Assay of von Willebrand Factor (vWF)-cleaving Protease Based on Decreased Collagen Binding Affinity of Degraded vWF

Summary

Patients with thrombotic thrombocytopenic purpura (TTP) have a deficiency of von Willebrand factor (vWF)-cleaving protease, whereas patients with hemolytic-uremic syndrome (HUS) show normal activity of this protease. Present methods for assaying vWF-cleaving protease by immunoblotting are time-intensive and cumbersome. We therefore developed a new functional assay based on the preferential binding of high-molecular-weight forms of vWF to collagen. In this assay, the diluted plasma sample to be tested is added to normal human plasma in which protease activity had been abolished. The vWF present in the protease-depleted plasma is digested by the vWF-cleaving protease in the test plasma. The proteolytic degradation leads to low-molecular-weight forms of vWF, which show impaired binding to microtiter plates coated with human collagen type III. The collagen-bound vWF is quantified using a peroxidase-conjugated rabbit antibody against human vWF. The values of vWF-cleaving protease activity in tested plasma samples are read from a calibration curve achieved by incubating the vWF-substrate with dilutions of a normal human plasma pool (NHP). Testing of plasma from patients with TTP and HUS showed that the assay can be used to distinguish between these two syndromes. The presence of an inhibitor can be detected by carrying out the test after incubation of NHP with the patient plasma sample, thus enabling differentiation of patients with familial TTP from those with non-familial TTP.

Keywords

von Willebrand factor-cleaving protease - collagen binding assay - TTP - HUS

• References

• 1 Neild GH. Hemolytic uremic syndrome/thrombotic thrombocytopenic purpura: Pathophysiology and treatment. Kidney Int 1998; 53 (Suppl. 64) S45-9.
• 2 Moake JL, Rudy CK, Troll JH, Weinstein MJ, Colannino NM, Azocar J, Seder RH, Hong SL, Deykin D. Unusually large plasma factor VIII: von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura. N Engl J Med 1982; 307: 1432-5.
• 3 Mannucci PM, Lombardi R, Lattuada A, Ruggenenti P, Vigano GL, Barbui T, Remuzzi G. Enhanced proteolysis of plasma von Willebrand factor in thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome. Blood 1989; 74: 978-83.
• 4 Chow TW, Turner NA, Chintagumpala M, McPherson PD, Nolasco LH, Rice L, Hellums JD, Moake JL. Increased von Willebrand factor binding to platelets in single episode and recurrent types of thrombotic thrombocytopenic purpura. Am J Hematol 1998; 57: 293-302.
• 5 Moake JL, Byrnes JJ, Troll JH, Rudy CK, Weinstein MJ, Colannino NM, Hong SL. Abnormal VIII: von Willebrand factor patterns in the plasma of patients with the hemolytic-uremic syndrome. Blood 1984; 64: 592-8.
• 6 Furlan M, Robles R, Lämmle B. Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis. Blood 1996; 87: 4223-34.
• 7 Tsai H-M. Physiologic cleavage of von Willebrand factor by a plasma protease is dependent on its conformation and requires calcium ion. Blood 1996; 87: 4235-44.
• 8 Furlan M, Robles R, Solenthaler M, Wassmer M, Sandoz P, Lämmle B. Deficient activity of von Willebrand factor-cleaving protease in chronic relapsing thrombotic thrombocytopenic purpura. Blood 1997; 89: 3097-103.
• 9 Furlan M, Robles R, Solenthaler M, Lämmle B. Acquired deficiency of von Willebrand factor-cleaving protease in a patient with thrombotic thrombocytopenic purpura. Blood 1998; 91: 2839-46.
• 10 Furlan M, Robles R, Galbusera M, Remuzzi G, Kyrle PA, Brenner B, Krause M, Scharrer I, Aumann V, Mittler U, Solenthaler M, Lämmle B. Von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome. N Engl J Med 1998; 339: 1578-84.
• 11 Tsai H-M, Lian EC-Y. Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura. N Engl J Med 1998; 339: 1585-94.
• 12 Santoro SA. Preferential binding of high molecular weight forms of von Willebrand factor to fibrillar collagen. Biochim Biophys Acta 1983; 756: 123-6.
• 13 Brown JE, Bosak JO. An ELISA test for the binding of von Willebrand antigen to collagen. Thromb Res 1986; 43: 303-11.
• 14 Duggan MJ, DiMichele DM, Christian MJ, Fink LM, Hathaway WE. Collagen-binding of von Willebrand’s factor antigen in the classification of von Willebrand’s disease. Am J Clin Pathol 1987; 88: 97-102.
• 15 Gilchrist M, Stewart MW, Etches WS, Gordon PA. Rapid diagnosis of von Willebrand’s disease using ELISA technology. Thromb Res 1990; 57: 659-64.
• 16 Siekmann J, Turecek PL, Schwarz HP. The determination of von Willebrand factor activity by collagen binding assay. Haemophilia 1998; 4 (Suppl. 03) 15-24.
• 17 Favaloro EJ, Dean M, Grispo L, Exner T, Koutts J. Von Willebrand’s disease: use of collagen binding assay provides potential improvement to laboratory monitoring of desmopressin (DDAVP) therapy. Am J Hematol 1994; 45: 205-11.
• 18 Chang P, Aronson DL. Plasma derived von Willebrand factor preparations: collagen binding and ristocetin cofactor activities. Thromb Haemost 1997; 78: 930-3.
• 19 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
• 20 Berkowitz SD, Dent J, Roberts J, Fujimura Y, Plow EF, Titani K, Ruggeri ZM, Zimmerman TS. Epitope mapping of the von Willebrand factor subunit distinguishes fragments present in normal and type IIA von Willebrand disease from those generated by plasmin. J Clin Invest 1987; 79: 524-31.