Willebrand disease, acquired

In patients with TTP, absent or severely reduced activity of ADAMTS 13 prevents timely cleavage of unusually large multimers of von Willebrand factor as they are secreted by endothelial cells. In flowing blood, the uncleaved multimers induce the adhesion and aggregation of platelets.

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CONTINUING EDUCATION

To earn CEUs, see test on page 20.

LEARNING OBJECTIVES

Upon completion of this article the reader will be able to:

1. Describe the clinical features of thrombotic thrombocytopenic purpura.

2. Define the pathogenesis of von Willebrand factor (vWF) and TTP.

3. Describe the application and interpretation of vWF-cleaving protease assays.

Thrombotic microangiopathies (TMAs) are a heterogeneous group of diseases characterized by hemolytic anemia and thrombocytopenia associated with platelet aggregation in the microcirculation and subsequent ischemic manifestations. TMAs are generally acute in onset, sometimes fatal and often difficult to diagnose. Thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) are the two main thrombotic microangiopathic disorders that until recently have been difficult to distinguish.

In patients with TTP, there are unusually large von Willebrand factor (ULvWF) multimeric proteins present in the plasma, which induce platelet aggregation, leading to the clinical manifestations. These ULvWF multimers 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, while patients with acquired TTP have been found to possess an antibody to this protease. 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 in the diagnostic work-up of patients with TMA. The measurement of the enzyme activity has involved the laborious procedure of multimeric analysis by gel electrophoresis until the recent development of a much simpler collagen-binding assay. Measurement of the protease is now becoming increasingly utilized in the critical diagnosis of TTP and is proving important in guiding therapeutic decisions. The current review describes the clinical features of TMAs, the proposed role of vWF-CP in TTP, as well as the laboratory methods for assay of the enzyme.

Thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura is a rare hematologic disorder involving the formation of platelet aggregates in the microvasculature. (1,2) The consumption of platelets in these aggregates results in severe thrombocytopenia. Occlusive platelet thrombi are formed in many organs, including the brain, heart, kidneys, spleen, adrenals and gastrointestinal tract, causing ischemia of these tissues and release of large amounts of lactate dehydrogenase (LDH). In addition, erythrocytes are fragmented as they transverse small vessels narrowed by platelet aggregates, leading to hemolysis and the formation of schistocytes.

Clinical features

The clinical features and severity of TTP are related to the extent of microvascular platelet aggregation and thrombi formation. The characteristic pentad of thrombocytopenia, hemolytic anemia, fevers, neurological symptoms and renal dysfunction have been associated with TTP. (1,2) These clinical features are present in varying percentages in patients with TTP. (3) More recently, the clinical criteria for suspicion of TTP have been simplified to the triad of thrombocytopenia, schistocytosis and elevated LDH. Thrombocytopenia is present in more than 90% of cases and can be moderate to severe with platelet counts typically below 20,000/[micro]L in acute episodes and an associated increase in megakaryocytes in the bone marrow. Coombs negative hemolytic anemia is present in virtually all patients with TTP and is usually severe. The hemolysis contributes to elevated LDH, as well as increased conjugated bilirubin and low or undetectable haptoglobin. Peripheral blood smears show schistocytes and helmet cells resulting from fragmentation of erythrocytes. Approximately 50% of patients with TTP present with fever, which is occasionally very high. Neurologic symptoms are present in as many as 90% of patients. Symptoms may initially be mild with headache, agitation and confusion, but can progress rapidly to sensory and motor deficits, seizures, coma and death. Patients with TTP usually have only mild renal dysfunction with moderate increases in serum creatinine. Acute renal failure is rare in patients with TTP.

Thrombotic thrombocytopenic purpura can be classified into different types, based predominantly on age at onset and clinical course. (1,2) 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 10% to 30% of patients. Familial TTP is rare and usually presents in infancy or childhood. TTP that follows a recurring course is also referred to as chronic relapsing TTP. In addition, acute episodes of TTP have been associated with other clinical conditions, such as pregnancy, bone marrow transplantation and HIV-1 infection. A small percentage of patients receiving the platelet ADP receptor inhibitors ticlopidine (Ticlid) and clopidogrel (Plavix) for prevention of arterial thrombosis (4-6) also have developed the syndrome.

