Hepatic veno-occlusive disease (VOD) is a life-threatening complication after stem cell transplantation (SCT), characterized by thrombus formation in hepatic venules leading to a symptom triad of hyperbilirubinemia, hepatomegaly, and ascites. Multifactorial defects in the hemostatic system may contribute to its pathogenesis, but its remains to be investigated. Unusually large VWF multimers (UL-VWFMs), produced in and released from vascular endothelial cells, are most biologically active in the interaction with platelets under a high shear stress. UL-VWFMs are cleaved and degraded into smaller VWFMs by a specific liver producing plasma protease, termed VWF-cleaving protease (VWF-CPase), which has recently been identified as a metalloprotease solely produced in liver, termed ADAMTS13. Herein, we studied the correlation between plasma VWF-CPase activity and UL-VWFMs in 21 patients who received SCT, seven patients with VOD and 14 patients without VOD. In non-VOD patients, activities (mean ± 1s.d.) of VWF-CPase were 78 ± 17% of the control before the conditioning regimen, 76 ± 18% on day 0, 64 ± 19% on day 7, 57 ± 23% on day 14, 68 ± 13% on day 21 and 79 ± 19% on day 28 after SCT. The respective values in VOD patients were 32 ± 19%, 27 ± 15%, 18 ± 11%, 22 ± 18%, 26 ± 22% and 12 ± 4%. Thus, VWF-CPase activity was significantly reduced in VOD patients, even before the conditioning regimen, and such a difference was not found in other laboratory tests. However, despite such a clear difference, UL-VWFMs were present in plasmas of both patient groups, together with the increase of VWF antigen and ristocetin cofactor activity. These results indicate that the measurement of this enzyme activity is extremely useful in predicting the occurrence of VOD prior to a demonstration of its direct involvement in its pathogenesis.
Hepatic veno-occlusive disease (VOD) is a hazardous complication which occurs within 30 days after hematopoietic stem cell transplantation (SCT).1,2,3,4,5,6 VOD is characterized by thrombus formation in hepatic venules leading to a symptom triad of hyperbilirubinemia, hepatomegaly, and ascites.1,2,3,4,5,6 Although the etiology of VOD remains unclear, it is assumed that injured endothelium lining sinusoids and pores connected to the hepatic venular lumen and the unbalanced hemostatic system may contribute to its pathogenesis.6 The frequency of VOD has been reported to vary by different researchers,2,3,5 because the diagnostic criteria proposed by McDonald et al1 and Jones et al4 on the basis of the symptom triad mentioned above is liberal.
von Willebrand factor (VWF) is a macromolecular plasma glycoprotein, and plays an essential role in primary hemostasis.7,8 VWF is also known as a marker of endothelial cell activation and an acute phase reactant,9,10,11 and thus its plasma level remains high over several months after hematopoietic SCT9,10,11. However, the relationship between an increased VWF level after SCT and thromboembolic complications including VOD remains to be investigated.
Unusually large VWF multimers (UL-VWFMs), produced in and released from vascular endothelial cells, have often been found in plasma of patients with congenital or acquired thrombotic thrombocytopenic purpura (TTP), a multiorgan disorder characterized by Moschcowitz's pentad of microangiopathic hemolytic anemia (MAHA), thrombocytopenia, fluctuating neurological symptom, renal dysfunction, and fever.12,13,14 Those UL-VWFMs are assumed to interact with circulating platelets and lead to platelet clumping under an elevated high shear stress.14,15 In the normal circulation, however, UL-VWFMs are rapidly degraded into smaller VWFMs by a specific plasma protease termed VWF-cleaving protease (VWF-CPase), which splits the Tyr842-Met843 bond of the subunit.15,16 Most recently, this enzyme has been purified as a single-chain glycoprotein with a molecular mass of ∼150 kDa by two different groups of investigators.17,18 Northern blotting based on the N-terminal amino acid sequence of this purified enzyme revealed that the full-length (4.6 kb) mRNA is only expressed in liver, and cDNA sequencing identified this enzyme as a new member of the metalloproteases belonging to the ADAMTS (a disintegrin-like domain, and metalloproteinase, with thrombospondin type-1 motif) family, designated ADAMTS13.18,19,20 The deduced amino acid residue number of this enzyme is 1427, and its gene is located on chromosome 9q34.18,19,20
We have studied two patient groups, with VOD and without VOD after SCT, with a special reference to plasma VWF-CPase activity and UL-VWFMs. VWF-CPase activity was significantly reduced in VOD patients throughout their clinical course, but it was almost at the normal level in non-VOD patients. The UL-VWFMs, however, were present in plasma of both the groups of patients collected on day 28 after SCT, suggesting that the amount of UL-VWFMs released into circulation was increased beyond that of plasma VWF-CPase activity in both the groups. The imbalance of a larger amount of UL-VWFMs over VWF-CPase activity may contribute in part to the pathogenesis of VOD.
