Margherita Punzo

Abnormal VWF modifies megakaryocytopoiesis: studies of platelets and megakaryocyte cultures from patients with von Willebrand disease type 2B

von Willebrand factor (VWF) is an essential mediator of platelet adhesion to the vessel wall, but little is known about its role in megakaryocytopoiesis. VWF and its platelet receptor, glycoprotein Ibalpha (GPIbalpha), are both expressed during megakaryocyte (MK) maturation. This study was designed to evaluate whether the enhanced VWF-GPIbalpha interactions typical of patients with von Willebrand disease type 2B (VWD2B) modify platelet production. Platelets from 9 patients with VWD2B with 7 different gain-of-function mutations were examined by electron microscopy (EM) and immunofluorescence labeling. For the patients with VWD2B, EM characteristically showed variable numbers of structurally abnormal giant platelets, sometimes in agglutinates. Cultures of MKs from controls performed with or without purified VWF confirmed a positive influence of VWF on platelet production with specific inhibition by an antibody blocking VWF binding to GPIbalpha. VWD2B MK cultures examined by EM showed a disorganized demarcation membrane system and abnormal granule distribution. They produced platelets with structural abnormalities typical of VWD2B. Confocal examination of MK revealed limited extension of pseudopods with few large proplatelets. These results confirm that megakaryocytopoiesis is modified by the enhanced VWF-GPIbalpha interactions. These data obtained for controls and patients with VWD2B suggest a novel regulatory role of VWF-GPIbalpha interactions in platelet production.

Expression studies of missense mutations p.D141Y, p.C275S located in the propeptide of von Willebrand factor in patients with type 3 von Willebrand disease.

Summary Missense mutations are not considered a common cause of type 3 von Willebrand’s disease (VWD), the most severe defect of von Willebrand factor (VWF) characterized by undetectable levels of this protein in plasma and platelets. Nevertheless, several missense mutations have been identified in these patients. In this study, we report the cases of two Italian patients with type 3 VWD, both compound heterozygotes for different missense mutations and null alleles, p.D141Y/c.2016_2019del and p.C275S/p.W222X. We performed in vitro expression studies of the candidate missense mutations, both located in the D1 domain of VWF propeptide, to confirm their link with the disease and to understand the mechanisms of type 3 VWD responsible in these patients. Mutant and wild‐type (WT) expression vectors were used for transient transfection and co‐transfection studies in COS‐7 cells. Single construct transfections of both missense mutations showed a strongly reduced but detectable secretion of recombinant (r)VWFs (approximately 15% of WT), with essentially only dimers being visualized on multimeric analysis. As expected, expression of a single construct of either mutation with the WT, showed mildly reduced secretion (approximately 40% of WT) and a full set of multimers. These expression studies indicate that the two amino acids D141 and C275 are key residues in the tertiary structure of the VWF propeptide. Their replacement with a tyrosine and a serine, respectively, might compromise propeptide folding, affecting both its intracellular survival and its capacity to mediate multimerization. Co‐expression of hybrid rVWFs confirmed the recessive inheritance pattern of these missense mutations.

Type 2A (IIH) von Willebrand disease is due to mutations that affect von Willebrand factor multimerization

Summary.  Background: Type IIH von Willebrand disease was reported 20 years ago as a novel variant characterized by the loss of the largest multimers in plasma and platelets and absence of the typical triplet structure. Objectives and methods: The propositus and his daughter have been reinvestigated and characterized at the molecular level. The identified mutations were expressed in COS‐7 cells to evaluate the mechanism of this variant. Results and Discussion: The propositus had normal von Willebrand factor (VWF):ristocetin cofactor activity (RCo) and high VWF antigen (VWF:Ag) values, with a low VWF:RCo/VWF:Ag ratio (0.51). No abnormalities were found in his daughter, except for the reduced triplet structure in plasma VWF and diminished ultralarge VWF (ULVWF) multimers in platelets. Three mutations were identified in the propositus: 604C>T (R202W), 4748G>A (R1583Q), and 2546G>A (C849Y). The amounts of secreted recombinant VWF (rVWF) were apparently increased for R202W (130%), R202W‐R1583Q (131%), and R202W‐R1583Q/WT (121%), reduced for C849Y (72%) and C849Y/WT (83%), and normal for R1583Q (107%) and R202W‐R1583Q/C849Y (102%). In cell lysates, higher values were found in association with the C849Y mutation. A normal multimeric pattern was found in R1583Q rVWF, mainly dimers in R202W rVWF, and intermediate molecular weight multimers in C849Y rVWF. Hybrid R202W‐R1583Q/WT and C849Y/WT rVWFs had a nearly normal multimeric pattern, whereas in hybrid R202W‐R1583Q/C849Y rVWF there was a loss of large/intermediate multimers. Conclusions: The propositus phenotype seems to be due to mutations R202W and C849Y, both affecting the VWF multimerization process and, for C849Y rVWF, intracellular survival. The absent triplet multimeric structure in the propositus and its reduction in his daughter appears to be related to the lack of ULVWF multimers, which mainly contribute to the formation of satellite bands.

