laboratory findings of the patients are summarized in the Table. The duration of ticlopidine therapy before diagnosis of TTP ranged from 2 to 7 weeks (median, 3 weeks). None of the patients had a history of autoimmune disorders, and none were receiving penicillins, antineoplastic chemotherapy, or oral contraceptives before onset of the disease. In all patients, remission occurred after ticlopidine therapy was discontinued and daily plasma exchange was instituted. The median number of plasma exchanges received by the patients was 10 (range, 5 to 30). None of the patients had relapse after plasma exchange was discontinued. Table. Clinical and Laboratory Findings von Willebrand Factor Binding to Single Platelets Binding of von Willebrand factor to platelets was studied in patients 1 to 3; the test was not available for the other 4 patients. Platelet-bound von Willebrand factor was 7.5, 4.5, and 4.5 arbitrary units, respectively (normal value<2.1 arbitrary units) in the initial blood samples; these values returned to normal when patients received plasma exchange and platelet counts increased. von Willebrand Factor Multimers In all seven patients, the largest multimers, which are found in normal plasma, were missing in the initial plasma samples. For patient 1, von Willebrand factor multimeric patterns in three of seven subsequent plasma samples were abnormal; one sample lacked the largest von Willebrand factor multimers, and two contained unusually large multimers. The initial plasma sample (obtained on day 1) for patient 3 was missing the largest multimers. The next two samples (obtained on days 2 and 4) contained unusually large multimers. The multimers were normal in the subsequent two samples (obtained on days 8 and 9). For patients 1, 2, and 6, plasma samples obtained upon remission were available for investigation; all samples showed normal multimeric patterns. von Willebrand Factor Metalloproteinase Activity For patient 1, only plasma samples obtained on days 4 to 6 after admission (when his platelet counts were 77 109/L, 70 109/L, and 76 109/L) were available for the study. These samples contained 28%, 17%, and 14%, respectively, of the proteinase activity found in plasma from normal controls. Proteinase activity was 0% in the initial plasma samples of patients 2 to 4 and 7%, 12%, and 4%, respectively, in patients 5 to 7. However, the plasma samples of these three patients were obtained from the plasmapheresis bags in amounts of 200 to 250 mL during the initial plasma exchanges. For patient 2, the protease activity increased to 100% on day 3, when the platelet count was 260 109/L. For patient 3, protease activity increased to 6%, 10%, 81%, and 77%, respectively, on days 2, 4, 8, and 9, when platelet counts were 25 109/L, 130 109/L, 280 109/L, and 325 108/L. Plasma proteinase levels in patient 5 increased to 23% and 55% on days 4 and 6, respectively, when platelet counts were 140 109/L and 180 109/L. A plasma sample obtained from patient 6 on day 5, when the platelet count had increased to 277 109/L, contained 94% proteinase activity. The mean (SD) plasma proteinase activity in the 7 controls receiving ticlopidine for 3 to 5 weeks was 114% 36%, which did not differ from the activity in the 10 randomly selected controls who were not treated with ticlopidine (97% 17%). In a previous study (16), 74 randomly selected patients without TTP had proteinase activity of 103% 23%. Inhibitors of von Willebrand Factor Proteinase To explore the causes of proteinase deficiency, the initial plasma sample of patient 2 was mixed with normal control plasma after treatment at 56 C for 30 minutes. The von Willebrand factor metalloproteinase activity in the mixture was suppressed to 23% of the activity found in a control mixture of heated normal pooled plasma and normal control plasma. Plasma samples from patients 3 to 7 were sufficient in volume for studies to determine whether their immunoglobulins inhibited the proteinase. The IgG isolated from patient 3 on day 1 exhibited a concentration-dependent inhibition of proteinase activity in normal control plasma. The concentration of the IgG molecules required to inhibit 50% of the protease activity in the mixture (IC50) was 2.2 mg/mL. The IgG isolated from the same patient on day 9 was not inhibitory. The IC50 of the IgG isolated from initial plasma samples of patients 4 to 7 was 5.5, 2.2, 4.4, and 2.2 mg/mL, respectively. In tests comparing susceptibility to inhibition, von Willebrand factor metalloproteinase in plasma from the normal controls and that in the plasma samples from controls who received ticlopidine were equally sensitive to inhibition by IgG isolated from patients with ticlopidine-associated TTP (data not shown). The inhibitory activity of IgG was abolished when it was incubated with antibodies to IgG Fab (data not shown). Discussion In two series of single-episode and intermittent idiopathic TTP (15, 16), inhibition of plasma von Willebrand factor proteinase by IgG autoantibodies was found to be characteristic. In support of a role of von Willebrand factor proteinase deficiency in the pathogenesis of platelet thrombosis, the deficiency was not observed in persons who did not have the disease. Furthermore, shear stress was found to increase the capacity of von Willebrand factor to support platelet aggregation (16). We now describe seven patients with ticlopidine-associated TTP who also had severely decreased levels of von Willebrand factor proteinase 2 to 7 weeks after initiation of ticlopidine therapy. The durations of ticlopidine therapy before the diagnosis of TTP are similar to the 2 to 12 weeks observed in 98 cases of TTP in a recently described series (17). The deficiency in our patients resolved after ticlopidine therapy was discontinued and plasmapheresis was instituted. The deficiency was not observed in randomly selected patients who had been receiving ticlopidine for similar durations but did not develop the disease. The absence or severe reduction of von Willebrand factor metalloproteinase was accompanied by binding of von Willebrand factor to platelets. Concurrently, the large von Willebrand factor multimers were missing. The level of von Willebrand factor proteinase activity required to prevent or decrease binding of von Willebrand factor to platelets and thrombosis was low (approximately 10% to 15%). Thus, even a slight increase in the proteinase activity was sufficient to suppress the values of platelet-bound von Willebrand factor. At this low level of proteinase activity, von Willebrand factor proteolysis remained defective. This explained the presence of unusually large von Willebrand factor multimers in the plasma. The decrease in von Willeb

