Thrombotic Thrombocytopenic Purpura From Platelet Aggregates to Plasma

Transcription

1 Pathology Patterns Reviews Thrombotic Thrombocytopenic Purpura From Platelet Aggregates to Plasma Marisa B. Marques, MD, 1 Charles A. Mayfield, MD, PhD, 1 and Douglas P. Blackall, MD 2 Key Words: Thrombotic thrombocytopenic purpura; TTP; Microangiopathic hemolytic anemia; ADAMTS-13; Plasmapheresis; Thrombotic microangiopathy Abstract Thrombotic thrombocytopenic purpura (TTP) is a syndrome of severe thrombocytopenia and microangiopathic hemolytic anemia without an alternative explanation. Although some patients also have a combination of fever and neurologic and/or renal manifestations, these are not required for the diagnosis. Thus, plasmapheresis should start as soon as TTP is placed high in the differential diagnosis to prevent significant mortality. Histopathologically, TTP is characterized by widespread platelet thrombi in the microcirculation. Ultralarge von Willebrand factor (vwf) multimers found in the patient s plasma are the basis for the platelet thrombi. Recent evidence has linked the abnormal fragments of vwf with deficiency of a plasma enzyme named vwf-cleaving protease, or ADAMTS-13. While a small percentage of patients with TTP have a constitutional defect in this enzyme, many with the acute idiopathic form have an antibody to ADAMTS-13, affecting its ability to cleave vwf. The determination of the enzyme activity and the presence of its inhibitor have emerged as a potential tool in the diagnosis and prognosis of TTP. Furthermore, it helps to differentiate TTP from the hemolytic uremic syndrome, in which the level of ADAMTS-13 is expected to be normal or only slightly decreased. Thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) are 2 thrombotic microangiopathies (TMAs) characterized by thrombocytopenia and hemolytic anemia secondary to platelet aggregation in the microcirculation with subsequent ischemic manifestations. TMAs generally have an acute onset, sometimes are fatal, and often are difficult to diagnose. TTP is caused by the deposition of hyaline thrombi in the arterioles of multiple organs, first described in an autopsy case by Moschkowitz 1 in Although rare, the severity of the clinical manifestations and the high mortality without proper treatment make TTP a remarkable and classic multiorgan disorder. In patients with TTP, unusually large (UL) von Willebrand factor (vwf) multimers in the plasma are responsible for inducing spontaneous platelet aggregation, which leads to the clinical manifestations. These UL vwf multimers normally are cleaved into a series of smaller vwf proteins by the enzyme vwf-cleaving protease (vwf-cp). Patients with familial forms of TTP have been shown to display severely decreased or absent vwf-cp activity, whereas patients with acquired TTP have an antibody to the enzyme. There have been no such alterations of the vwf-cp observed in patients with HUS. These important observations have led to increased use of vwf-cp activity and inhibitor identification assays in the diagnostic workup of patients with TMAs. The present review describes the clinical and laboratory features of TTP and its treatment, the proposed role of vwf-cp in the pathogenesis of TTP, and laboratory methods for assay of the enzyme. Case Study A 39-year-old woman was admitted to the hospital with complaints of weakness and headache and was found to be S89 S89

2 Marques et al / THROMBOTIC THROMBOCYTOPENIC PURPURA REVIEW anemic and profoundly thrombocytopenic. Her admission laboratory data are shown in Table 1. A diagnosis of TTP was made, and daily plasma exchanges of 1.5 plasmavolumes replaced with fresh frozen plasma (FFP) were started. After 27 plasmaphereses, there was no clinical or laboratory evidence of active TTP, and the patient was discharged (Table 1). She returned 18 days later with similar complaints and was found to have recurrent thrombocytopenia, albeit without evidence of hemolysis. A bone marrow biopsy demonstrated only megakaryocytic hyperplasia, and serologic test results were negative for platelet antibodies. With a presumptive diagnosis of TTP relapse, plasmapheresis was reinitiated, and the platelet count normalized after only 3 procedures. The patient again was discharged without signs of TTP. She returned 2 weeks later with upper respiratory infection symptoms and thrombocytopenia. Before reinitiating plasma exchanges, a blood specimen was obtained to assess vwf-cp activity. She subsequently was found to be severely deficient in the protease, caused by a strong