Abnormal Large Platelets Are Associated Not Only with von Willebrand Disease (VWD) Type 2B (Including Type 2B/1 New York) but Also with Severe Type 3: A Potential Role for von Willebrand Factor (VWF) in Megakaryocytopoiesis.

Background: VWD type 2B results from mutations in exon 28 of the VWF gene. Gain of function of this adhesive protein results in an increased affinity for the platelet glycoprotein (GP) Ib-IX-V complex. Recently we reported that impaired megakaryocytopoiesis results from an abnormal interaction between GPIb with newly synthesized VWF in megakaryocytes of a family with the R1308P mutation (

Aim of the Study: to further examine the potential consequences of VWF abnormalities on platelet production we have studied a series of patients with different types of VWD.

Patients, Methods: 13 VWD patients were enrolled in the study after informed consent. Diagnoses of VWD were performed according to the criteria of the ISTH-SSC-SC. Included were 8 VWD 2B patients from 6 families with the following mutations: R1306W (n=1), R1308C (n=1), I1309V (n=1), V1326M (n=2), R1341Q (n=2) and P1266L (n=1, 2B/1NY). Also studied were 5 additional VWD cases characterized by low/absent VWF in their platelets: VWD 2M (n=1, D1277-E78delInsL), VWD 3 without inhibitors against VWF (n=2, 276delT/257delA and 6182delT/6182delT) and VWD with isoantibodies against VWF (n=2, large deletions of the VWF gene). The platelet count was decreased at the time of examination for 6/8 VWD 2B patients and normal for 2/8 (n=1 V1316M and n=1 P1266L). Platelet counts were normal in the remaining 5 patients with VWD types 3, and 2M. Electron microscopy (EM) of platelets and immunolocalization of VWF were performed.

Results: EM showed the presence of an increased population of giant platelets (15 to 40% versus <10% for controls) in all VWD 2B. Characteristics of these platelets were the presence of large vacuoles often filled with material and the presence of numerous membrane complexes. Additional abnormalities were observed in the patient with 2B/1NY; alpha-granule morphology was different with a population of enlarged granules, sometimes giant. There was also a heterogeneneous distribution with some platelets almost devoid of alpha-granules. Immunogold staining for all type 2B patients showed that VWF was present not only inside the granules but also in the surface-connected canalicular system. For 3/8 patients with VWD 2B, cleaved caspase was present in the platelets indicating abnormal caspase activity at least for R1341Q and V1316M. In VWD 2M (mutation D1277-E78delInsL) characterized by low platelet VWF content as well as in the VWD 3 (n=2) with a premature stop codon, no significant modification of platelet morphology was found. Some residual VWF was also seen in the alpha-granules of these 2 VWD 3 patients. In contrast, a significant number of enlarged platelets with numerous vacuoles were found in the 2 VWD 3 with large deletions and isoantibodies directed against VWF. Immunogold labelling for platelet VWF was completely negative for these two patients.

Conclusions: Patients with VWD types 2B and 3 (undetectable VWF) show platelet production defects of varying severity, suggesting a major role of VWF in the fine regulation of megakaryocytopoiesis. Up-regulation or loss of the interaction between VWF and GPIb may lead to a variable proportion of giant platelets with or without thrombocytopenia.