Pathogenesis

The occlusive thrombi in TTP are composed predominantly of platelets without underlying perivascular inflammation or other vessel wall damage. Immunohistochemical studies have demonstrated that the platelet thrombi also contain a large amount of von Willebrand factor with little or no fibrin or fibrinogen. (7) vWF mediates the adhesion of platelets to vascular endothelial lesions, in addition to being the carrier protein for coagulation factor VIII. vWF circulates in plasma as multimers, ranging in size from 500 to 20,000 kilodaltons. The multimers are composed of 280-kilodalton monomers linked by disulfide bonds and are released predominantly from Weibel-Palade bodies in endothelial cells. The largest multimers are referred to as ULvWF multimers and appear to bind with higher affinity to the platelets, inducing aggregation under conditions of increased fluid shear stress. (8,9,10) Increased amounts of these highly adhesive ULvWF multimers have been found in patients with chronic relapsing TTP. (8,11,12) In these patients, the ULvWF multimers disappear as the acute episode progresses, consistent with incorporation into platelet aggregates, reappear during remission and are predictive of relapse. Indeed, patients with both a single acute episode of TTP and those with recurrent TTP have been shown to display increased binding of vWF to platelets and to contain increased numbers of circulating platelet aggregates compared to periods of remission. (13)

[FIGURE 1 OMITTED]

In normal plasma, a vWF-CP has been identified that cleaves the ULvWF multimers between tyrosine 842 and methionine 843 within the monomeric vWF subunits, producing a series of smaller multimers (Figure 1). (14,15) The action of vWF-CP may serve to regulate multimer size, preventing the release or accumulation of ULvWF multimers in plasma and, thus, averting the spontaneous interaction of the UL multimers with platelets. vWF-CP is a member of the ADAMTS family of metalloprotease enzymes and is designated ADAMTS-13. (16-18) It is a 190-kilodalton zinc and calcium-dependent protein that is synthesized predominantly in the liver: A severe deficiency of vWF-CP in TTP was demonstrated by Furlan, et al, in 1997 in four patients with chronic relapsing TTP. (19)

Subsequently, a number of studies by Furlan and others have shown extremely low vWF-CP activity in both familial and acquired forms of TTP. (3,20-22) vWF-CP in these patients is usually <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 9q34. (23) In contrast, the decreased vWF-CP activity in acute idiopathic or acquired TTP is due to inhibition by IgG antibodies present in as much as 83% of patients (Figure 1). (3,20-22) An antibody inhibitor of vWF-CP has also been found in patients with ticlopidine- and clopidogrelinduced TTP. (5,6)

Diagnosis

Despite the characteristic pentad (or triad) of clinical features found in some patients with TTP, it is often difficult to distinguish this disease from other clinical conditions, including other thrombotic microangiopathies. The triad of thrombocytopenia, hemolysis and schistocytosis may also be present in many other conditions. There are certain laboratory findings useful in differentiating TTP from other disorders, which can have similar clinical presentation (Table 1). The distinction between TTP and HUS, however, can be much more obscure as these two disorders can have similar clinical and laboratory presentations. This distinction, though, is critical as the treatments for TTP and HUS differ significantly. Without prompt initiation of plasmapheresis, there is a 90% 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 has been based largely on a few clinical features. Patients with TTP are more likely to present with neurologic symptoms, while patients with HUS usually have more severe renal dysfunction with anuric renal failure.

While numerous studies have demonstrated a dramatic decrease or absence of vWF-CP activity in patients with TTP, several of these studies have shown that the protease activity is only moderately decreased and often normal in HUS. (3,20-22,24) Thus, measurement of vWF-CP activity has emerged as a valuable diagnostic means to distinguish TTP and HUS. Interestingly, most patients with TTP associated with bone marrow transplantation have normal or only slightly decreased vWF-CP activity. (3,25) vWF-CP activity has been found to be moderately decreased in patients with liver cirrhosis. (26) The most severe deficiencies in vWF-CP activity are in those individuals with decompensated cirrhosis and acute inflammatory states in which the average protease activity is 40% to 50% of normal. This is in comparison to a vWF-CP activity of 0% to 10% of normal in most patients with TTP. Thus, an extremely low or absent vWF-CP activity can still be a valuable criteria in helping to establish a diagnosis of TTP. In addition, the presence or absence of an inhibitor will further differentiate the type of TTP and allow more specific treatment options (Table 2).

vWF-CP assays

vWF-CP activity can be determined by measurement of the ability of the enzyme from 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 [Ba.sup.+2] and urea. (6,19-22) The appearance of the vWF-CP degradation products (dimers of 176-kilodalton fragment and dimers of 140-kilodalton fragment resulting from cleavage at the tyrosine 842-methionine 843 site) were measured by immunoblotting after separation by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Alternatively, the disappearance of the ULvWF multimers could be checked by immunoblotting of fragments separated on a SDS-agarose gel.