inhibitor. After the initiation of plasmapheresis, the patient s primary team offered her the option of splenectomy or a trial of rituximab. She chose the latter and received the first dose of 375 mg/m 2 immediately following the first plasma exchange. After 3 more plasmaphereses, the platelet count was more than /µl ( /L). She received another dose of rituximab a week from the first and 2 more as an outpatient. At the time of discharge, she had received 6 plasma exchanges. She has been in TTP remission for more than 300 days. Clinical Manifestations The clinical features and severity of TTP are related to the extent of microvascular platelet thrombi and subsequent tissue hypoxia. The characteristic pentad of thrombocytopenia, hemolytic anemia, fever, neurologic symptoms, and renal dysfunction have been associated with the syndrome, 1-4 but they are present in varying percentages in patients with TTP. 5,6 The criteria for diagnosing TTP have been simplified to thrombocytopenia and microangiopathic hemolytic anemia without an alternative explanation. 7 The latter also were the criteria used for enrollment in a landmark multicenter trial conducted by the Canadian Apheresis Study Group in which plasmapheresis was proven superior to plasma infusion. 8 Only approximately 50% of patients with TTP initially have fever, but if present, it may be very high. Furthermore, neurologic manifestations might include headache, agitation, and confusion and can progress rapidly to sensory and motor deficits, seizures, coma, and death. Although the true incidence of TTP is unknown, underestimation is likely because most deaths occur during the first 2 days of clinically evident disease that might not be diagnosed as TTP. 9 TTP can manifest in multiple forms based predominantly on age at onset and clinical course. 3,4 The most common form of TTP is acquired idiopathic TTP, which occurs in adults and adolescents. Although usually limited to a single acute episode, acquired TTP can recur at unpredictable intervals in 30% to 60% of patients. 9,10 Familial TTP is rare and usually manifests in infancy or childhood. Most patients with familial TTP have recurring episodes at regular intervals of 3 to 4 weeks. TTP that follows this recurring course also is referred to as chronic relapsing TTP. 4 In addition, acute episodes of TTP have been associated with various clinical conditions such as pregnancy, bone marrow transplantation, and HIV-1 infection. The syndrome also has developed in a small percentage of patients receiving the platelet adenosine diphosphate receptor inhibitors ticlopidine (Ticlid) and clopidogrel (Plavix) for prevention of arterial thrombosis Table 1 Laboratory Data for a Patient With Acute Idiopathic Thrombotic Thrombocytopenic Purpura Initial Diagnosis First Relapse Second Relapse Variable Reference Range Admission Discharge Admission Discharge Admission Discharge Platelet count, ( ) 12 (12) 258 (258) 33 (33) 475 (475) 38 (38) 472 (472) 10 3 /µl ( 10 9 /L) Hemoglobin, ( ) 6.4 (64) 10.1 (101) 10.9 (109) 12.2 (122) 11.9 (119) 9.9 (99) g/dl (g/l) Schistocytes None Moderate Rare None None None None LDH, U/L (U/L) ( ) 2,020 (2,020) 259 (259) 259 (259) ND 428 (428) 183 (183) Haptoglobin, ( ) <7 (<0.1) ND 85 (0.9) ND 119 (1.2) ND mg/dl (g/l) Bilirubin, total, (0-17) 3.2 (55) ND 0.8 (14) ND 1.3 (22) ND mg/dl (µmol/l) No. of TPEs during hospitalization LDH, lactate dehydrogenase; ND, not done; TPEs, total plasma exchanges. S90 S90

3 Pathology Patterns Reviews Laboratory Findings The widespread deposition of platelets in the microcirculation leads to severe thrombocytopenia, which is present in most patients. Although the decrease in platelet count can be moderate, it typically is severe, yielding counts of less than /µl ( /L) at initial examination with a corresponding megakaryocytic hyperplasia in the bone marrow. Furthermore, while microangiopathic hemolytic anemia is a hallmark of TTP, some patients initially are not anemic, as shown in the 2 exacerbations of the patient described herein. When present, hemolysis contributes to elevated lactate dehydrogenase (LDH) levels, hyperbilirubinemia, and a low or an undetectable level of haptoglobin. Abundant schistocytes often are evident on the peripheral blood smear. LDH also is released from multiple organs owing to generalized tissue ischemia resulting from the occlusive platelet thrombi. In 10 patients with TTP, Cohen et al 14 demonstrated that the high serum levels of LDH were due mainly to isoenzyme LDH5 (tissue) as opposed to LDH1 or LDH2 present in RBCs. Prothrombin time, partial thromboplastin time, and fibrinogen values are expected to be normal because TTP is not a disorder of secondary hemostasis; however, the levels of fibrin degradation products are elevated in a majority of patients. 