Page 284 - Haematologica April 2020

V. Daidone et al.
Figure 5. Multimer analysis of recombinant von Willebrand factor (rvWF). No rvWF was found in the conditioned medium from cells expressing the P1 vWF mutation. Both P2 and P3 rvWF were produced instead, and showed: (i) unusually large vWF multimers associated with the accumulation of multimers on the boundary of the running and stacking gel; (ii) an oligomer pattern that had shifted towards the high-molecular-weight forms; and (iii) oligomers with a slower migration pattern. P1: p.Leu757Valfs*22; P2: p.L757_R763delinsVSSQ; P3: p.L757_761delinsVSSQG.
enzymatic activity (p.L757_761delinsVSSQG, P3).30 Despite bearing a PTC, RNAI seems to escape the NMD control mechanism, as it accounts for more than 80% of total mRNA expression.19,31
The proband was homozygous for the c.2269_2270del mutation, and carried no other mutations in his vWF gene. He had a very complex and severe phenotype character- ized by both quantitative and qualitative vWF alterations. Both plasma and platelet vWF levels were significantly decreased, leading to a diagnosis of severe type 1 vWD. The multimer organization of his circulating vWF revealed a peculiar pattern involving the absence of any oligomers except for the vWF dimer, and an unusually ultra large band, without any intermediate components. This pattern did not change after DDAVP infusion, which increased the pre-DDAVP oligomers, without any other intermediate components being observed. On the other hand, the patient’s platelet vWF showed a multimer organization (albeit lacking in the large components), with each oligomer characterized by a delayed migration, a picture suggestive of a persistence of vWFpp.28 A similar vWF multimer pattern had previously been reported in a patient with systemic lupus erythematosus, who had the two above-described circulating vWF oligomers, but a nor- mal platelet vWF content and multimer pattern in the presence of an anti-vWF antibody.26 In contrast, no anti- vWF antibodies were detectable in our patient.
The delayed migration of the proband’s platelet vWF oligomers was attributable to the presence of the P2 and P3 vWF forms, both resistant to the action of furin, and consequently characterized by the persistence of the vWFpp. This was confirmed by our patient’s undetectable vWF:FVIIIB values, as well as by the P2 and P3 rvWF forms, which point to his vWF having an impaired capac- ity to carry FVIII.32 The persistence of vWFpp is known to disrupt the capacity of vWF to bind FVIII because of a steric hindrance that makes the FVIII binding site in the D3 domain of vWF inaccessible.28
The DDAVP-induced release of vWF stored in the Weibel-Palade bodies, or a drop in ADAMTS13 concentra- tion in the bloodstream are usually associated with the appearance of ultra large vWF multimers in the circulation. In both cases, all vWF oligomers are still present, however, whereas this was not so in our patient. There are also vWF mutations that interfere with the factor’s recognition and/or binding by ADAMTS13, giving rise to the presence of unusually large vWF bands33,34 that are not normally organized, a picture not seen in our patient either. Indeed, the multimer pattern of the proband’s circulating vWF appeared to be more similar to that of vWF inside normal endothelial cells.35 We know that the cysteine-linked vWF dimers grow in a concatemer via the formation of disulfide bonds at the N-terminal of the vWF molecule, with the contribution of vWFpp. After its secretion from endothe- lial cells, vWF remains anchored to the vessel wall and is then uncoiled by the action of the blood flow. This makes the Tyr1605-Met1606 bond accessible to ADAMTS13 for cleavage. The persistence of vWFpp could interfere with the unraveling of vWF during its release from endothelial cells;36-39 alternatively, the P2 and P3 vWF forms might be unable to remain anchored to the vessel wall during their release. Intriguingly, p-selectin protein (which is known to anchor vWF to the endothelial vessel wall under normal conditions) binds vWF in the D’D3 region, very near where our P2 and P3 mutations occur.40 While circulating vWF needs dynamic flow conditions for it to be cleaved by ADAMTS13 after its release, this might not be the case for vWF inside alpha-granules, and this would explain the different multimer organization in the two biological dis- tricts. A different contribution of aberrant vWF mRNA in endothelial cells and platelets is less plausible, but mRNA in endothelial cells would need to be explored to be able to verify this.
In vitro expression experiments confirmed that the c.2269_2270del mutation, hitherto considered a type 3 vWF defect,29,41 is actually associated with a residual syn-
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