Thrombotic Thrombocytopenic Purpura and ADAMTS-13: New Insights into Pathogenesis, Diagnosis, and Therapy

Thrombotic Thrombocytopenic Purpura and ADAMTS-13: New Insights into Pathogenesis, Diagnosis, and Therapy Janis Wyrick-Glatzel, MS, MT(ASCP) (University of Nevada Las Vegas, Las Vegas, NV) DOI: 10.1309/77KRKLJW0EA75T2R Received 08.12.04 Revisions Received 09.17.04 Accepted 09.24.04 The etiology of thrombotic thrombocytopenic purpura (TTP) has been linked to abnormalities of the von Willebrand factorcleaving metalloprotease known as ADAMTS-13. Congenital deficiencies of the protease or acquired autoantibody formation against the protein cause the presence of ultra large von Willebrand factor (ULVWF) multimers in circulation. Early diagnosis and prompt initiation of plasma exchange is critical to the clinical outcome in patients diagnosed with TTP. Thrombotic thrombocytopenic purpura was first described by Moschcowitz in 1925 as an acute febrile pleiochromic anemia. Dr. Moschcowitz described the rapid onset and progression of petechiae, pallor, paralysis, coma, and death in a 16-year-old female. 1 Characteristic microvascular hyaline thrombi of terminal arterioles and capillaries were detected at autopsy. Today, these thrombi remain the hallmark of pathologic diagnosis. As first described, the natural course of TTP was a rapid and fatal progression resulting in death shortly after onset of symptoms. In 1966, Amorosi and Ultmann established the pentad of clinical features that became the diagnostic criteria for TTP. Their studies detailed the variety of clinical manifestations and the diagnostic features of similar diseases that remain today. 2 In 1991, Rock and colleagues documented the efficacy of extensive plasma exchange in reducing mortality to approximately 25% in clinical trials. 3 Moake and colleagues, in 1982, focused their research on the large multimers of von Willebrand factor (vwf) found in the plasma of 4 patients with relapsing TTP. Their studies linked the large multimeric structures with the capacity to agglutinate platelets. 4 Furlan and colleagues in Switzerland, and Tsai in New York, isolated a von Willebrand factor cleaving protease in 1996. The studies supported the theory that patients with TTP are deficient in a plasma metalloprotease that cleaves the vwf subunit at a specific peptide bond, decreasing the multimer size and its biologic activity. It was recognized early in the depiction of the disease, that a deficiency of a plasma protein or a circulating inhibitor might be responsible for the disease. 5,6 Several studies showed that patients with TTP had severe deficiencies of the vwf protease through congenital deficiencies of the protease or acquired conditions with autoantibody formation against the protease. 6,7 Today, research has characterized the von Willebrand factor protease as a protein in the metalloprotease family now known as ADAMTS-13. 8,9 The pathogenesis of TTP appears to result from a deficiency of ADAMTS-13, the protease responsible for the proteolytic cleavage of large hemostatically hyperactive vwf multimers, in plasma into smaller less adhesive multimers. ADAMTS-13 prevents inappropriate microvascular platelet aggregation. Deficiency of ADAMTS-13 is a finding for TTP, a disorder that manifests as thrombocytopenia, microangiopathic hemolytic anemia, renal failure, neurologic dysfunction, and fever, the classic pentad of clinical features. Platelet-rich microvascular thrombi are responsible for the renal and cerebral infarcts as well as damage to other organ systems. In the absence of the protease, large, hyperactive, multimers of vwf circulate in the plasma. The large hyperactive multimers are secreted from the endothelial cells and cause disseminated platelet clumping in the microvasculature. In patients with a congenital deficiency of ADAMTS-13, the protease is supplied by plasma infusion. In patients with an acquired deficiency, the inhibitor of ADAMTS- 13 is cleared from the plasma by plasmapheresis and is linked to the presence of an autoantibody. Recent molecular studies have both purified and sequenced the protease as well as clone the corresponding gene and cdna. Clinical research has characterized TTP as increasingly complex. Evidence of this complexity is reflected when individuals with severely deficient ADAMTS-13 activity often do not present with clinical findings characteristic of a diagnosis of TTP. Thrombotic microangiopathic syndromes are variably termed TTP, hemolytic uremic syndrome (HUS), secondary TTP, or idiopathic TTP. Hemolytic uremic syndrome is a closely related disorder and was originally diagnosed in 1953 in children with acute renal failure, cerebral symptoms, hemolytic anemia, and thrombocytopenia. Hemolytic uremic syndrome is usually associated with a preceding gastrointestinal syndrome and ingestion of enterohemorrhagic Escherichia coli that produce a Shiga-like toxin. Many of the clinical symptoms associated with HUS are common to that of TTP. The diagnosis of TTP has been described in patients during pregnancy or postpartum period, in association with drugs such as mitomycin C, cyclosporine, and quinine, after exposure to chemotherapy, total body irradiation, and organ transplantation, in patients with December 2004 Volume 35 Number 12 LABMEDICINE 733