5 Patients with TTP usually have only mild renal dysfunction; acute renal failure is rare and suggests HUS. Despite the characteristic pentad (or dyad) of clinical features classic for TTP, it often is difficult to distinguish this disease from other clinical conditions, including other TMAs. Thrombocytopenia, hemolysis, and schistocytosis also might be seen in disseminated intravascular coagulation, preeclampsia or eclampsia, malignant hypertension, Evans syndrome, severe vasculitis, and scleroderma with hypertension and renal failure and in patients with a dysfunctional prosthetic heart valve. Laboratory findings (eg, prothrombin time and partial thromboplastin time, fibrinogen level, and the direct antiglobulin test) are useful in differentiating TTP from other disorders with similar clinical manifestations. However, the distinction between TTP and HUS can be much more obscure because these disorders have overlapping clinical and laboratory findings. This differentiation is critical, though, because the treatments for TTP and HUS differ significantly. Without the prompt initiation of plasmapheresis, there is a substantial risk of mortality associated with TTP. On the other hand, there has been no randomized clinical trial demonstrating the efficacy of plasmapheresis (or plasma infusion) in patients with HUS. Until recently, the distinction between TTP and HUS was based largely on clinical manifestations: patients with TTP are more likely to have neurologic symptoms, whereas patients with HUS usually have more severe renal dysfunction, often with anuric renal failure. In 1982, Moake et al 15 described UL vwf multimers in the plasma of patients with chronic relapsing TTP. They proposed the existence of an enzyme responsible for depolymerizing vwf in healthy people. The purification of such a protease later was accomplished and named vwf-cleaving protease. 16,17 More recently, the protease has been cloned and determined to be a member of the ADAMTS family of metalloproteases. Thus, vwf-cp is now ADAMTS The ability to measure ADAMTS-13 activity has provided a new clinical laboratory tool for the diagnosis of TTP. Since the availability of assays for ADAMTS-13 activity, numerous studies have demonstrated a dramatic deficiency in patients with TTP, as opposed to patients with HUS in whom the protease activity is decreased only moderately and, often, is normal. 6,23-26 However, a recent review of the Oklahoma TTP-HUS registry pointed out that only 13% of 142 adult patients with TTP-HUS had severe ADAMTS- 13 deficiency (<5% activity). 27 Furthermore, the clinical manifestations, outcomes, and response to plasmapheresis were not different between the group with severe deficiency and the group with higher levels of enzyme activity. 27 Thus, the determination of ADAMTS-13 activity has only a limited role in the diagnosis of TTP. It also is noteworthy that partial and sometimes even complete deficiencies of ADAMTS-13 have been reported in patients with TMAs secondary to various other clinical conditions such as neoplasia, infection, immunologic disorders, and pregnancy. 6 On the other hand, most patients with TTP associated with bone marrow transplantation have normal or slightly decreased ADAMTS-13 activity. 6,28 Furthermore, ADAMTS-13 activity is moderately deficient in conditions such as metastatic neoplasms, decompensated liver cirrhosis, acute inflammatory conditions (mostly bacterial respiratory infections), chronic renal insufficiency/ hemodialysis, and pregnancy (second and third trimesters) and in neonates and during the early postoperative period after major surgeries. 29 Mild deficiency also can be observed in older patients (>65 years). 29 The most severe deficiencies in ADAMTS-13 activity occurring outside of TTP are found in patients with decompensated cirrhosis and acute inflammatory states, in which the average protease activity is 40% to 50% of normal. This is in comparison with ADAMTS-13 activity of 0% to 10% seen almost exclusively in patients with TTP. Thus, an extremely low or absent ADAMTS-13 activity is specific for TTP, 4,30,31 but the sensitivity ranged between 33% and 100% in 5 large cohort studies. 6,24,25,27,32 Finally, the distinction between a primary enzymatic defect and acquired deficiency due to an autoantibody inhibitor might further differentiate the type of TTP and permit more specific treatment options. S91 S91