Patients and methods
Subjects enrolled in this study were 21 patients (16 boys and five girls) from 1 to 15 years of age (mean 6 years), who received SCT from October 1993 to December 1999 in the Department of Pediatrics at Osaka Medical Center and Research Institute for Maternal and Child Health or at Nara Medical University. Those patients who were consecutively chosen, were diagnosed as ALL (n = 11), CML (n = 3), severe aplastic anemia (SAA) (n = 2), AML (n = 2), juvenile chronic myelogenous leukemia (JCML) (n = 1), chronic granulomatous disease (CGD) (n = 1) and neuroblastoma (n = 1) (Table 1). Of them, 17 patients received allogeneic bone marrow transplantation (BMT) (from 10 HLA-identical siblings, six HLA-matched unrelated donors and one member of an HLA-mismatched family), two received autologous BMT and two received cord blood stem cell transplantation (CBSCT) (from one HLA-mismatched sibling and one unrelated donor). For the conditioning regimen, total body irradiation (TBI) of 12 Gy + thiotepa (TEPA) (600 mg/m2) + melphalan (LPAM) (140 mg/m2) was conducted in nine patients, TBI 12 Gy + LPAM (210 mg/m2) in three, TBI 12 Gy + TEPA (800 mg/m2) in one, thoraco-abdominal irradiation (TAI) 10 Gy + antithymoglobulin (ATG) (10 mg/kg) + cyclophosphamide (CY) (200 mg/kg) in one. Busulphan (BU) (16 mg/kg) + CY (200 mg/kg) was administered to four patients, BU (16 mg/kg) + LPAM (210 mg/m2) to two and ATG (10 mg/kg) + CY (200 mg/kg) to one. Eleven received BMT from HLA-matched siblings or HLA-mismatched family members and were treated with cyclosporin A (CsA) alone or with additional methotrexate (MTX) as a prophylaxis for acute graft-versus-host-disease (GVHD), but two of them developed severe (grade III–IV) acute GVHD. Six with BMT from HLA-matched unrelated donors were treated with FK506, but three developed severe acute GVHD. Two with CBSCT from HLA-mismatched donors were treated with FK506, and did not develop severe acute GVHD. Heparin (100 U/kg/day) was continuously infused into all the patients from −8 to 30 days after SCT to prevent thrombotic complications. A diagnosis of VOD was made based on the criteria of McDonald et al.1 Citrated plasma from these patients was consecutively collected before the conditioning regimen and on days 0, 7, 14, 21 and 28 after SCT and stored in aliquots at −80°C until use.
The following assays were performed: VWF antigen (VWF:Ag),21 ristocetin cofactor (VWF:Rco),22 SDS–1.2% agarose gel electrophoresis,23 and luminographic detection24 of VWFM using Kodak XRP–1 film and Renaissance kit (NEN Life Science, Boston, MA, USA).
VWF-CPase activity and its inhibitor
Plasma VWF-CPase activity was assayed by the modified method of Furlan et al25 based on VWFM analysis, as described in recent publications.26,27,28 One hundred percent of VWF-CPase activity was defined as the amount contained in 1 ml of pooled normal plasma (NP). Detection limit of VWF-CPase activity in this assay was ∼3%,26,27 and that of the normal range (n = 60; 30 females and 30 males, 20–39 years of age) was 102 ± 23% (mean ± 1 s.d.).28 The inhibitor activity against VWF-CPase was measured using the heat-inactivated patient plasma or IgGs purified with a protein A sepharose CL–6B column as described.29,30 One unit of the inhibitor was defined as the amount destroying by 50% of VWF-CPase activity of the control based on the Bethesda method,31 originally developed to measure factor VIII inhibitor.
The difference of plasma VWF-CPase activity between VOD and non-VOD patients was remarkable (Figure 1a). In non-VOD patients, the level of plasma VWF-CPase activity was within the normal range or slightly decreased throughout the clinical course after SCT. A representative VWF-CPase activity in a non-VOD patient is shown in Figure 1b. Mean values of VWF-CPase activity (mean ± 1 s.d.) in 14 non-VOD patients were: 78 ± 17% at the pre-conditioning stage, 76 ± 18% on day 0, 64 ± 19% on day 7, 57 ± 23% on day 14, 68 ± 13% on day 21 and 79 ± 19% on day 28 after SCT. The inhibitor titers against VWF-CPase activity in these plasmas were uniformly <0.1 U/ml throughout the clinical course. In VOD patients, however, VWF-CPase activity was remarkably reduced throughout the clinical course after SCT. A representative VWF-CPase activity in a VOD patient is shown in Figure 1b. Mean values of VWF-CPase activity (mean ± 1 s.d.) in seven non-VOD patients were: 32 ± 19% in the pre-conditioning stage, 27 ± 15% on day 0, 18 ± 11% on day 7, 22 ± 18% on day 14, 26 ± 22% on day 21 and 12 ± 4% on day 28 after SCT. Throughout the clinical course, the enzyme activity in VOD patients was statistically significantly lower than that in non-VOD patients (Mann–Whitney U test: P <0.01). The inhibitor titer against VWF-CPase activity in plasmas of VOD patient was also uniformly <0.1 U/ml throughout the course. Furthermore, all the purified IgGs (3.4∼6.2 mg/ml, final) from these VOD patient plasmas showed no significant inhibition on VWF-CPase activity.