autoimmune disorders, and as an idiopathic mechanism. Patients with idiopathic TTP are generally ADAMTS-13 deficient and appear to be at greater risk for a prolonged clinical course with relapse. This review article will describe the pathophysiology of TTP and the ADAMTS-13 molecule, the methodology in assaying for the ADAMTS-13 protein, the TTP syndromes associated with a defective or absent function of the ADAMTS-13 molecule, and the evaluation and management of patients who present clinically with a suspected diagnosis of TTP. Pathology of TTP Thrombotic thrombocytopenic purpura is characterized by severe thrombocytopenia, often with counts below 20 x 10 9 /L, peripheral blood schistocytes as an indicator of microangiopathic hemolytic anemia, systemic platelet aggregation causing occlusive thrombi of the microvasculature, and organ ischemia of the brain, gastrointestinal tract, and the kidney. In the past, diagnosis was made on the pentad of clinical manifestations: thrombocytopenia, neurologic abnormalities, microangiopathic hemolytic anemia, fever, and renal failure. Currently, clinical diagnosis may be made based on thrombocytopenia, schistocytes, and an elevated serum lactate dehydrogenase (LDH). Lactate dehydrogenase is released as a consequence of ischemic or necrotic tissue not hemolysis. Commonly, coagulation studies are essentially normal. The systemic microthrombi involve capillaries and arterioles, and appear to spare the venuoles for reasons not yet elucidated. Immunofluorescent and electron microscopy show the microthrombi to consist of fibrin and platelets. Endothelial cells within the occluded vessels are hyperplastic showing cellular organelle changes. However, the involved vessel wall shows no inflammatory changes or cellular infiltration. In vivo studies have shown that microvascular endothelial cells derived from the heart, kidneys, brain, and spleen are susceptible to damage by plasma derived from patients with TTP. 10 Damage to the endothelial cells translate to functional abnormalities to include impaired prostacyclin activity, 11 loss of fibrinolytic activity, 11 and apoptosis due to an upregulation of the Fas receptor. 12 Multimers of vwf are made in megakaryocytes and endothelial cells, and are stored within the platelet alpha granule and the Weibel-Palade bodies of endothelial cells. The vwf produced by endothelial cells and platelets are larger multimers than that found circulating in normal plasma. The large polypeptide multimer known as ultralarge vwf (ULVWF) is cleaved in circulation at the peptide bond between tyrosine at position 842 and methionine at position 843 by a 200-kd plasma metalloprotease identified as ADAMTS-13. Cleavage reduces the size of the factor to dimers of 176-kd and 140-kd fragments. The vwf-cleaving process produces a smaller vwf form that circulates after cleavage and does not induce the adhesion or aggregation of platelets during normal blood flow. Upon activation, endothelial cells and platelets release the contents of their storage granules into circulation. These ULVWF multimers avidly bind to the extracellular matrix of the vascular bed and to various platelet receptors, thus effectively promoting vwf-mediated platelet adhesion. The ULVWF multimers are extremely large and adhesive as a result of the increased number of binding sites for platelets as compared to the smaller plasma forms. The ULVWF multimers efficiently bind to glycoprotein Ib components of platelet glycoprotein Ib-IX-V receptors. The initial attachment of ULVWF to glycoprotein Ib receptors and the subsequent attachment to platelet glycoprotein IIb-IIIa complexes cause in vitro platelet adhesion and aggregation. Under normal physiology, plasma is devoid of ULVWF due to the processing of these multimers as they emerge from the surface of the endothelial cell by ADAMTS-13. Studies have documented, that less than 5% normal activity of ADAMTS-13 is found in most patients during an acute episode of acquired idiopathic TTP. 13 In the absence of ADAMTS-13, failure to cleave the ULVWF multimers as they emerge from endothelial cells allows the multimers to remain anchored to the cells forming beads-on-a-string structures. The anchor activity appears to be partially modulated by P-selectin molecules with transmembrane domains. P-selectin, a type I receptor, is synthesized by the cells that also synthesize vwf and is stored in the same endothelial cell and platelet alpha granule as vwf. P-selectin is thought to serve a crucial role in vwf anchorage to endothelial cells as well as induce detachment of platelet strings from the endothelial cell surface. Circulating platelets appear to initially adhere by interaction of vwf with the glycoprotein Ib membrane receptor. Additional platelets aggregate during blood flow probably by means of activated IIb-IIIa complexes, forming potentially occlusive microvascular platelet thrombi. The platelet adhesion and aggregation appears in vitro in the presence of elevated levels of fluid shear stress. 