A simpler test currently being utilized is the collagen-binding assay. (27) This assay is based on the ability of the largest vWF multimers to selectively bind collagen, compared with smaller forms of vWF. The principle of this assay is that the degradation of the largest vWF multimers by vWF-CP gives rise to the smaller vWF multimers that bind less to collagen. Dilutions of patient's plasma are incubated with normal human plasma in which the protease has been inactivated, and the products resulting from proteolytic degradation of vWF in the normal plasma by vWF-CP in patient's sample are captured on microtiter plates coated with human collagen type III. The vWF multimers bound to the collagen can then be detected with a peroxidase-conjugated antibody to human vWF and quantitated from standard curves. Inhibitor levels are determined by measuring the ability of patient's plasma to inhibit vWF-CP activity in normal human plasma mixed with protease-inactivated patient plasma using the same collagen-binding assay. The reference range for vWF-CP activity is 67% to 177% in this assay. Depending on the reference laboratory, a vWF-CP activity of <6% indicates a severe deficiency, which has a sensitivity of 70% to 100% for the diagnosis of idiopathic TTP. (28) Inhibitor levels are calculated in Bethesda units with a reference interval of [less than or equal to]0.3 inhibitor units in at least one reference laboratory. The collagen-binding assay is reportedly more sensitive in distinguishing very low vWF-CP activity (0% to 5%), while the immunoblot assay measuring the disappearance of large multimers on SDS-agarose gels is more accurate in the normal and moderately low ranges of protease activity. (27) Table 3 depicts our experience in three patients with TTP whose plasmas were tested for the vWF-CP activity and inhibitor by both available methods described above. Comparison of the results shows agreement between the two methods in the measurement of both the protease activity and inhibitor level. The assay for vWF-CP is not yet widely available and is still a relatively lengthy procedure so that it does not influence initial clinical decisions at this time. The assay does, however, currently have a role in the diagnosis and subsequent management of patients with TTP.

Use of vWF-CP in clinical practice

vWF-CP activity levels and the presence or absence of an antibody inhibitor are proving to be valuable laboratory data in establishing a diagnosis in patients with suspected TTP. A vWF-CP activity of 0% to 10% strongly supports a diagnosis of TTP over other TMAs and can differentiate TTP from HUS, even in the absence of other distinguishing clinical features. In cases of suspected TTP, low or absent vWF-CP activity or the presence of an inhibitor can confirm the diagnosis.

A 38-year-old woman presented to our institution with severe thrombocytopenia (platelet count of 12,000/[micro]L), hemolytic anemia with moderate schistocytosis and high LDH (2,020 IU/L). A definitive diagnosis could not be established, and she was treated empirically with plasmapheresis (27 procedures) for presumable TTP. She gradually improved and was discharged approximately one month after admission with normalized laboratory values. During the following two-month period, she required readmission twice. Her only presenting sign during both hospitalizations was severe thrombocytopenia. Work-up for the isolated thrombocytopenia included platelet IgG antibody levels, heparin antibody levels and a bone marrow aspirate/biopsy, which were all nonrevealing.