Advances in the Pathogenesis, Diagnosis, and Treatment of Thrombotic Thrombocytopenic Purpura

Thrombotic thrombocytopenic purpura (TTP) and the hemolytic uremic syndrome (HUS) are both characterized by thrombocytopenia, microangiopathic hemolysis, and organ dysfunction. Other disorders occasionally present with similar manifestations. Recent studies have demonstrated that deficiency in the von Willebrand factor cleaving protease ADAMTS13, due to genetic mutations or autoimmune inhibitors, causes TTP. Molecular cloning of ADAMTS13 elucidates the structure of the protease, raising the prospect for advances in diagnosis and treatment of the disease. Assay of ADAMTS13 activity distinguishes TTP from HUS and other types of thrombotic microangiopathy (TMA); therefore, the term TTP/HUS should be avoided because it obscures the known or potential differences among the various types of TMA.

ADAMTS13 Is expressed in hepatic stellate cells

ADAMTS13 is a circulating zinc metalloprotease that cleaves the hemostatic glycoprotein von Willebrand factor (VWF) in a shear-dependent manner. Deficiency in ADAMTS13, owing to genetic mutations or autoimmune inhibitors, causes thrombotic thrombocytopenic purpura (TPP). Northern blot analysis has shown that ADAMTS13 is expressed primarily in the liver. By using real-time RT-PCR, we confirmed that in mice the liver had the highest level of the ADAMTS13 transcript. To identify the liver cell-type-specific origin of ADAMTS13, we used in situ hybridization techniques to investigate the pattern of ADAMTS13 expression in the liver; analyzed the ADAMTS13 proteolytic activity in the culture media of fractionated liver cells; and confirmed ADAMTS13 expression with RT-PCR analysis and cloning of the mouse ADAMTS13 gene. The results revealed that ADAMTS13 was expressed primarily in cell fractions enriched in hepatic stellate cells. The mouse ADAMTS13 cloned from primary hepatic stellate cells was similar to its human counterpart in digesting VWF and was susceptible to suppression by EDTA or the IgG inhibitors of patients with TTP. Since hepatic stellate cells are believed to play a major role in the development of hepatic fibrosis and cirrhosis, the identification of the liver cell-type expressing ADAMTS13 will have important implications for understanding pathophysiological mechanisms regulating ADAMTS13 expression.