14 Many of the platelet strings are as long as 3 millimeters. These strings may detach from the endothelial cell in the absence of ADAMTS-13 activity and may embolize to blood vessels thus creating organ ischemia. A 2-hit model has been proposed to explain the variability associated with TTP. Conditions that may cause increased vwf secretion and decreased ADAMTS-13 activity may favor the clinical condition of TTP. ADAMTS-13 deficiency may trigger the sequence of events, so that the thrombotic microangiopathic condition occurs after activation or injury of the microvascular endothelial cells causing secretion of ULVWF. 15 It has been reported that the administration of desmopressin, DDAVP, exacerbates TTP by the release of ULVWF into circulation. 16 Under conditions of fluid shear stress, platelet aggregation depends on vwf. The threshold for shear-induced platelet aggregation is shifted to lower values of shear stress when ADAMTS-13 activity is decreased. 17 Plasma thrombospondin-1 has also been shown to have biologic activity toward the intersubunit disulfide bonds of vwf multimers. 18 It is uncertain if plasma thrombospondin-1 is a second responder in ADAMTS- 13 deficiency thus preventing TTP, or if deficiencies in this protein also contribute to TTP. Structure, Biosynthesis and Genetics of ADAMTS-13 The family ADAMTS represents A Disintegrin-like And Metalloprotease with ThromboSpondin type 1 motif. (Figure 1). Similar to other large extracellular proteins, ADAMTS-13 consists of 1,427 amino acid residues and contains a signal peptide, a short propeptide, a metalloprotease domain of the reprolysin/adamalysin type, a disintegrin domain, several thrombospondin type 1 motif (TSP1), a cysteine-rich domain, a spacer domain, and 2 domains. domains contain peptide sequences similar to complement components C1r.C1s, embryonic sea urchin protein egf, and bone morphogenic protein-1. Several of the domains found in ADAMTS-13 play a role in binding to other macromolecules, 734 LABMEDICINE Volume 35 Number 12 December 2004 labmedicine.com Downloaded from https://academic.oup.com/labmed/article-abstract/35/12/733/2504349

NORMAL ADAMTS-13 Cleaved ULVWF Multimers ADAMTS-13 Weibel-Palade Body ADAMTS-13 A1 domain A3 domain 842-3 (A2 domain) n P-section Endothelial Cell ULVWF monomeric subunit TTP Weibel-Palade Body Uncleaved ULVWF Multimers Platelets Weibel-Palade Body Figure 1_(A) In normal individuals, ADAMTS-13 enzyme molecules from the plasma attach to, and then cleave, ULVWF multimers that are secreted in long strings from stimulated endothelial cells. (B) The ULVWF multimeric strings may be anchored to the endothelial cell by P- selectin molecules. ADAMTS-13 molecules attach to exposed domains and proteolytically cleave ULVWF multimers. The smaller VWF forms that circulate after cleavage do not induce adhesion and aggregation of platelets during normal blood flow. (C) Absent or severely reduced activity of ADAMTS-13 in patients with TTP. Non-cleaved ULVWF multimers induce platelet adhesion and aggregation. Either congenital deficiencies or acquired defects of ADAMTS-13 result in TTP. Taken in part from: Moake JL. Von Willebrand Factor, ADAMTS-13, and Thrombotic Thrombocytopenic Purpura. Semin in Hematol. 2004; 41(1); with permission. cell surfaces, and extracellular matrixes. To assess the functional importance of each domain, truncated fragments of ADAMTS- 13 molecule were expressed and studied for vwf-cleaving activity. Neither the disintegrin domain, nor the first TSP1 motif, nor the cysteine-rich domain was highly active. Restoration of biologic activity with vwf was observed with the addition of the spacer domain as well as further addition of the remaining TSP1 motif and domains. 