Finally, prior to starting a new series of plasma exchanges on her third admission, a sample for vWF-CP assay was collected and, subsequently, showed complete absence of protease activity due to the presence of a strong inhibitor. The vWF-CP assay was critical in establishing the diagnosis in this patient with an unusual presentation of recurrent TTP. Following additional plamaphereses and immunosuppressive treatment, she improved dramatically and was discharged with a platelet count of 472,000/[micro]L, stable hemoglobin and no evidence of hemolysis.

vWF-CP assays can also allow differentiation between chronic relapsing TTP due to an intrinsic deficiency of the protease and acquired idiopathic TTP resulting from the presence of an antibody inhibiting the protease. This distinction has important therapeutic implications and may indicate 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 provides vWF-CP. Adults with acute episodes of TTP require daily plasmapheresis in which the patient's plasma is exchanged with fresh frozen plasma. Treatment with plasmapheresis improves survival dramatically (90% survival) in acute TTP presumably by removing ULvWF multimers and replacing vWF-CP. Plasmapheresis may also remove the IgG antibodies against vWF-CP in patients with idiopathic TTP, which could explain some cases of improved response to plasmapheresis in this form of TTP. Unfortunately, the presence of a high titer of antibody inhibitor to vWF-CP is associated with refractoriness to plasmapheresis (29) and may indicate the need for adjuvant therapy. Glucocorticoids, cyclosporine and splenectomy have been used successfully in combination with plasmapheresis in the treatment of TTP, perhaps through suppression of antibody production. More recently, rituximab, a monoclonal antibody against CD20 on B-lymphocytes, has emerged as a potential therapy for refractory TTP.

A recent case report describes a patient with a 19-month history of relapsing TTP, despite treatment with plasmapheresis and multiple other therapies including splenectomy. (30) She was found to have a complete deficiency of vWF-CP activity and the presence of an 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 over a 13-month follow-up period.

Summary

TTP is a rare but potentially fatal disease unless plasmapheresis is initiated promptly. Although most hospitals will only treat such patients sporadically, unless the possibility of TTP is explored, the diagnosis may be missed. In patients with unexplained severe thrombocytopenia (usually less than 10,000 platelets/[micro]L) and hemolytic anemia with schistocytes, TTP must be ruled out. The presence of other clinical and laboratory features as described in Table 1 may aid in the differential diagnosis. Often, however, TTP remains a possibility and the patient should be transferred to a facility where plasmapheresis is available. Further, a sample of citrated plasma should be collected prior to any plasma transfusion, and sent frozen to a reference laboratory for determinations of the vWF-CP activity and the presence of an inhibitor to the enzyme. The clinical laboratory should be aware of this test to help ensure that the sample is correctly collected and shipped. Although the patient should be treated as if having TTP until the result of the tests is available, the assays may help determine the specific diagnosis, as well as the potential need for further therapy as discussed above. We anticipate that continued accumulation of vWF-CP data with clinical correlation will further define the role of this enzyme in the diagnosis and management of patients with TMAs.

CE test on von WILLEBRAND FACTOR-CLEAVING PROTEASE IN THROMBOTIC THROMBOCYTOPENIC PURPURA

MLO and Northern Illinois University (NIU), DeKalb, IL, are co-sponsors in offering continuing education units (CEUs) for this issue's article on von WILLEBRAND FACTOR-CLEAVING PROTEASE IN THROMBOTIC THROMBOCYTOPENIC PURPURA. CEUs or contact hours are granted by the College of Health and Human Sciences at NIU, which has been approved as a provider of continuing education programs in the clinical laboratory sciences by the ASCLS P.A.C.E.[R] program (Provider No. 0001) and by the American Medical Technologists Institute for Education (Provider No. 121019; Registry No. 0061). Approval as a provider of continuing education programs has been granted by the state of Florida (Provider No. JP0000496), and for licensed clinical laboratory scientists and personnel in the state of California (Provider No. 351). Continuing education credits awarded for successful completion of this test are acceptable for the ASCP Board of Registry Continuing Competence Recognition Program. After reading the article on page 10, answer the following test questions and send your completed test form to NIU along with the nominal fee of $20. Readers who pass the test successfully (scoring 70 percent or higher) will receive a certificate for 1 contact hour of P.A.C.E.[R] credit. Participants should allow four to six weeks for receipt of certificates.

The fee for each continuing education test will be $20.

All feature articles published in MLO are peer-reviewed.

This test was prepared by Jeanne M. Isabel, MSEd, CLSpH(NCA), MT(ASCP), associate professor, School of Allied Health Professions, College of Health and Human Sciences, Northern Illinois University, DeKalb, IL.