4 Marques et al / THROMBOTIC THROMBOCYTOPENIC PURPURA REVIEW ADAMTS-13 Assays ADAMTS-13 enzymatic activity can be determined by measuring the ability of the patient s plasma to degrade vwf multimers. Initial assays involved the incubation (for up to 24 hours) of large vwf multimers purified from normal human cryoprecipitate or supernatant from endothelial cell cultures, with dilutions of patient plasma in the presence of barium and urea. 13,23-25,32 The appearance of ADAMTS-13 degradation products (dimers of 176 and 140 kd) was measured by immunoblotting after protein separation using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Alternatively, the disappearance of the UL vwf multimers also could be determined by the same electrophoresis procedure and immunoblotting. Three faster assays for the determination of ADAMTS- 13 activity and the presence of its inhibitor have been described Of these, the one presently available for clinical use in the United States is the collagen binding assay. 33 This test is based on the preferential binding of the largest vwf multimers to collagen compared with that of the smaller forms of the protein. The principle of this assay is that the degradation of the largest vwf multimers by ADAMTS-13 generates smaller vwf multimers that bind less well to collagen. Dilutions of patient s plasma are incubated with normal human plasma in which the protease has been inactivated. The products resulting from proteolytic degradation of vwf in the normal plasma by ADAMTS-13 in the patient s sample are captured on microtiter plates coated with human collagen type III. The vwf multimers bound to the collagen then can be detected with a peroxidase-conjugated antibody to human vwf and quantitated from standard curves. The presence of an inhibitor also can be determined by testing the capability of the patient s plasma to inhibit ADAMTS-13 activity in normal plasma after mixing with protease-inactivated patient plasma followed by the collagen binding assay. In this assay, an ADAMTS-13 activity of less than 6% indicates a severe deficiency, which has been reported to have a sensitivity of 70% to 100% for the diagnosis of idiopathic TTP. 30 The quantitation of an ADAMTS-13 inhibitor is calculated in Bethesda units in a way similar to that used for specific coagulation factor inhibitors such as factor VIII. The other 2 assays described thus far also are indirect measurements of ADAMTS-13 activity and are based on residual ristocetin cofactor activity of degraded vwf or the immunoradiometric measurement of residual vwf antigen after its cleavage by the protease. 34,35 A recent multicenter study compared the sensitivity of these 3 assays in identifying severe ADAMTS-13 deficiency and the presence of inhibitors. 36 Five European laboratories participated in the study, and the investigators found good interassay and interlaboratory agreement in the ability to detect very low levels of ADAMTS-13 activity and enzyme inhibitors. They concluded that all assays might be useful in the workup of patients with suspected TTP. 36 However, at this time, these assays are available only through large reference laboratories, and their results should not influence initial therapeutic decisions. 27 Pathogenesis Our present understanding of TTP pathogenesis is based largely on 2 observations reported more than 20 years ago: (1) Plasma is beneficial in treating patients with TTP. 37 (2) UL vwf multimers are seen in the plasma of patients with TTP. 15 Based on these observations, it was concluded that patients with TTP must have a defect in processing the largest vwf multimers, those that are most active in mediating platelet adhesion and aggregation. The occlusive thrombi in TTP are composed predominantly of platelets without underlying perivascular inflammation or other vessel wall damage. Immunohistochemical analysis and electron microscopy reveal that the thrombi are composed of degranulated and altered platelets and vwf with scarce fibrin or fibrinogen. 38 In normal plasma, vwf mediates the adhesion of platelets to vascular endothelial lesions, is the carrier protein for factor VIII, and contains binding sites for platelet glycoprotein (Gp) Ibα, GpIIb-IIIa, and collagen. Under physiologic conditions, vwf, released predominantly from Weibel-Palade bodies in vascular endothelial cells, circulates as multimers ranging in size from 500 to 20,000 kd, composed of 280-kd monomers linked by disulfide bonds. The largest multimers bind with higher affinity to the GpIbα portion of the GpIbα-IX-V complex and to the GpIIb-IIIa complexes on platelets and induce agglutination under conditions of increased fluid shear stress Increased amounts of these highly adhesive UL vwf multimers have been found in patients with chronic relapsing TTP Figure 1. 