19 Although, certain functions have been attributed to the various domains of the ADAMTS- 13 molecule, the lack of a quantitative ADAMTS-13 antigen assay hinders the exact knowledge of the structure-function relationship. The ADAMTS-13 molecule depends upon both zinc and calcium ions for activity. Enzyme activity is optimal at ph 8.0 and lower ionic strength enhances enzyme activity. ADAMTS-13 appears to be resistant to the usual protease inhibitors, perhaps consistent with its long circulatory half-life of approximately 2 to 3 days. A specific plasma inhibitor may not be necessary due to the unique mechanism of activation of ADAMTS-13. To date, the only known physiologic substrate for ADAMTS-13 is vwf multimers. In plasma, there is no degradation of fibrinogen, collagen, or albumin during proteolysis. The rate of vwf cleavage by ADAMTS-13 is increased markedly by fluid sheer stress and low concentrations of urea or guanidine hydrochloride. Plasma ADAMTS-13 activity in healthy adults ranges from 50% to 178% using static nonphysiologic assays. Normal plasma concentration of ADAMTS-13 is not exactly known but is estimated to be 1 µg/ml. Because it is a stable protein in circulation, patients with a congenital ADAMTS-13 deficiency may be treated with plasma infusion on a 2 to 3 week protocol to prevent recurrence of thrombotic microangiopathic episodes. The human ADAMTS-13 gene is mapped to chromosome 9q34, spans approximately 37kb, contains 29 exons, and encodes for a propeptide with 1,427 amino acid residues. 20 Northern blotting techniques depict a 4.7kb mrna transcript found primarily in the perisinusoidal cells of the liver, and a 2.4kb mrna found in skeletal muscle, the placenta, and in tumor cell lines from colon cancer and malignant melanoma. 22 December 2004 Volume 35 Number 12 LABMEDICINE 735

Post-translation modification of the ADAMTS-13 protein involves O-linked and N-linked glycosylation as well as proteolysis. Glycosylation most likely accounts for the differences between the apparent masses of intracellular and secreted forms of ADAMTS-13. It is proposed that ADAMTS-13 is synthesized as a zymogen and most likely secreted as a protease with the propeptide domain removed. A variety of nonsense, missense, deletions, and splice site mutations attributed to the ADAMTS-13 gene on chromosome 9q34 are putatively responsible for severely decreased vwf-cleaving protease activity as seen in hereditary TTP. Patients studied thus far have had mutations to ADAMTS-13 with no vwf mutations identified. There appears to be striking variability of the clinical phenotype seen in hereditary TTP. Characteristically, half of patients initially present with the first acute episode of TTP in the neonatal and childhood period, while other patients present early in adulthood, and still others with a late onset of TTP at age 35 and older. Currently, it is difficult to correlate genotype and phenotype with age of onset and severity of symptoms for patients with hereditary TTP. Further clinical genetic studies will assist in determining if genetic variation in ADAMTS-13 is associated only with hereditary not acquired TTP, the most common clinical form associated with autoantibodies to ADAMTS-13. In summary, the genetic analysis characterizing a mutation in the gene suggests a molecular mechanism for TTP. This finding may provide a means for screening as well as a therapeutic application for the treatment of TTP. ADAMTS-13 Activity Assays Currently, there are 5 reference assays established for determining the activity of ADAMTS-13 in plasma. Basic to each assay is 2 distinct steps. The first step employs proteolysis of a substrate by ADAMTS-13 containing plasma. Substrates are either exogenous, endogenous, recombinant, or purified fragments of vwf. Required within the assay is a process in which the substrate is unfolded using either urea or guanidine. Various dilutions of test plasma are used with the activation of ADAMTS-13 by divalent cations. The second step quantifies the remaining substrate, or the residual vwf after proteolytic digestion, by such means as electrophoresis, immunologic properties, or the measurement of functional activity. Electrophoretic techniques utilize the principle of detection of proteolytic fragments or the generation of smaller multimers. Enzyme linked immunosorbent assays (ELISA), or immunoradiometric assays (IMRA), using monoclonal antibodies to vwf, constitute the immunologic techniques utilized in ADAMTS-13 assays. Functional assays employ ristocetin cofactor activity of vwf or the decrease of collagen binding activity. Since 1998, these techniques have been used to demonstrate deficiencies of ADAMTS-13 activity in thrombotic thrombocytopenic purpura. In comparison of the reference assays, only 1 method measures the generation of vwf degradation products by ADAMTS-13 proteolysis. 21 The normal range of ADAMTS-13 activity is estimated at 68% to 126%, in the control group. The other methods are based on the degradation of vwf multimers, 22 decrease in collagen binding activity, 23 decreased in ristocetin cofactor activity, 24 or a decrease in vwf antigen. 