Despite a significant decrease of VWF-CPase activity in VOD patients, SDS–1.2% agarose gel electrophoretic analysis of both the groups of patient plasma, collected on day 28 after SCT, showed a high frequency of the presence of UL-VWFMs, which was almost indistinguishable from one another (Figure 2). Levels of VWF:Ag and VWF:Rco in these patient plasmas were also very high as compared to the normal plasma (Figure 2).
Results by routine laboratory tests of the patients enrolled in this study that included hemolytic markers are shown in Table 2. A significantly higher level of T-Bil was observed on days 21 and 28 after SCT in VOD patients. However, the other results including the platelet count were not statistically significantly different between the two patient groups. Furthermore, no schistocytes were found in the specimens of peripheral blood from either patient group (data not shown).
The high mortality of VOD after SCT has remained almost unchanged in the past few decades, despite the introduction of anticoagulant therapy by heparin with or without antithrombin.32 Several other therapeutic approaches are currently under investigation. In this regard, two reports by Bearman et al33 and Espigado et al34 are particularly interesting. They describe a reversal of severe hepatic VOD after a bolus infusion of recombinant tissue plasminogen activator (rt-PA) or by a combined therapy of plasma exchange and the rt-PA. These results suggest that thrombi formed in hepatic venules play an important role in the pathogenesis of VOD.
On the other hand, we recently reported that a deficient activity of plasma VWF-CPase in patients with congenital biliary atresia with cirrhosis can be fully restored by living-related liver transplantations,35 and suggested that the liver is a major organ for synthesizing plasma VWF-CPase.35 This speculation became consistent with more recent reports on the cDNA cloning of this enzyme, which demonstrated that its mRNA is uniquely expressed in the liver.19,20 Along these lines, we have clearly shown that VWF-CPase activity is significantly reduced in VOD patients even before the conditioning regimen, but it is in the almost normal range in non-VOD patients (32 ± 19% in VOD vs 78 ± 17% in non-VOD). Furthermore, on day 28 after SCT, the most striking difference in enzyme activity between the two groups has been observed (12 ± 4% in VOD vs 79 ± 19% in non-VOD). Comparing the two patient groups on the clinical side, the majority of VOD patients were refractory or relapsed and had been treated with intensive cytoreductive therapy just before the conditioning regimen. Furthermore, all but one donor was unrelated to the VOD group, but none of them was unrelated to the non-VOD group. A high-risk conditioning regimen with TBI, TEPA and LPAM was given to six out of seven VOD patients, but three out of 14 non-VOD patients. These differences, in fact, reflect the following results: four out of seven VOD patients suffered from acute severe (grade III–IV) GVHD as compared to only one out of 14 non-VOD patients. Thus, it is likely that the cytoreductive drugs concentrated in the liver during therapy for SCT impair liver tissue more selectively than other organs.
Of several liver-produced enzymes, the reason why VWF-CPase activity is more impaired at the early stage of therapy in the VOD group is presently unknown. Despite a clear difference in VWF-CPase activity between the two patient groups, UL-VWFMs are present in almost all plasmas of both groups. This result is in sharp contrast with the cases of congenital TTP, termed Upshaw–Schulman syndrome (USS), in which a ∼10% increase of plasma VWF-CPase activity by the infusion of fresh frozen plasma totally eliminates UL-VWFMs.26,27 Furthermore, in both patient groups, plasma VWF:Ag and VWF:Rcof levels are significantly high. This indicates that the relative amount of UL-VWFMs released into circulation is increased beyond that of plasma VWF-CPase activity in both the groups. A lower ratio of the enzyme to the substrate, namely VWF-CPase: UL-VWFMs, is presumably not the only reason for the pathogenesis of VOD, but may contribute to it. In fact, no significant difference in hemolytic markers except for total bilirubin are found between these two groups. Furthermore, platelet counts between the two groups could not be properly evaluated, because platelet concentrates were regularly transfused into these patients to prevent bleeding due to SCT-associated thrombocytopenia. Thus, again it should be emphasized that VOD is caused by multifactorial defects in hemostatic system.1 The use of cytoreductive drugs and TBI apparently induces the destruction of antithrombotic function of vascular endothelial cells that accelerates disease progression. Further investigation will be required to address these issues. Nevertheless, our present data indicate that VWF-CPase activity can be used as the best predictor for the occurrence of VOD, known to date.
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We would like to thank Professor Emeritus Koiti Titani (Fujita Health University) for his encouragement and critical reading of this manuscript. The work was supported in part by Grants-in-Aid for Scientific Research (C) from the Japanese Ministry of Education Culture and Science (to YF and MM) and for Blood Coagulation Abnormalities from the Ministry of Health and Welfare of Japan (to YF).
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Park, Y., Yoshioka, A., Kawa, K. et al. Impaired activity of plasma von Willebrand factor-cleaving protease may predict the occurrence of hepatic veno-occlusive disease after stem cell transplantation. Bone Marrow Transplant 29, 789–794 (2002) doi:10.1038/sj.bmt.1703544
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