Prevalence of type 2b 'Malmö/New York' von Willebrand disease in Italy: the role of von Willebrand factor gene conversion.

von Willebrand disease type 2B (VWD2B) is due to a unique gain-of-function variant of von Willebrand factor (VWF) that spontaneously interacts with circulating platelets, usually resulting in loss of VWF high molecular weight multimers (HMWM) in plasma and, in most cases, low platelet counts, especially after stress situations [1,2]. Diagnosis of VWD2B is based on heightened ristocetin-induced platelet aggregation (RIPA) in platelet-rich plasma (PRP). VWD2B Malmö [3] or New York [4] , previously reported as type I, is associated with increased RIPA at low concentrations of ristocetin but normal HMWMand no thrombocytopenia after stress situations. This peculiar VWD2B variant is caused by the mutation 3797C>T (P1266L) [5] in the VWF gene [6]. However, the majority of previously reported patients carrying the mutation P1266L show more than one nucleotide substitution [5,7–10], all matching the published VWF pseudogene sequence [11]. This finding, first described in VWD by Eikenboom et al. [7], has been explained by amechanism of gene conversion between the VWF gene and its pseudogene. The aims of this study were to determine the prevalence of this mutation in a large cohort of VWD2B patients enrolled in the Italian Registry of VWD and to evaluate whether or not gene conversions play a role in generating the mutations identified. Criteria for VWD2B were those recommended by the International Society on Thrombosis and Haemostasis Scientific and StandardizationCommittee VWFSubcommittee (ISTH-SSC on VWF) [2]. RIPA and multimeric analyses were performed as previously reported [12,13]. Patients bleeding severity score was obtained using a detailed questionnaire [14]. Platelet counts had been obtained over 2 years at baseline and after physiologic (pregnancy) or pathological (infections, surgeries) situations or by the use of desmopressin. Among 1234 patients enrolled in the Italian Registry, 66 (35 unrelated families) were diagnosed with VWD2B because of RIPA < 0.7 mg mL (normal range 0.7–1.2 mg mL). DNA sequence analysis was performed using oligonucleotides specifically designed to amplify selectively the VWF gene. The 5¢ portion of exon 28 encoding for the VWF A1 domain was sequenced in all VWD2B cases with a normal VWFmultimeric pattern. Four unrelated families (13 individuals) had a normal multimeric pattern in plasma and no thrombocytopenia before and after stress situations. Mutation 3923G>T (R1308L) was responsible for this phenotype in one family (five patients), and has been documented in a previous study [15]. In the remaining three families (seven patients) more than one mutation was found, but all patients shared a substitution of proline 1266. The following nucleotide changes were identified: 3686T>G (V1229G), 3692A>C (N1231T), 3735G>A, 3789G>A and 3797C>T (P1266L) in family A (three patients), 3789G>A and 3797C>T (P1266L) in family B (two patients) and 3692A>C (N1231T), 3789G>A and 3797C>A (P1266Q) in family C (two patients) (Fig. 1). Nucleotide numbers are reported according to the VWF cDNA sequence [16]. The P1266L/Q genotypes correlate with the patients bleeding history and laboratory tests. Patients bleeding severity score was high (mean 2.85, range 1–6), although less than in other VWD2B variants (8, range 4–14; normal range 0 to )1)[14]. These lower bleeding severity scores are consistent with the mildly reduced or normal VWF levels and activities, reported herewith as mean values (ranges): VWF: Ag 56.4 (30– 73) IU dL, and VWF:RCo 47 (21–66) IU dL, increased RIPA 0.53 (0.40–0.70) mg mL, presence of the HMWMand normal platelet count. We have further investigated additional causes for the bleeding tendency of these patients. However, only in family A did two patients show mildly reduced platelet secretion after stimuli with ADP, as reported by others [17]. VWF:CB (mean values 56.8, 48–64 IU dL) could be tested only in patients with mutation P1266L, and were similar to those of VWF:RCo (56.8, 45–66 IU dL), at variance with the discrepant values reported in VWD2B patients carrying the R1308L mutation [15]. Correspondence: Luciano Baronciani, A. Bianchi Bonomi Hemophilia and Thrombosis Center, Foundation IRCCS Maggiore Policlinico Hospital, Mangiagalli, Regina Elena Via Pace 9, 20122 Milan, Italy. Tel.: +39 2 55 03 53 41; fax: +39 2 55 03 53 47. E-mail: luciano.baronciani@unimi.it

Platelet morphological changes in 2 patients with von Willebrand disease type 3 caused by large homozygous deletions of the von Willebrand factor gene

Platelet morphological defects have previously been described in von Willebrand disease type 2B (VWD2B), we now describe that they may also occur in patients with VWD3 lacking both platelet and plasma von Willebrand factor (VWF). Electron microscopy (EM) and immunofluorescence labeling (IF) were

Relevance of chloride binding to von Willebrand factor in type 2B von Willebrand disease patients

L . BARONCIANI , * F . PEYVANDI ,* M. PUNZO,* S . LANCELLOTT I , M. T . CA NC IA NI ,* A . B . FEDER IC I * and R . DE CR ISTOFARO *A. Bianchi Bonomi Hemophilia and Thrombosis Center and Fondazione Luigi Villa, Università degli Studi di Milano, Department of Medicine and Medical Specialties, Foundation IRCCS Maggiore Policlinico Hospital, Mangiagalli, Regina Elena, Milan; and Hemostasis Research Centre, Institute of Internal Medicine and Geriatrics, Catholic University School of Medicine, Rome, Italy