Masanori Matsumoto

von Willebrand Factor—Cleaving Protease and Upshaw-Schulman Syndrome

Vascular endothelial cell (EC)-produced plasma von Willebrand factor (vWF) plays a critical role in primary hemostasis through its action of anchoring platelets onto the injured denuded subendothelial matrices under high shear stress. Unusually large vWF multimers (UL-vWFMs), present in plasma immediately after release from ECs, are most biologically active, but they are soon cleaved and degraded into smaller vWFMs by a specific plasma protease, termed vWF-cleaving protease (vWF-CPase), in normal circulation. Recent studies on the relationship between UL-vWFMs and vWF-CPase, together with its autoantibody (inhibitor) have brought about a clear discrimination between thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. Furthermore, a congenital deficiency of this enzyme activity has been shown to cause Upshaw-Schulman syndrome, a complex constitutional bleeding diathesis. Successful purification of vWF-CPase revealed that this enzyme is composed of a single polypeptide with a molecular mass of approximately 190 kd, and its complementary DNA cloning unambiguously indicated that it is uniquely produced in the liver and its gene is located on chromosome 9q34. The messenger RNA of vWF-CPase had a span of 4.6 kb, and its enzyme was designated ADAMTS 13. The predicted complete amino acid sequence of this enzyme consisted of 1427 residues, including a signal peptide, a short propeptide terminating in the sequence RQRR, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin-1 repeat (TSP1), a cysteine-rich domain, an ADAMTS spacer, 7 additional TSP1 repeats, and 2 CUB domains.

Impaired activity of plasma von Willebrand factor-cleaving protease may predict the occurrence of hepatic veno-occlusive disease after stem cell transplantation.

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.

Prophylactic fresh frozen plasma may prevent development of hepatic VOD after stem cell transplantation via ADAMTS13-mediated restoration of von Willebrand factor plasma levels.

We initially conducted a multicenter, randomized trial (n=43), and subsequently a questionnaire study (n=209) of participating hospitals, to evaluate whether infused fresh frozen plasma (FFP) could prevent the occurrence of hepatic veno-occlusive disease (VOD) after stem cell transplantation (SCT). Forty-three patients were divided into two groups: 23 receiving FFP infusions and 20 not receiving it. VOD developed in three patients not receiving FFP. Plasma von Willebrand factor (VWF) antigen levels were lower at days 0, 7 and 28 after SCT in patients receiving FFP than in those not receiving it, whereas plasma ADAMTS13 activity (ADAMTS13:AC) did not differ between them. Plasma VWF multimer (VWFM) was demonstrated to be defective in the high∼intermediate VWFM during the early post-SCT phase, but there was a significant increase in high VWFM just before VOD onset. This suggests that a relative enzyme-to-substrate (ADAMTS13/high-VWFM) imbalance is involved in the pathogenesis of VOD. To strengthen this hypothesis, the incidence of VOD was apparently lower in patients receiving FFP infusions than in those not receiving it (0/23 vs 3/20) in the randomized trial. Further, the results combined with the subsequent questionnaire study (0/36 vs 11/173) clearly showed the incidence to be statistically significant (0/59 vs 14/193, P=0.033).

Platelets Treated with Ticlopidine Are Less Reactive to Unusually Large von Willebrand Factor Multimers than Are Those Treated with Aspirin under High Shear Stress

Much attention has recently been focused on the interaction between unusually large von Willebrand factor multimers (UL-VWFM) and platelets under high shear stress in pathological thrombus formation. The antiplatelet drugs acetylsalicylic acid (aspirin) and a thienopyridine derivative (ticlopidine) are commonly used to treat cerebral ischemia but exert different effects on high-shear-stress-induced platelet aggregation (H-SIPA) in the plasma. To examine the effects of these drugs in the absence of plasma factors, we studied H-SIPA using washed platelets (WPs) and purified UL-VWFM. WPs were prepared from the blood of 9 aspirin-treated and 11 ticlopidine-treated patients with cerebral ischemia, and H-SIPA in the presence of UL-VWFM was measured using a cone plate aggregometer. Plasma levels of VWF antigen with its multimer analysis, ristocetin cofactor and VWF-cleaving protease (ADAMTS13) activity were also measured. Forty-six healthy volunteers from 2 age groups, 20–40 years (n = 20) and 41–60 years old (n = 26), were also tested as controls. H-SIPA was significantly inhibited for ticlopidine-treated platelets, but it was observed to a lesser extent for aspirin-treated platelets. For both groups, no difference in the plasma levels of VWF antigen, ristocetin cofactor and ADAMTS13 activity was noted. All patients possessed UL-VWFM, and it was detected in healthy volunteers with increasing frequency with increasing age. Under plasma-free conditions, platelets from aspirin-treated patients exhibit marginal but significant inhibition of H-SIPA. Furthermore, the presence of UL-VWFM in the plasma of patients and normal volunteers is directly related to their age rather than being a consequence of underlying disease.

