Xinglong Zheng

Remission of Chronic Thrombotic Thrombocytopenic Purpura after Treatment with Cyclophosphamide and Rituximab

Context Many adults with thrombotic thrombocytopenic purpura (TTP) have autoantibodies that promote clotting by inhibiting a von Willebrand factorcleaving protease (ADAMTS13) in plasma. Contribution This 42-year-old woman had chronic relapsing TTP despite plasma exchange; splenectomy; and treatment with vincristine, prednisone, and cyclosporine. She had low to absent ADAMTS13 activity and an IgG inhibitor. After immunosuppressive therapy with rituximab and cyclophosphamide, the TTP remitted, ADAMTS13 levels normalized, and the inhibitor was undetectable. Cautions Intensive immunosuppressive therapy aimed at the autoimmune aspect of TTP should be tested in prospective trials before being used widely. The Editors Most adults with idiopathic thrombotic thrombocytopenic purpura (TTP) have autoantibodies that inhibit a von Willebrand factorcleaving protease in plasma (1-3). This protease recently was cloned and identified as a new member of the ADAMTS metalloproteases, ADAMTS13 (4-8). Plasma exchange therapy has increased survival in patients with TTP from less than 10% to approximately 75% (9). However, plasma exchange does not directly address the underlying autoimmune process, and up to one third of patients have relapse after initially successful treatment (10). Anecdotal data suggest that immunosuppressive therapy can sometimes be beneficial, but this approach has not been tested systematically in patients known to have ADAMTS13 deficiency. We report the case of a woman with chronic relapsing TTP who had undetectable ADAMTS13 activity and elevated ADAMTS13 inhibitor titers. Treatment with rituximab and cyclophosphamide resulted in prompt disappearance of ADAMTS13 antibodies, normalization of ADAMTS13 activity, and remission of thrombotic microangiopathy. Case Report A 42-year-old woman was hospitalized in May 1999 with gait disturbance, disorientation, and lethargy. She had been healthy, with three uncomplicated pregnancies and deliveries and no previous symptoms of autoimmune disease. Computed tomography showed many cerebral infarcts. The platelet count was 60 109 cells/L, the hemoglobin level was 96 g/L, and the fibrinogen level was normal. The serum lactate dehydrogenase level was 1157 U/L. The patient received intravenous immunoglobulin but did not respond. She was transferred to our institution in June 1999 (Figure, day 1). Figure. Platelet count ( top ) and ADAMTS13 activity ( bottom ) in a patient with thrombotic thrombocytopenic purpura during 2.5 years of observation. On admission, hematocrit was 0.231, platelet count was 24 109 cells/L, haptoglobin level was 0.09 g/L (normal range, 0.27 to 2.2 g/L), and serum creatinine concentration was 141 mol/L (1.6 mg/dL) (normal range, 50 to 125 mol/L [0.6 to 1.4 mg/dL]). Prothrombin time and partial thromboplastin time were normal. Results of direct and indirect antiglobulin tests were negative. The peripheral smear contained more than 10 schistocytes per high-power field. Thrombotic thrombocytopenic purpura was diagnosed, and plasma exchange (1.5 volumes daily) was begun (Figure, top). The platelet count normalized but decreased to 34 109 cells/L by day 22. Prednisone (1 mg/kg of body weight per day) and aspirin (325 mg/d) were added without sustained benefit. The platelet count improved transiently after laparoscopic splenectomy on day 36 but decreased to less than 100 109 cells/L by day 43. Two intravenous doses of vincristine, 2 mg each, resulted in gradual improvement. Frequency of plasma exchanges was decreased to every other day, and the patient was discharged on day 85 with a normal platelet count. Plasmapheresis was discontinued on day 97. On day 105, the platelet count was 43 109 cells/L. Plasma exchange was resumed, and cyclosporine, 75 mg twice per day, was started. The platelet count increased, and plasma exchange was tapered over 3 weeks. During the next 4 months, blood counts were relatively stable. The daily prednisone dose was decreased to 40 mg. On day 248, the patient was admitted with cortical blindness and a platelet count of 58 109 cells/L. Plasma exchange was followed by an increase in platelet count but no improvement in vision. The patient was discharged after 12 days, and plasma exchange was tapered over 2 weeks. During the next 11 months, the patient was hospitalized four times for relapsed TTP and each time was treated with plasma exchange. The prednisone dose was tapered to 5 mg/d. On day 572, the patient was hospitalized with deafness and thrombocytopenia. The platelet count increased rapidly with plasma exchange. The serum creatinine concentration increased to 221 mol/L (2.5 mg/dL), and cyclosporine therapy was discontinued. The patient received two doses of intravenous rituximab (375 mg/m2 weekly). The creatinine concentration decreased to 159 mol/L (1.8 mg/dL), the platelet count remained above 100 109 cells/L, and symptoms were stable for 5 months. On day 740, the