1. The hematologic disorder involving platelet aggregates in the vasculature is:

a. chediak-higashi disease

b. idiopathic thrombocytopenic purpura

c. may-hegglin anomaly

d. thrombotic thrombocytopenic purpura

2. Occlusive platelet thrombi formed in the organs release large amounts of:

a. CK

b. LDH

c. ALT

d. potassium

3. Erythrocytes that transverse small narrowed vessels are transformed into:

a. acanthocytes

b. codocytes

c. depranocytes

d. schistocytes

4. Clinical features of TTP include thrombocytopenia with platelet counts below:

a. 500/[micro]L

b. 5000/[micro]L

c. 10,000/[micro]L

d. 20,000/[micro]L

5. Hemolytic anemia can often be detected by low or undetectable levels of plasma haptoglobin.

a. True

b. False

6. Familial TTP is the most common form of thrombotic thrombocytopenic purpura.

a. True

b. False

7. The occlusive thrombi of TTP are composed predominantly of:

a. fibrin

b. fibrinogen

c. platelets

d. von Willebrand factor

e. both c and d

8. von Willebrand factor is the carrier protein for coagulation factor:

a. I

b. V

c. VIII

d. IX

9. In normal plasma, von Willebrand factor-cleaving protease (vWF-CP) serves to:

a. cleave the unusually large vWF multimers

b. regulate multimer size

c. avert spontaneous interaction of UL multimers and platelets

d. all of the above

10. Decreased vWF-CP activity in acute idiopathic or acquired TTP is due to:

a. mutations in the vWF-CP gene

b. inhibition by IgG antibodies

c. platelet activators

d. heparin

11. Clinical findings of thrombocytopenia, hemolysis, and schistocytosis are only found in TTP.

a. True

b. False

12. Heparin-induced thrombocytopenia (HIT) can be distinguished from TTP because HIT findings include:

a. platelet antibodies

b. positive DAT

c. no anemia

d. high D-dimers

13. The two disorders with similar clinical presentations and laboratory findings are:

a. TTP and HUS

b. TTP and DIC

c. HUS and DIC

d. HIT and HUS

14. Measurement of vWF-CP has become a valuable diagnostic tool to distinquish:

a. DIC and HUS

b. DIC and TTP

c. DIC and HIT

d. HUS and TTP

15. Different types of TTP can be identified by the presence of an antibody inhibitor. All of the following types have the inhibitor except:

a. acute idiopathic

b. drug associated

c. chronic relapsing

d. recurrent

16. Measurement of vWF-CP activity is currently determined by:

a. collagen-binding assay

b. enzyme immunoassay

c. Southern Blot

d. gel electrophoresis

17. Treatment for TTP includes:

a. blood transfusions

b. plasmapheresis

c. platelet transfusions

d. vitamins with iron

18. A monoclonal antibody against CD20 on B lymphocytes which may be used as potential therapy for refractory TTP is:

a. glucocorticoids

b. cyclosporine

c. rituximab

d. ticlopidine

CLARIFICATION: Please note that the CE test for October 2003 (p. 23) was prepared by Shirley Richmond, PhD, Dean, College of Health and Human Sciences, MT(ASCP), CLS, NCA, Northern Illinois University, DeKalb, IL.

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References

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25. van der Plas RM, Schiphorst ME, Huizinga EG, et al. von Willebrand factor proteolysis is deficient in classic, but not in bone marrow transplantation-associated, thrombotic thrombocytopenic purpura. Blood. 1999;93:3798-3802.

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28. Bianchi V, Robles R, Alberio L, et al. von Willebrand factor-cleaving protease (ADAMTS13) in thrombocytopenic disorders: a severely deficient activity is specific for thrombotic thrombocytopenic purpura. Blood. 2002;100:710-713.

29. Tsai HM. High titers of inhibitors of von Willebrand factor-cleaving metalloproteinase in a fatal case of acute thrombotic thrombocytopenic purpura. Am J Hematol. 2000;65:251-255.

30. Zheng X, Pallera AM, Goodnough LT, et al. Remission of chronic thrombotic thrombocytopenic purpura after treatment with cyclophosphamide and rituximab. Ann Intern Med. 2003;138:105-108.

Charles A. Mayfield, MD, PhD, and Marisa B. Marques, MD, are members of the Department of Pathology at the University of Alabama at Birmingham.

By Charles A. Mayfield, MD, PhD, and Marisa B. Marques, MD

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