Pathophysiology of thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a disorder with characteristic von Willebrand factor (VWF)-rich microthrombi affecting the arterioles and capillaries of multiple organs. The disorder frequently leads to early death unless the patients are treated with plasma exchange or infusion. Studies in the last decade have provided ample evidence to support that TTP is caused by deficiency of a plasma metalloprotease, ADAMTS13. When exposed to high shear stress in the microcirculation, VWF and platelets are prone to form aggregates. This propensity of VWF and platelet to form microvascular thrombosis is mitigated by ADAMTS13, which cleaves VWF before it is activated by shear stress to cause platelet aggregation in the circulation. Deficiency of ADAMTS13, due to autoimmune inhibitors in patients with acquired TTP and mutations of the ADAMTS13 gene in hereditary cases, leads to VWF–platelet aggregation and microvascular thrombosis of TTP. In this review, we discuss the current knowledge on the pathogenesis, diagnosis and management of TTP, address the ongoing controversies, and indicate the directions of future investigations.

Von Willebrand Factor and Von Willebrand Factor-Cleaving Metalloprotease Activity in Escherichia coli O157:H7-Associated Hemolytic Uremic Syndrome

Hemolytic uremic syndrome (HUS) usually occurs after infection with Shiga toxin-producing bacteria. Thrombotic thrombocytopenic purpura, a disorder with similar clinical manifestations, is associated with deficient activity of a circulating metalloprotease that cleaves von Willebrand factor at the Tyr842-Met843 peptide bond in a shear stress-dependent manner. We analyzed von Willebrand factor-cleaving metalloprotease activity and the status of von Willebrand factor in 16 children who developed HUS after Escherichia coli O157:H7 infection and in 29 infected children who did not develop this complication. Von Willebrand factor-cleaving metalloprotease activity was normal in all subjects, but von Willebrand factor size was decreased in the plasma of each of 16 patients with HUS. The decrease in circulating von Willebrand factor size correlated with the severity of thrombocytopenia and was proportional to an increase in von Willebrand factor proteolytic fragments in plasma. Immunohistochemical studies of the kidneys in four additional patients who died of HUS demonstrated glomerular thrombi in three patients, and arterial and arteriolar thrombi in one patient. The glomerular thrombi contained fibrin but little or no von Willebrand factor. A decrease in large von Willebrand factor multimers, presumably caused by enhanced proteolysis from abnormal shear stress in the microcirculation, is common in HUS.

Rituximab for chronic recurring thrombotic thrombocytopenic purpura: a case report and review of the literature

Deficiency of von Willebrand factor (VWF) cleaving protease ADAMTS13 has been demonstrated to be the proximate cause of a subset of thrombotic microangiopathic haemolytic anaemias (MAHA) typical for thrombotic thrombocytopenic purpura (TTP). ADAMTS13 gene mutations cause the hereditary form; acquired deficiency has been attributed to presence of an autoantibody, which may represent a specific subset of MAHA best termed ‘autoimmune thrombotic thrombocytopenic purpura’. We describe a patient with relapsing TTP because of ADAMTS13 inhibitors, who failed to achieve sustained remission despite therapies with plasma exchange, steroids, vincristine, staphylococcal protein A and splenectomy. The ADAMTS13 inhibitor titre remained elevated and clinical stability was only maintained by plasma exchange every 2–3 d over a period of 268 d. The patient then received rituximab therapy (eight doses of 375 mg/m2 weekly), during which she required five plasma exchanges in the first 10 d, two exchanges in the next 3 weeks, and none thereafter for 450 d and ongoing. The ADAMTS13 inhibitor titre decreased and enzyme activity increased. We compared this case with that of seven previously reported TTP cases also treated with rituximab; experience suggests that rituximab therapy deserves further investigation for patients with either refractory or relapsing TTP caused by ADAMTS13 inhibitors.

Rituximab induces remission of cerebral ischemia caused by thrombotic thrombocytopenic purpura

Objective: This report describes the experience of a case of atypical thrombotic thrombocytopenic purpura (TTP) whose diagnosis was based on severe deficiency of the von Willebrand factor (vWF) cleaving metalloprotease ADAMTS13.