platelet count was 69 109 cells/L. The patient received daily plasma exchange for 16 days, responded well, and was discharged. She was admitted twice during the next 4 months to receive plasma exchange for relapsing TTP. Beginning on day 849, the patient received one dose of intravenous cyclophosphamide, 1 g/m2, and four doses of intravenous rituximab, 375 mg/m2, every 7 to 14 days. No side effects of rituximab were noted. During 10 months of follow-up, the patient has had a normal platelet count, stable hematocrit, normal lactate dehydrogenase level and creatinine concentration, and rare schistocytes. No other signs or symptoms of TTP have been noted. Her neurologic defects (blindness and deafness) persist unchanged. Methods Informed consent was obtained from the patient and her family. ADAMTS13 was assayed as described elsewhere (11); however, the substrate was 10 g of von Willebrand factor per mL in 5 mmol of TrisHCl per L (pH, 8.0), 1.5 mol of urea per L, and 1 mmol of phenylmethanesulfonyl fluoride per L (Sigma, St. Louis, Missouri). Plasma samples were heat-treated at 56 C for 30 minutes and were serially diluted in phosphate-buffered saline. Diluted plasma (5 L) was added to normal human plasma (5 L) and incubated at 37 C for 30 minutes. Residual ADAMTS13 activity was measured as described earlier. One unit of inhibitor reduces the ADAMTS13 activity of an equal volume of normal plasma by 50%. Recombinant ADAMTS13 (7) truncated after the metalloprotease (residues 1 to 289) was cloned in pcDNA3.1/V5-His TOPO (Invitrogen, Carlsbad, California), expressed in transiently transfected Chinese hamster ovary cells (CHO-S), and purified from conditioned medium on TALON metal affinity resin (BD Biosciences Clontech, Palo Alto, California). ADAMTS13 was immunoprecipitated with IgG adsorbed from plasma samples onto protein A agarose. Proteins were analyzed by using sodium dodecyl sulfatepolyacrylamide gel electrophoresis and Western blot with monoclonal anti-V5 antibody (Invitrogen). Results Early in her disease, the patient had complete deficiency of plasma ADAMTS13 activity and an inhibitor was detected (Figure, bottom). Plasma therapy over the next 19 months resulted in several short periods of remission, even though ADAMTS13 inhibitor titers decreased minimally and plasma ADAMTS13 activity remained low. The inhibitory activity was recovered in IgG purified from the patients plasma and immunoprecipitated the metalloprotease domain of recombinant ADAMTS13 (data not shown). For continuing autoimmune TTP, immunosuppressive therapy with rituximab was initiated. Rituximab is usually given at a dose of 375 mg/m2, repeated weekly for four doses (12, 13). The patient received an abbreviated course of two doses, which was followed by prompt disappearance of ADAMTS13 inhibitor, normalization of ADAMTS13 activity, and a normal platelet count. The patient had the first of three additional clinical relapses 5 months after receiving rituximab. Before the second course of rituximab plus cyclophosphamide, ADAMTS13 activity was 33% of normal and no ADAMTS13 inhibitor was detected. These values were obtained after 9 days of daily plasmapheresis, which may have partially corrected the ADAMTS13 deficiency. Subsequently, symptoms of TTP resolved, platelet count and plasma ADAMTS13 activity increased, and the ADAMTS13 inhibitor remained undetectable. The most recent ADAMTS13 level was 17%, with no inhibitor detected. Discussion Although idiopathic TTP is usually an autoimmune disease (1-3), standard therapy does not address this underlying mechanism. Plasma exchange may increase patient survival because it removes deleterious antibodies and replenishes ADAMTS13 protein. Most patients have self-limited disease that remits after 1 to several weeks of plasma exchange. However, approximately one third of patients have a chronic relapsing course (10). Thus, TTP often behaves as an aggressive autoimmune disease, and some patients might benefit from additional therapy directed at B cells and antibody production. Many immunosuppressive regimens have been tried in TTP, with encouraging but inconclusive results. Corticosteroids are often administered at the time of plasma exchange (14), but their efficacy has not been systematically evaluated (9, 10). Case reports and small series have described sustained responses to splenectomy (15), and responses to cyclosporine (16), vincristine, cyclophosphamide, and azathioprine (17, 18) have also been described. This anecdotal experience provides a rationale for testing additional immunosuppressive strategies in patients with relapsing or refractory TTP. Rituximab is a chimeric anti-CD20 monoclonal antibody developed for treatment of non-Hodgkin lymphoma; CD20 is expressed on B cells. Rituximab has emerged as a promising treatment for autoimmune disorders, including autoimmune hemolytic anemia (12) and idiopathic thrombocytopenic purpura (13). Common side effects observed during the treatment of lymphoma include transient hypotension and fever during the first