25 The estimated ADAMTS-13 activity in normal subjects tested using these methods is greater than 50%, 29% to 119%, 52% to 134%, and 44% to 178% activity, respectively. It is believed that ADAMTS-13 activity as low as 5% to 10% is sufficient for the prevention of microvascular platelet thrombi, and that parents of TTP patients, with ADAMTS-13 activity as low as 6% to 20% are generally asymptomatic. 26 Data generated from numerous studies validate the ability of these methods to screen for ADAMTS-13 deficiency and to distinguish between hereditary and acquired deficiencies. This distinction is quite relevant due to the difference in patients therapeutic management. Further work however is necessary to improve functional methods as well as the comparison and standardization of ADAMTS-13 activity and to develop an antigenic assay to better characterize all patients with ADAMTS-13 deficiency. The ULVWF multimers appear to undergo proteolytic processing to smaller vwf multimers directly on the endothelial cell surface. As a result of either a structural defect of the ADAMTS- 13 molecule or of autoantibody formation, blocking the binding of ADAMTS-13 to the endothelial cell receptor can result in abnormal processing of the ULVWF multimers and microthrombi formation. Current ADAMTS-13 assays do not detect defective ADAMTS-13 binding. The development of endothelial cell based assays of ADAMTS-13 activity will better define those cases of TTP without severe ADAMTS-13 deficiency that result from impaired binding. Clinical Manifestations Thrombotic thrombocytopenic purpura is considered a rare disorder with an estimated annual incidence of 3.7 per 1 million individuals. 27 The median age at diagnosis is 35; however, there is a wide range of incidence from the neonatal period to as old as 90 years of age. The disorder is characterized by microvascular platelet thrombi causing a microangiopathic hemolytic anemia with thrombocytopenia. Schistocytes typically present on the peripheral blood smear. Organ dysfunction such as fever, neurologic abnormalities, and renal failure accompany the diagnosis of TTP and thus define the expression of the classic pentad of clinical manifestations that was originally described by Moschcowitz. Despite the rarity of the disorder, early recognition and proper medical intervention is critical to patient outcome. Prior to 1960, fewer than 3% of patients survived the disease. The use of plasma exchange in the therapeutic protocol has increased the efficacy of treatment and thus the survival rate to between 82% reported in 1 study 28 and as high as 90% reported in yet another study. 29 Various studies support the clinical diagnosis of TTP when ADAMTS-13, the vwf-cleaving protease, activity is less than 5% of the activity in normal human plasma. 30 Neurologic abnormalities include such symptoms as deficits in motor or sensory function, seizures, headaches, altered mental status, visual impairment, and coma. It is suggested that these changes result from either microocclusive or microhemorrhagic changes in the vasculature. Fevers and other nonspecific symptoms such as general malaise, fatigue, and weakness are considered common presenting symptoms in patients with TTP. Renal involvement is present in an estimated 88% of patients diagnosed with TTP. 11 Acute renal failure with gross hematuria and proteinuria result due to the microvascular obstruction of the glomerular capillaries. Hematologic changes include a moderately severe thrombocytopenia (platelet counts of less than 20 x 10 9 /L) with the presentation of petechial hemorrhages commonly found of the lower extremities. Pathologic hemorrhages may also include the CNS, the gastrointestinal tract, the oro-nasal pharynx, the genitourinary tract, and the lung. Despite the 736 LABMEDICINE Volume 35 Number 12 December 2004 labmedicine.com Downloaded from https://academic.oup.com/labmed/article-abstract/35/12/733/2504349

thrombocytopenia, severe bleeds are usually not a typical clinical presentation. A moderately severe anemia, however, with hemoglobin values of less than 10g/dL and a peripheral blood film with schistocytes present is common. Red cell hemolysis is not immune in nature but results from the passage of red cells in the microvasculature occluded from platelet thrombi. Typical indicators of hemolysis such as increases in LDH, unconjugated bilirubin, and reticulocyte levels are generally monitored to evaluate the degree of hemolysis. Coagulation studies are essentially normal in TTP, and are often used as a means to differentially diagnoses TTP from disseminated intravascular coagulation (DIC). Table 1_ADAMTS-13 and Classification of Thrombotic Thrombocytopenic Purpura (TTP) ADAMTS-13 Metalloprotease ADAMTS-13 Mutations Infancy/childhood Adult onset ADAMTS-13 Deficiencies Autoantibodies Underlying diseases No known etiology Classification of TTP Upshaw-Schulman Syndrome Congenital/familial: