Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura

Abstract

Background: Thrombotic thrombocytopenic purpura is a potentially fatal disease characterized by widespread platelet thrombi in the microcirculation. In the normal circulation, von Willebrand factor is cleaved by a plasma protease. We explored the hypothesis that a deficiency of this protease predisposes patients with thrombotic thrombocytopenic purpura to platelet thrombosis.

Methods: We studied the activity of von Willebrand factor-cleaving protease and sought inhibitors of this protease in plasma from patients with acute thrombotic thrombocytopenic purpura, patients with other diseases, and normal control subjects. We also investigated the effect of shear stress on the ristocetin cofactor activity of purified von Willebrand factor in the cryosupernatant fraction of the plasma samples.

Results: Thirty-nine samples of plasma from 37 patients with acute thrombotic thrombocytopenic purpura had severe deficiency of von Willebrand factor-cleaving protease. No deficiency was detected in 16 samples of plasma from patients with thrombotic thrombocytopenic purpura in remission or in 74 plasma samples from normal subjects, randomly selected hospitalized patients or outpatients, or patients with hemolysis, thrombocytopenia, or thrombosis from other causes. Inhibitory activity against the protease was detected in 26 of the 39 plasma samples (67 percent) obtained during the acute phase of the disease. The inhibitors were IgG antibodies. Shear stress increased the ristocetin cofactor activity of von Willebrand factor in the cryosupernatant of plasma samples obtained during the acute phase, but decreased the activity in cryosupernatant of plasma from normal subjects.

Conclusions: Inhibitory antibodies against von Willebrand factor-cleaving protease occur in patients with acute thrombotic thrombocytopenic purpura. A deficiency of this protease is likely to have a critical role in the pathogenesis of platelet thrombosis in this disease.

Figure 1

Von Willebrand Factor–Cleaving Protease Activity in Plasma Samples from 37 Patients with Thrombotic Thrombocytopenic Purpura and 74 Control Subjects. The protease activity in each sample was reflected by the intensity of the 350-kd band generated from purified von Willebrand factor and expressed as a percentage of the activity in control plasma. Panel A shows the protease activity of 39 plasma samples obtained during acute episodes of thrombotic thrombocytopenic purpura (TTP) before plasma-exchange therapy, 23 samples obtained after therapy, and 16 samples obtained during remission. Diamonds represent five samples, and circles one sample. Panel B shows the protease activity of plasma samples obtained from 35 randomly selected subjects without thrombotic thrombocytopenic purpura, 21 patients with miscellaneous autoimmune or blood disorders, and 18 patients with heparin-induced thrombocytopenia. Panel C shows the protease activity of plasma samples obtained from seven patients with thrombotic thrombocytopenic purpura during both an acute episode and remission. Panel D shows a composite immunoblot of 200-kd, 350-kd, and larger fragments generated from purified von Willebrand factor in control plasma (lanes 1 and 2) but not in plasma samples from two patients with acute thrombotic thrombocytopenic purpura (lanes 3, 4, 5, and 6). Each sample was assayed in duplicate. EDTA was absent from the buffer in lanes 1, 3, and 5 and present in lanes 2, 4, and 6.

Figure 2

Detection of an Inhibitor of von Willebrand Factor–Cleaving Protease. In Panel A, von Willebrand factor–cleaving protease activity was measured in a mixture of control plasma and an equal volume of a plasma sample from 21 patients with miscellaneous autoimmune or blood disorders, 37 patients with acute thrombotic thrombocytopenic purpura (39 samples), and 16 patients with thrombotic thrombocytopenic purpura in remission. Panel B shows the results of autoradiography. Von Willebrand factor was cleaved in control plasma incubated with heated control plasma (lanes 3 and 4), whereas there was minimal cleavage in control plasma incubated with a plasma sample from a patient with thrombotic thrombocytopenic purpura (TTP) (lanes 1 and 2). EDTA was absent from the buffer in lanes 1 and 3 and present in lanes 2 and 4. In Panel C, protease activity was measured in 3:1 mixtures of various dilutions of a plasma sample from a patient with thrombotic thrombocytopenic purpura and control plasma.

Figure 3

Inhibitory Activity of IgG from 12 Patients with Acute Thrombotic Thrombocytopenic Purpura (TTP) and from 11 Patients after Plasma-Exchange Therapy Was Instituted as a Function of the Inhibitory Activity of Plasma Samples from Which IgG Was Isolated. IgG antibody was isolated from plasma samples from the patients and mixed with control plasma, and protease activity was plotted against the protease activity measured in the mixture of control plasma and the patients’ plasma samples.

Figure 4

Inhibition of von Willebrand Factor–Cleaving Protease Activity by an IgG Antibody Isolated from Plasma from a Patient with Thrombotic Thrombocytopenic Purpura (TTP) (Panel A) and Neutralization of the Inhibitory Activity by the Antibody against Human Fab Region (Panel B). In Panel A, the protease activity of control plasma and IgG antibody isolated from the patient was plotted against the final IgG concentration in the mixture (log scale). The protease activity of the mixture of control plasma and control IgG is also shown. In Panel B, the protease activity of a mixture of control plasma, IgG antibody isolated from the patient, and IgG antibodies to the Fab region (anti-Fab) or the Fc region (anti-Fc) was plotted against the ratio of anti-Fab or anti-Fc to IgG in the mixture (log scale).

Figure 5

Effects of Shear Stress on the Proteolysis of Purified von Willebrand Factor Multimers. Purified von Willebrand factor was dissolved in the cryosupernatant of control plasma or a plasma sample from a patient with thrombotic thrombocytopenic purpura (TTP). Panel A shows the sizes of the von Willebrand factor multimers on agarose-gel electrophoresis in the cryosupernatants in the presence and absence of shear stress. Panel B shows the frequency of the cleaved 350-kd and 200-kd fragments on polyacrylamide-gel electrophoresis and immunoblotting in the presence and absence of shear stress. Shear stress at a rate of 6404 per second decreased the size of the multimers and increased the numbers of cleaved fragments only in control cryosupernatant. The von Willebrand factor in the cryosupernatant of control plasma had an increase in the normalized peak distance (Panel C) and the amount of 350-kd fragments (Panel D) that was dependent on the rate of shear stress. A normalized peak distance of more than 1 indicates that the size of the multimers was decreased as compared with that measured in the absence of shear stress. The amount of 350-kd fragments was expressed as a percentage of the amount in the absence of shear stress.

Figure 6

Effect of Shear Stress on Ristocetin Cofactor Activity of Purified von Willebrand Factor. Shear stress decreased the ristocetin cofactor activity of von Willebrand factor in the cryosupernatant of control plasma, but increased it in the cryosupernatant of plasma from a patient with thrombotic thrombocytopenic purpura (TTP). The ristocetin cofactor activity is expressed as a percentage of the activity in the absence of shear stress.