15,39,42 In these patients, the UL vwf multimers disappear as the acute episode progresses, consistent with incorporation into platelet aggregates, reappear during remission, and are predictive of relapse. Indeed, during a single acute episode of TTP and during TTP recurrences, vwf displays increased binding to platelets, and the patient s plasma contains increased numbers of circulating platelet aggregates compared with periods of remission. 43 In normal plasma, a vwf-cp cleaves the UL vwf multimers between tyrosine 1605 and methionine 1606 within the monomeric vwf subunits, producing a series of smaller multimers. 16,17 The action of vwf-cp might regulate multimer size, preventing the release or accumulation of UL vwf multimers in plasma and averting the spontaneous interaction of vwf with platelets. vwf-cp, or ADAMTS-13, S92 S92

5 Pathology Patterns Reviews Normal ADAMTS-13 activity UL vwf multimers released from endothelial cells Deficient ADAMTS-13 activity thrombi leading to TTP are a result of the presence or persistence of high-affinity UL vwf multimers secondary to severely deficient vwf-cp activity with or without an antibody inhibitor. However, evidence also has shown that not all patients with TTP have a deficiency of ADAMTS ,24,25,27,32 Thus, other pathogenetic mechanisms must have a role in the development of such a syndrome. Treatment Figure 1 von Willebrand factor cleaving protease (ADAMTS-13) activity in normal plasma degrades unusually large (UL) von Willebrand factor (vwf) multimers preventing spontaneous platelet agglutination. In thrombotic thrombocytopenic purpura, UL vwf persists due to lack of the enzyme or its inhibition by an antibody, leading to the formation of platelet thrombi in the absence of endothelial damage. is a 190-kd zinc- and calcium-dependent protein that is synthesized predominantly in the liver. A severe deficiency of vwf-cp in TTP was first demonstrated by Furlan et al 32 in 1997 in 4 patients with chronic relapsing TTP. The plasma of these patients contained UL vwf multimers and low vwf-cp activity without any identifiable inhibitor of the protease. Two patients were brothers whose asymptomatic parents both had below-normal vwf- CP activity, suggesting the heritable nature of this type of TTP. Subsequently, a number of studies by Furlan et al 23,24 and others 6,25 have shown extremely low vwf-cp activity in the familial and acquired forms of TTP. vwf-cp in these patients usually is less than 5% and often undetectable during single acute episodes and in recurrences. In familial and chronic relapsing TTP, the deficient protease activity has been attributed to mutations in the vwf-cp gene located on chromosome 9q In contrast, the decreased vwf-cp activity in acute idiopathic or acquired TTP is due to inhibition by IgG antibodies present in as many as 83% of patients. 6,23-25 An antibody inhibitor of vwf-cp also has been found in patients with ticlopidine- and clopidogrel-induced TTP. 12,13 These findings strongly support the hypothesis that platelet aggregates and The availability of assays for ADAMTS-13 activity and inhibitor has contributed to the determination of the best therapeutic approach for patients with different forms of TTP. These assays permit differentiation between chronic relapsing TTP due to an intrinsic deficiency of the protease and acquired idiopathic TTP that results from the presence of an antibody inhibitor. This distinction has important therapeutic implications and might predict the likely course of the disease. Infants or young children with familial chronic relapsing TTP generally respond well to transfusion with plasma or cryosupernatant, which supplies vwf-cp. Adults with acute episodes of TTP require daily plasmapheresis in which the patient s plasma is exchanged with FFP. 8 Treatment with plasmapheresis confers 90% survival in patients with acute TTP, presumably by removing UL vwf multimers and replacing vwf-cp. Furthermore, plasmapheresis also might remove IgG antibodies against ADAMTS-13 in patients with idiopathic TTP. The latter is suggested by the observation that plasma infusion alone is inferior to plasma exchange. 