chronic relapsing Acquired: transient or recurrent/intermittent Secondary Idiopathic Differential Diagnosis of Patients with TTP and Related Syndromes Various diseases on initial presentation can manifest similar to TTP. These disorders include: childhood HUS, pregnancyassociated microangiopathy, transplant-associated thrombocytopenic purpura, drug-induced purpura, autoimmune disorders, hemolysis, elevated liver function tests and low platelets (HELLP) syndrome and underlying malignancies (Table 1). The sudden development of clinical symptoms that define TTP in patients with no known underlying disease is referred to as idiopathic TTP. In patients with a variety of clinical conditions, the associated symptoms of TTP may be found and therefore is commonly referred to as secondary TTP. To date, there is no absolute for the diagnosis of TTP. A severe deficiency of less than 5% ADAMTS-13 activity, with clinical symptoms of an acute thrombocytopenia and evidence of a microangiopathic hemolytic anemia, appropriately defines a diagnosis of TTP. Because the sensitivity of ADAMTS-13 deficiency it is not well established, a deficiency of the protein alone does not constitute a diagnosis of TTP. The clinical uncertainty in making a diagnosis of TTP is evident when severely deficient ADAMTS-13 activity does not always produce the clinical entity of TTP; nor does severely deficient ADAMTS-13 activity diagnose those individuals with TTP who should be clinically managed by effective therapy. Childhood HUS is a diagnostic term used for the development of systemic complications resembling TTP in children usually less than 4-to-5 years of age who present with a prodrome of bloody diarrhea caused by an enterohemorrhagic strain of E. coli 0157:H7. This organism produces a Shiga-toxinrelated enterocolitis. Five to ten percent of childhood patients infected with E. coli 0157:H7 present with acute HUS. 22 In contrast to the pentad that typically defines TTP, HUS is characterized by a triad of clinical manifestations, which include thrombocytopenia, microangiopathic hemolytic anemia, and renal failure. Because HUS is often difficult to diagnosis clinically, the term TTP-HUS or thrombotic microangiopathy may be used. Studies show that once the organism attaches to the epithelium of the colon it secretes the toxin. The toxin enters the bloodstream and binds to a glycolipid surface receptor on endothelial cells under the influence of inflammatory cytokines. It is believed that the binding of the Shiga toxin results in platelet clumping. In a study of childhood HUS cases, changes in the fibrinolytic system were noted as seen by an increase in tpa antigen and tpa-pai-1 complex in response to intravascular fibrin formation. 31 Acute renal failure is the most critical clinical symptom to be managed in HUS. Despite the severity of renal insufficiency, mortality is low and most Shiga-toxin-associated HUS patients respond well to plasma exchange. The association of pregnancy with TTP may account for some of the hypercoagulable risk in women near term and postpartum. In a large study of TTP cases, 10% of all cases were diagnosed during pregnancy or postpartum, with the majority of episodes occurring near delivery. 32 There appears to be a decreased plasma concentration of ADAMTS-13 activity during the second and third trimesters of pregnancy with a concomitant increase of plasma von Willebrand factor perhaps as a contributing factor. 33 The diagnosis of TTP from other complications such as pre-eclampsia, eclampsia, and HELLP syndrome is often obscured. To make the clinical distinction between TTP, HELLP, or HUS, it should be recognized that neurologic changes are common in TTP, renal failure in HUS, and neither neurologic nor renal changes are present in HELLP. A diagnosis of HELLP is usually made in a clinical setting of preeclampsia with microangiopathic hemolytic anemia and severe thrombocytopenia. This syndrome usually resolves within days following delivery. The risk of TTP in subsequent pregnancies is rather vague and hard to determine. Few studies have defined the etiology of post bone marrow transplantation and TTP. Schriber and colleagues reviewed all cases of TTP associated with post-bone marrow transplantation (BMT) through 1996. The study recognized that fatal outcomes in patients were observed when (1) TTP developed within 120 days posttransplant, (2) patients were treated with cyclosporine for graft-vs-host disease, (3) received an allogeneic BMT, and (4) had renal and neurologic abnormalities. 