The interaction between von Willebrand factor (VWF) and platelets plays a key role in hemostasis. VWF gene abnormalities result in the most common inherited bleeding disorder, von Willebrand disease (VWD). VWD2B, characterized by gain-of-function binding of VWF to platelet glycoprotein (GP)Ib, is due to mutations located within a portion of exon 28 of the VWF gene, coding for the VWFA1 domain. We have previously shown in a family with a R1308P mutation that the enhanced interaction between VWF and GPIb can also occur in the bone marrow leading to a premature release of platelets from megakaryocytes (MKs) with circulating large platelets and agglutinates (Nurden et al, Blood 2006). We have now widened our study to see if these changes are common in VWD2B. As a control, we also included platelets from patients lacking VWF. In total, we have analyzed the platelets from 9 VWD2B patients with the following mutations: P1266L, R1306W, R1308C, I1309V, V1316M, R1341Q, R1341W; platelets from 2 VWD3 patients with a large deletion of the VWF gene were also included. We have used electron microscopy (EM) and immunofluorescence (IF) to evaluate platelet morphological characteristics; included in the morphometric studies was the measure of platelet size. We have also analyzed platelet VWF content and performed plasma/platelet VWF multimeric analyses. PMCA4B belonging to the SERCA proteins (a substrate of caspase) and GRP78 a stress protein were evaluated as markers of abnormal megakaryocytopoiesis. At the time of blood withdrawal, 4 patients were thrombocytopenic; nevertheless, for all patients with VWD2B a pool of giant platelets was found. Agglutinates were often but not always present. Globally, the percentage of platelets with the longest diameter (LD) > than 3μm was 58 ±16% (controls 24 ± 13%) and the shortest diameter (SD) > 2μm was 33% ± 13 (controls 4 ± 0.6%). Thus platelets were enlarged without being round. The largest platelets were found for the patient with the V1316M mutation. The content of alpha granules appeared normal, as was the quantity of internal membrane pools. To our surprise, giant platelets were also present for the two VWD3 patients; 66 and 57% of the platelets have a LD > 3μm and 21 and 19% a SD > than 2μm. The EM evaluation for both type 3 patients suggested that some giant platelets resembled large fragments prematurely detached from MKs. Examining further by IF and EM coupled with immunogold labelling with a mixture of anti-GPIIb-IIIa and GPIb-IX MoAbs revealed occasional circulating MKs. Nuclei surrounded by cytoplasmic remnants were also seen. Analysis of PMCA4B and GRP 78, showed that their levels were increased for VWD2B patients with the R1341Q mutation but borderline for the patient with the R1308C mutation (contrasting with increased levels for R1308P) showing that premature apoptosis was not a constant feature. These results were normal for the VWD3 patients. Our results confirm that megakaryocytopoiesis can be affected by both an abnormal gain-of-function (VWD2B) and completely deficient (VWD3) VWF. However, the consequences of up-regulated VWF and VWF deficiency may be different.

Disclosures: No relevant conflicts of interest to declare.

Author notes

Corresponding author