Effect of von Willebrand disease type 2B and type 2M mutations on the susceptibility of von Willebrand factor to ADAMTS-13.

BACKGROUND: von Willebrand disease (VWD) type 2 is associated with mutations in von Willebrand factor (VWF) that affect its secretion, multimeric pattern, affinity for platelet receptors and clearance of the protein. While increased proteolysis by a disintegrin-like and metalloprotease with thrombospondin type 1 motifs-13 (ADAMTS-13) has been clearly established for VWF type 2A, only little is known about VWF types 2B and 2M in this regard. OBJECTIVES: Sensitivity of wild-type (WT) and mutated recombinant (r) VWF to proteolysis by ADAMTS-13 was investigated to better understand the role of this process in the pathophysiology of VWD. METHODS: We used human rADAMTS-13-WT to digest 11 full-length recombinant forms of VWF carrying molecular abnormalities identified in patients with VWD type 2A (E1638K and P1648S), type 2B (InsM1303, R1306W, R1308P and V1314F) and type 2M (G1324A, E1359K, K1362T, R1374H and I1425F). RESULTS: Using low ionic strength conditions, all mutations induced increased proteolysis of rVWF by rADAMTS-13 as compared with rVWF-WT. The susceptibility of mutants decreased in the following order: type 2A > type 2B > type 2M > rVWF-WT. At physiological salt concentration (150 mm NaCl) the sensitivity of all rVWF to rADAMTS-13 was significantly decreased. However, type 2A and type 2B mutants still exhibited a significantly higher susceptibility to rADAMTS-13 than rVWF-WT, whereas type 2M mutants normalized. CONCLUSIONS: Type 2M mutants and rVWF-WT exhibit a similar sensitivity to rADAMTS-13-mediated proteolysis, in agreement with the normal multimeric pattern in vivo. In VWD type 2B, the spontaneous binding to platelets and excessive degradation by ADAMTS-13 of VWF high-molecular-weight multimers may account for their clearance from plasma.

Mapping and functional studies of two alloantibodies developed in patients with type 3 von Willebrand disease.

Inhibitors against von Willebrand factor (vWF) developed in two unrelated multitransfused patients (patients 1 and 2) with severe (type 3) von Willebrand disease (vWD) were analyzed. Both inhibitors were identified as antibodies of the IgG class by ELISA using immobilized purified vWF and either serum or purified Ig from the patients. Typing, mapping and functional studies of both antibodies revealed significantly distinct properties. Patient 1 antibody contained all subclasses of IgG (1, 2, 3 and 4) whereas antibody from patient 2 was a mixture of only IgG1 and 4. By ELISA using a series of immobilized purified proteolytic fragments of vWF, patient 1 antibody mainly bound to fragment SpIII and, to a lower extent, to fragments SpII and SpI; it poorly bound to P34 and the 39/34 kDa fragment. In contrast, patient 2 antibody only bound to fragments corresponding to the N-terminal portion of vWF but failed to bind to SpII. Functional studies were performed by testing the capacity of each antibody to inhibit vWF binding to its various ligands. Both antibodies blocked vWF binding to Factor VIII (FVIII), fibrillar type III collagen, bitiscetin and the subsequent induced binding to GPIb. Patient 1 antibody also blocked vWF binding to platelet GPIb when induced by ristocetin. However it failed to block vWF binding to GPIb when induced by botrocetin as well as the binding of botrocetin itself to vWF. Our data thus suggest that this inhibitor does not recognize the GPIb-binding site on vWF but the sites of vWF involved in its interaction with ristocetin. In contrast, we observed that patient 2 antibody blocked vWF binding to platelet GPIb induced by either agonist as well as vWF binding to botrocetin. Finally, the effect of the antibodies was tested on vWF binding to GPIIb/IIIa. As expected from the mapping experiments, only IgG from patient 1 blocked the interaction while IgG from patient 2 had no effect. In conclusion, we have shown that two multitransfused patients with type 3 vWD have developed alloantibodies with similar properties to those of polyclonal antibodies but with distinct effects on the functions of vWF.

Estimation of the von Willebrand factor-cleaving protease in plasma using monoclonal antibodies to vWF.

A protease present in plasma cleaves von Willebrand factor (vWF) at the peptide bond 842Tyr-843Met of the mature subunit. To quantify this vWF-cleaving protease activity in plasma we have developed a simple method based on the estimation by IRMA of the degradation of a constant amount of wild type recombinant vWF used as substrate, by serial dilutions of test plasma used as protease provider. vWFAg was estimated by two-site IRMA using as first coating antibody a monoclonal antibody (MoAb) whose epitope is localized on the C-terminal side of the cleavage site, and as second labeled antibody a pool of MoAbs specific for the N-terminal side. Because the proteolytic process leads to the progressive separation of the C- and N-terminal portions of the vWF subunit such an IRMA also shows a progressive apparent loss of vWFAg. In contrast, the levels of vWFAg estimated after proteolysis by regular IRMA remained essentially constant. Results obtained with this new method were compared with the analysis by SDS-agarose gel electrophoresis of the multimeric pattern of proteolyzed WT-rvWF and no significant difference was noted testing a series of 28 plasmas. As compared with normal pooled plasma, 14 normal individuals and 13 patients with various types of vWD had normal levels of protease activity (44-178%) by both methods. The validity of the method was confirmed by showing a lack of detectable protease activity in a patient with chronic relapsing thrombotic thrombocytopenic purpura. In conclusion our method appears as a useful tool for the quantification of the vWF-cleaving protease activity in plasma. Its sensitivity and specificity are similar to those of SDS-gel electrophoresis. However, this new IRMA has the major advantages of being much simpler and faster, and open to most research laboratories in the field.