34 Cyclosporine has a cytotoxic effect on endothelial cells and produces an increase in vwf release. Following BMT, however, many patients do not develop TTP yet are on clinical regimes that include immunosuppressive drugs. Other factors most likely contribute to the development of TTP. Many therapeutic agents have been associated with TTP- HUS like syndromes. Some of the more common drugs implicated are quinine, mitomycin, penicillin, oral contraceptives, and anti-platelet agents such as ticlopidine and clopidogrel. The mechanism by which TTP develops may follow either an acute immune-mediated response with the development of drug dependent antibody formation or an insidious dose-related toxic effect. Quinine is commonly associated with thrombocytopenia. Initially, the development of quinine-dependent platelet antibodies occurs with subsequent development of antibodies to multiple target tissues, which result in the systemic involvement seen in TTP. Mitomycin initiation of the TTP syndrome appears to be a dose-related effect and is associated with microangiopathic hemolytic anemia and thrombocytopenia. The onset may be sudden due to the initial toxicity or may appear gradually due to the cumulative toxicity of the drug. Most patients in this category responded well to plasma exchange. In some December 2004 Volume 35 Number 12 LABMEDICINE 737

patients receiving the antiplatelet agents, ticlopidine and clopidogrel, a reported deficiency of ADAMTS-13 and an inhibitor of ADAMTS-13 activity has been noted. 11 Despite the development of TTP within 14 days following the administration of clopidogrel, most patients responded to plasma exchange. 11 Thrombotic thrombocytopenic purpura may clinically complicate the course of patients with such autoimmune diseases as antiphospholipid syndrome, systemic lupus, polyarteritis nodosa, and scleroderma. In some patients with these multiorgan involvement diseases, the associated pathologic features are indistinguishable from those of TTP. The commonality between the disorders may complicate the clinical diagnosis and thus may prevent initiation of effective treatment. The association of various malignancies with altered coagulation is well documented. Adenocarcinomas are commonly associated with TTP-HUS; and of these cancers, gastric adenocarcinoma has a high association with TTP. Defining the predisposing mechanism of TTP in the cancer population is difficult at best since many chemotherapeutic agents are also associated with the initiation of TTP. Also, similarity exists between the clinical features of cancer and TTP. However, evaluating the degree of anemia and thrombocytopenia relative to the malignancy may help identify and diagnose TTP in patients in this clinical setting. In a study by Murgo and colleagues 11 characteristics to differentiate cancer-related TTP from chemotherapyrelated TTP were examined. The study defined those patients who developed TTP while in remission as chemotherapyinduced cases. In patients with cancer, TTP may be treated by means of plasmapheresis, immunosuppressive agents, and immunoadsorption with varying degrees of efficacy. Acute idiopathic TTP is difficult to recognize and diagnose clinically. A large percentage of cases of acquired idiopathic TTP are due to autoantibody formation. The autoantibody mediates a severe ADAMTS-13 protease deficiency. These cases often show remission associated with the absence of the autoantibody and normal ADAMTS-13 activity. Reappearance of the autoantibody generally precedes the clinical relapse and results in a subsequent episode of severe ADAMTS-13 deficiency. Relapses are considered common and frequent in patients with acute idiopathic TTP. ULVWF multimers are detected during acute episodes in some patients with acute idiopathic TTP. Focus is now pointed to the antigenic epitope of the autoantibody related to the efficacy of standard plasma exchange with replacement FFP in these patients. It is thought that this therapy removes the inhibitory autoantibody by plasma exchange and supplies the ADAMTS-13 protease during FFP replacement. Therapeutic Modalities in the Treatment of TTP Prior to the use of plasma exchange, the mortality rate of patients diagnosed with TTP surpassed 90%. At that time, TTP was considered a fatal disorder. Byrnes and Khurana in 1977 35 reported recurrence of the disorder could be prevented by the infusion of fresh-frozen plasma (FFP) or cryosupernatant, devoid of ULVWF multimers, without concomitant plasmapheresis. In 1985, studies performed by Moake and colleagues showed that normal ADAMTS-13 activity was found in FFP, cryosupernatant and solvent/detergent-treated plasma (SD-FFP) 36 and that the use of these components was effective therapy in patients with congenital relapsing TTP. Over the years, various cases of TTP were managed by the use of plasma infusion and/or plasma exchange as treatment protocol. However, in a randomized prospective controlled study, the Canadian Apheresis Group documented that plasma exchange was more effective than plasma infusion as a therapeutic modality for TTP. 35 Numerous