Low-Dosage Intravenous Immunoglobulin in the Management of a Patient with Acquired von Willebrand Syndrome Associated with Monoclonal Gammopathy of Undetermined Significance

We report herein the case of a 69-year-old Japanese man with acquired von Willebrand syndrome associated with monoclonal gammopathy of undetermined significance who developed IgG1-ĸ antibodies against von Willebrand factor (VWF). The patient was urged to undergo tooth extractions because of alveolar pyorrhea, and a low-dosage intravenous immunoglobulin (IV-Ig) therapy (0.3 g of IgG/kg/day for 3 days) was chosen for him. On the 4th day after the infusion, VWF antigen and VWF ristocetin cofactor increased to 40 and 78% of the control, respectively, and dental extractions were performed successfully. On the 7th day, these values reached a maximum, i.e. 95 and 160% of the control, respectively. Then, they quickly decreased to 35 and 75% on the 10th day, and 6 months later, they became 16 and <3% of the control, respectively. Upon analysis of plasma VWF multimers (VWFMs) in this patient, those with large to medium molecular masses more selectively disappeared before the IV-Ig infusion than did those with small molecular masses. On the 4th day, the pattern of VWFMs was completely normalized and appeared to persist until the 10th day. Six months later, a small amount of large to medium-sized VWFMs was still present, but at 7–8 months, the pattern of VWFMs became almost the same as that before infusion. Throughout the patient’s clinical course, the activity of plasma VWF-cleaving protease, which specifically cleaves the Tyr842-Met843 bond of the subunit and reduces its multimeric sizes, was quite normal (95–119%). These results provided consistent evidence that the selective absence of VWFMs with large to medium molecular masses in this patient is caused by the heightened clearance of a complex of IgG inhibitor and VWFMs from the circulation, presumably through IgG binding to the Fc receptor of macrophages. Furthermore, these results also indicated that a low-dosage IV-Ig therapy is effective enough for hemostatic management for programmed surgery.

Diffuse neurodeficits in intravascular lymphomatosis with ADAMTS13 inhibitor

Inhibitory antibody to von Willebrand factor (vWF)-cleaving protease (ADAMTS13) was detected in a patient with intravascular lymphomatosis. The increased serum level of the antibody paralleled an increase in the expression of uncleaved vWF, which might cause microvascular thrombosis and platelet consumption. Malignant cell proliferations with superimposed thrombosis within the lumina throughout the entire vasculature account for diffuse neurodeficits observed in the patient.

Simple plasma exchange reduced autoantibody to von Willebrand factor-cleaving protease in a Japanese man with ticlopidine-associated thrombotic thrombocytopenic purpura

DE A R SI R, Ticlopidine has been used in the prophylactic treatment of patients with thrombotic stroke or following coronary artery stent placement all over the world. Thrombotic thrombocytopenic purpura (TTP) is characterized by extensive platelet thrombi in the arterioles and capillaries, which has been associated with 1/1600 to 1/4800 patients receiving ticlopidine [1, 2]. Recently, autoantibody to von Willebrand factor-cleaving protease (vWFCPase) has been discovered in seven patients with ticlopidine-associated TTP [3] as well as patients with idiopathic TTP described before [4, 5]. We report a Japanese patient with ticlopidine-associated TTP who had autoantibody to vWF-CPase.

Persistent Gastrointestinal Angiodysplasia in Heyde's Syndrome after Aortic Valve Replacement

A 67-year-old woman with recurrent gastrointestinal bleeding and aortic valve stenosis presented with spurting bleeding from angiodysplasia of the upper jejunum. As electrophoresis revealed decreased levels of high-molecular-weight (HMW) von Willebrand factor (VWF) multimers, she was diagnosed with Heydes syndrome. After aortic valve replacement, her HMW VWF levels quickly recovered to normal, and the gastrointestinal bleeding ceased. However, capsule endoscopy still revealed gastrointestinal angiodysplasia six months later. This case shows that minute analyses of VWF multimers enable the diagnosis and confirmation of the resolution of Heydes syndrome, and implies that gastrointestinal angiodysplasia can be attributed to unknown factors other than decreased VWF multimers.