8 Alternative plasma-based products also have had an important role in the TTP therapeutic armamentarium. The first of these products, cryoprecipitate-reduced plasma or CRP, originally came into favor as salvage therapy when it was first determined that some patients with TTP had UL vwf multimers in their circulation. 44 It was not known whether this finding was the cause or a by-product of the disease, but CRP quickly became an important alternative therapy when FFP failed. The rationale for its use is that CRP is depleted of the largest multimers of vwf, those that are the most effective platelet aggregators. 10,45 Although CRP and FFP have never been compared directly in a randomized, case-control study, there is ample evidence of the clinical efficacy of CRP. In fact, the US Food and Drug Administration licensed CRP in 1997 with a requirement that product labeling denote that the component might be indicated for treating patients with TTP who do not have a response to standard therapy with FFP. Without the appropriate controlled trials, however, it will remain unknown whether CRP is equivalent or superior therapy for TTP. A report from the US TTP Apheresis Study Group, published in 1998, indicated that the response, relapse, and S93 S93

6 Marques et al / THROMBOTIC THROMBOCYTOPENIC PURPURA REVIEW mortality rates for patients treated with a combination of 5% albumin for the first half of the plasma exchange followed by plasma (FFP, CRP, or both) for the second half of the procedure were comparable to the rates for patients treated with plasma-only replacement fluid. 9 Their analysis also revealed that 40% of 20 surveyed centers used a plasma exchange taper. However, they did not find a statistical difference in the rate of relapse between the taper and the nontaper groups. 9 Unfortunately, the presence of a high-titer inhibitor to ADAMTS-13 has been associated with refractoriness to plasmapheresis 46 and might indicate the need for adjuvant therapy. Cyclosporine and splenectomy have been used successfully in combination with plasmapheresis in the treatment of TTP, perhaps through suppression of antibody production. 47,48 More recently, rituximab, a monoclonal antibody against CD20, has emerged as a potential therapy for refractory TTP. The first case report of its use described a patient with a 19-month history of relapsing TTP, despite treatment with plasmapheresis and multiple other therapies, including splenectomy. 49 The patient had a complete deficiency of ADAMTS-13 activity owing to a strong inhibitor. After initiation of therapy with rituximab and cyclophosphamide, the inhibitor promptly disappeared, protease activity normalized, and symptoms of TTP resolved. The patient subsequently had complete remission with no required treatment during a 13-month follow-up period. Since this case was published, rituximab has been used in other centers, but its role in the treatment of TTP has not been defined. 50,51 Response to treatment in TTP is variable, and the platelet count is the most important parameter to monitor. Although thrombocytopenia might persist for several days after the initiation of daily plasma exchanges, this is still the standard of care and should be continued for as long as needed, because some patients might need weeks to recover fully. 7 Prevention and New Horizons Accumulated experience clearly suggests that plasma infusion can prevent relapses in patients with chronic relapsing TTP. 52 However, there is no equivalent strategy to prevent relapse in patients with autoimmune-based TTP. Rituximab might represent a therapeutic option for this form of the disease. 49,50 The future likely will provide other examples of translational research that can be applied to TTP prevention, therapy, and diagnosis. With respect to the latter, new assays for ADAMTS-13 that more closely resemble the physiologic flow conditions found on endothelial cell surfaces under fluid shear forces are being developed, and their role in TTP is still uncertain Conclusions TTP remains a rare, life-threatening illness that once was almost uniformly fatal. The earliest case report provided a tantalizing hint that abnormal platelet aggregation was at the heart of the TTP disease process. However, the scientific underpinnings of this understanding were long preceded by an empiric knowledge regarding therapy: plasma saves lives. From serendipity to science, we now have a much more evolved understanding of TTP: its manifestations, therapy, and pathogenesis. Only the future knows what additional surprises will be discovered as new generations of investigators plumb the mysteries of TTP. 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