studies have documented that FFP, cryosupernatant, and SD-FFP all contain biologically active ADAMTS-13, the vwfcleaving protease. Currently, plasma exchange is considered efficacious in the treatment of TTP with a subsequent and significant drop in the mortality rate to between 10% and 20%. In defining ADAMTS-13 deficiency as the pathogenesis of TTP, it is believed that therapeutic plasma exchange is efficacious in replacing the vwf-cleaving protease. Severe thrombocytopenia with platelet counts below 20 x 10 9 /L and microangiopathic hemolytic anemia without an underlying cause are considered sufficient clinical findings to make a diagnosis of TTP and to initiate plasma exchange as standard treatment. Ideally, treatment with plasma exchange should occur within the first 24 hours of clinical presentation. The normal treatment protocol for TTP encompasses a single plasma volume exchange on a daily basis and should continue for a period of 1 to 2 weeks and gradually taper off. This therapy should continue for a few days beyond achieving a normal platelet count, stabile hemoglobin value, normal LDH level, and normal neurologic findings, until remission is achieved. Renal insufficiency, however, remits later in the course of the disease. The associated renal failure may require hemodialysis. When plasma exchange cannot be performed than plasma infusion is recommended to bridge the hiatus in therapy. It is felt that removal of toxic substances and the replacement of an abnormal or deficient factor by means of plasma infusion is an important adjunct to the treatment of TTP. In cases of severe ADAMTS-13 deficiency, or where the response to a daily course of plasma exchange is less than adequate, plasma exchange twice daily appears to be effective. Administration of corticosteroids in conjunction with plasma exchange may be of benefit in patients if response is not achieved through the normal course of plasma exchange. A percentage of adults and older children with acquired TTP exhibit a high-affinity autoantibody that inhibits ADAMTS-13 activity. These patients often do not respond to a daily course of plasma exchange and are treated with corticosteroids. Immunoadsorption in lieu of or in conjunction with plasma exchange may reduce treatment duration for this group of patients. Currently, there is no clinical data that predicts remission or the duration of the remission, highlighting the heterogeneity of clinical disorders defined as TTP. The use of platelet concentrates in the treatment of TTP is contraindicated. In a cohort of patients, treated with platelet concentrates during the course of therapy for TTP, extensive platelet aggregation in the CNS was noted postmortem. 11 Numerous studies suggest that patients have greater morbidity and higher mortality rates when thrombocytopenia is managed with platelet transfusion. Desmopressin, or DDAVP, is also contraindicated as a therapeutic modality in the treatment of TTP. Desmopressin is known to cause increased release of vwf multimers from endothelial cells into the circulation. The use of intravenous immunoglobulin (IVIG) and antiplatelet agents have also been investigated as possible therapeutics for TTP. The potential benefits from use of these agents remain inconclusive. In patients refractory to plasma exchange, splenectomy has been reported to achieve remission, but is considered to be a last recourse in treatment. Plasma exchange is generally considered safe; however, because of the prolonged course of treatment and the general health of the patient diagnosed with TTP, there are adverse complications associated with therapy. Approximately 10% of patients 738 LABMEDICINE Volume 35 Number 12 December 2004 labmedicine.com Downloaded from https://academic.oup.com/labmed/article-abstract/35/12/733/2504349

treated by plasma exchange experience adverse reactions with a higher rate of complications reported in those treated by plasma infusion. 35 Transfusion-related acute lung injury (TRALI) is the most severe complication and in the majority of cases is associated with infusion of plasma containing components. The estimated incidence of TRALI is 0.02% to 0.03% per plasma containing unit transfused. 35 Adverse reactions also associated with therapy may include: hemorrhage, pneumothorax, central venous catheter thrombosis, systemic infection, allergic reactions, hypoxia and hypotension. When evaluating the therapeutic management of patients with TTP-HUS, these risks should be taken into consideration. LM 1. Moschowitz E. An acute febrile pleiochromic anemia with hyaline thrombosis of the terminal arterioles and capillaries. Arch Intern Med. 1925;36:89-93. 2. Amorosi EL, Ultmann JE. Thrombotic thrombocytopenic purpura: Report of 16 cases and review of the literature. 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