Bovine proenteropeptidase is activated by trypsin, and the specificity of enteropeptidase depends on the heavy chain

Enteropeptidase, also known as enterokinase, initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. Enteropeptidase is synthesized as a single-chain protein, whereas purified enteropeptidase contains a ≈47-kDa serine protease domain (light chain) and a disulfide-linked ≈120-kDa heavy chain. The heavy chain contains an amino-terminal membrane-spanning segment and several repeated structural motifs of unknown function. To study the role of heavy chain motifs in substrate recognition, secreted variants of recombinant bovine proenteropeptidase were constructed by replacing the transmembrane domain with a signal peptide. Secreted variants containing both the heavy chain (minus the transmembrane domain) and the catalytic light chain (pro-HL-BEK (where BEK is bovine enteropeptidase)) or only the catalytic domain (pro-L-BEK) were expressed in baby hamster kidney cells and purified. Single-chain pro-HL-BEK and pro-L-BEK were zymogens with extremely low catalytic activity, and both were activated readily by trypsin cleavage. Trypsinogen was activated efficiently by purified enteropeptidase from bovine intestine (K m = 5.6 μm and k cat = 4.0 s−1) and by HL-BEK (K m = 5.6 μm and k cat = 2.2 s−1), but not by L-BEK (K m = 133 μm and k cat = 0.1 s−1); HL-BEK cleaved trypsinogen at pH 5.6 with 520-fold greater catalytic efficiency than did L-BEK. Qualitatively similar results were obtained at pH 8.4. In contrast to this striking difference in trypsinogen recognition, the small synthetic substrate Gly-Asp-Asp-Asp-Asp-Lys-β-naphthylamide was cleaved with similar kinetic parameters by both HL-BEK (K m = 0.27 mm and k cat = 0.07 s−1) and L-BEK (K m = 0.60 mm and k cat = 0.06 s−1). The presence of the heavy chain also influenced the rate of reaction with protease inhibitors. Bovine pancreatic trypsin inhibitor preferred HL-BEK (initial K i = 99 nm and final K i * = 1.8 nm) over L-BEK (K i = 698 nm andK i * = 6.2 nm). Soybean trypsin inhibitor exhibited a reciprocal pattern, inhibiting L-BEK (K i * = 1.6 nm), but not HL-BEK. These kinetic data indicate that the enteropeptidase heavy chain has little influence on the recognition of small peptides, but strongly influences macromolecular substrate recognition and inhibitor specificity.

Activation of Thrombin-activable Fibrinolysis Inhibitor Requires Epidermal Growth Factor-like Domain 3 of Thrombomodulin and Is Inhibited Competitively by Protein C*

Thrombomodulin is a cofactor protein on vascular endothelial cells that inhibits the procoagulant functions of thrombin and enhances thrombin-catalyzed activation of anticoagulant protein C. Thrombomodulin also accelerates the proteolytic activation of a plasma procarboxypeptidase referred to as thrombin-activable fibrinolysis inhibitor (TAFI). In this study, we describe structures on recombinant membrane-bound thrombomodulin that are required for human TAFI activation. Deletion of the N-terminal lectin-like domain and epidermal growth factor (EGF)-like domains 1 and 2 had no effect on TAFI or protein C activation, whereas deletions including EGF-like domain 3 selectively abolished thrombomodulin cofactor activity for TAFI activation. Provided that thrombomodulin EGF-like domain 3 was present, TAFI competitively inhibited protein C activation catalyzed by the thrombin-thrombomodulin complex. A thrombomodulin construct lacking EGF-like domain 3 functioned normally as a cofactor for protein C activation but was insensitive to inhibition by TAFI. Thus, the anticoagulant and antifibrinolytic cofactor activities of thrombomodulin have distinct structural requirements: protein C binding to the thrombin-thrombomodulin complex requires EGF-like domain 4, whereas TAFI binding also requires EGF-like domain 3.

ADAMTS13 and TTP.

Thrombotic thrombocytopenic purpura (TTP) has been a mysterious and deadly disease that often could be treated effectively by plasma exchange, but without real understanding of the underlying pathophysiology. Recent advances now suggest that deficiency of a specific von Willebrand factor (VWF) cleaving protease promotes tissue injury in TTP. VWF multimers participate in the formation of platelet thrombi. Proteolytic cleavage of VWF multimers normally limits platelet thrombus growth, and failure to cleave VWF appears to encourage microvascular thrombosis. The VWF cleaving protease proves to be a new member of the ADAMTS family of metalloproteases, designated ADAMTS13. Autoantibodies that inhibit ADAMTS13 cause sporadic TTP, and mutations in the ADAMTS13 gene cause an autosomal recessive form of chronic relapsing TTP. Further studies of ADAMTS13 seem likely to change our approach to the diagnosis and treatment of TTP and other thrombotic microangiopathies.

Apical Sorting of Bovine Enteropeptidase Does Not Involve Detergent-resistant Association with Sphingolipid-Cholesterol Rafts

Enteropeptidase is a heterodimeric type II membrane protein of the brush border of duodenal enterocytes. In this location, enteropeptidase cleaves and activates trypsinogen, thereby initiating the activation of other intestinal digestive enzymes. Recombinant bovine enteropeptidase was sorted directly to the apical surface of polarized Madin-Darby canine kidney cells. Replacement of the cytoplasmic and signal anchor domains with a cleavable signal peptide (mutant proenteropeptidase lacking the amino-terminal signal anchor domain (dSA-BEK)) caused apical secretion. The additional amino-terminal deletion of a mucin-like domain (HL-BEK) resulted in secretion both apically and basolaterally. Further deletion of the noncatalytic heavy chain (L-BEK) resulted in apical secretion. Thus enteropeptidase appears to have at least three distinct sorting signals as follows: the light chain (L-BEK) directs apical sorting, addition of most of the heavy chain (HL-BEK) inhibits apical sorting, and addition of the mucin-like domain (dSA-BEK) restores apical sorting. Inhibition of N-linked glycosylation with tunicamycin or disruption of microtubules with colchicine caused L-BEK to be secreted equally into apical and basolateral compartments, whereas brefeldin A caused basolateral secretion of L-BEK. Full-length BEK was not found in detergent-resistant raft domains of Madin-Darby canine kidney cells or baby hamster kidney cells. These results suggest apical sorting of enteropeptidase depends on N-linked glycosylation of the serine protease domain and an amino-terminal segment that includes an O-glycosylated mucin-like domain and three potential N-glycosylation sites. In contrast to many apically targeted proteins, enteropeptidase does not form detergent-resistant associations with sphingolipid-cholesterol rafts.

Structure of murine enterokinase (enteropeptidase) and expression in small intestine during development

Enterokinase (enteropeptidase) is expressed only in proximal small intestine, where it initiates digestive enzyme activation by converting trypsinogen into trypsin. To investigate this restricted expression pattern, mouse enterokinase cDNA was cloned, and the distribution of enterokinase mRNA and enzymatic activity were determined in adult mice and during gestation. Analysis of enterokinase sequences showed that a mucinlike domain near the NH2 terminus is composed of repeated ∼15-amino acid Ser/Thr-rich motifs. By Northern blotting and trypsinogen activation assays, enterokinase mRNA and enzymatic activity were undetectable in stomach, abundant in duodenum, and decreased distally until they were undetectable in midjejunum, ileum, and colon. By in situ mRNA hybridization, enterokinase mRNA was localized to the enterocytes throughout the villus. Expression was not observed in goblet cells, Paneth cells, or Brunners glands. Enterokinase mRNA and enzymatic activity were not detected in the duodenum of fetal mice but were easily detected in the duodenum on postnatal days 2-6. Both enterokinase mRNA and enzymatic activity decreased to very low levels after day 7 but increased after weaning and reached a high level characteristic of adult life by day 60. Therefore, in mice, duodenal enterocytes are the major type of cells expressing enterokinase, which appears to be regulated at the level of mRNA abundance.Enterokinase (enteropeptidase) is expressed only in proximal small intestine, where it initiates digestive enzyme activation by converting trypsinogen into trypsin. To investigate this restricted expression pattern, mouse enterokinase cDNA was cloned, and the distribution of enterokinase mRNA and enzymatic activity were determined in adult mice and during gestation. Analysis of enterokinase sequences showed that a mucinlike domain near the NH2 terminus is composed of repeated approximately 15-amino acid Ser/Thr-rich motifs. By Northern blotting and trypsinogen activation assays, enterokinase mRNA and enzymatic activity were undetectable in stomach, abundant in duodenum, and decreased distally until they were undetectable in midjejunum, ileum, and colon. By in situ mRNA hybridization, enterokinase mRNA was localized to the enterocytes throughout the villus. Expression was not observed in goblet cells, Paneth cells, or Brunners glands. Enterokinase mRNA and enzymatic activity were not detected in the duodenum of fetal mice but were easily detected in the duodenum on postnatal days 2-6. Both enterokinase mRNA and enzymatic activity decreased to very low levels after day 7 but increased after weaning and reached a high level characteristic of adult life by day 60. Therefore, in mice, duodenal enterocytes are the major type of cells expressing enterokinase, which appears to be regulated at the level of mRNA abundance.