Studies with the Nanobody That Detects the Gain-of-Function of von Willebrand Factor in a Cohort of Patients with Type 2B von Willebrand Disease: Correlation with Platelet Count, VWF Multimers and Molecular Defects.
Authors: Federici Augusto B. , Baronciani Luciano , Canciani Maria T. , Moroni Barbara , Balduini Carlo , Mannucci Pier M. , Lenting Peter J. , de Groot Philip G. 
Published: April 2020

Abstract
Background: Type 2B von Willebrand disease (VWD) is an inherited bleeding disorder caused by abnormal von Willebrand factor (VWF) that displays increased affinity to the platelet glicoprotein 1b alpha (GpIba) and is due to a group of mutations clustered within VWF A1 domain. Such an enhanced 2BVWF-GpIba binding usually result in loss of large VWF multimers and moderate-mild thrombocytopenia. A llama-derived antibody fragment (AuVWFa11) recognizing the GpIba-binding conformation has been recently developed (Blood2005;106:3035).
Aims and design of the study: to further explore the usefulness of AuVWFa11 in type 2B diagnosis, we have prospectively tested AuVWFa11 in our cohort of 16 patients previously characterized by platelet count, VWF multimers and mutations.
Methods: Data of platelet count with mean platelet volume (MPV) and morphologic evaluation of the blood smear to search for giant platelets or aggregates were associated with the history of physiologic or pathologic stress conditions such as pregnancy, infections, surgery or use of DDAVP. All patients were diagnosed by ristocetin induced platelet agglutination (RIPA) in the Platelet Rich Plasma (PRP), ristocetin cofactor activity (VWF:RCo) with VWF antigen (VWF:Ag), multimeric structure of VWF. Mutations within VWF A1 domain were searched for and confirmed by sequencing exon 28. AuVWFa11 was tested in 40 normal individuals (expressed as % of active VWF in normal pool plasma =0.70±0.13) and in type 2B.
Results: Data (mean ± SD) of the AuVWFa11 tested in the 16 patients with type 2B VWD are correlated with the main phenotypic data and genotype (Table1). Platelet count &lt; 140,000 was found at baseline in only 3/16 (%), but was observed after stress conditions in 12/16 cases (%); no reduced platelet counts was found in 4/16 patients (%) from two different families (R1308L, R1341Q). An increased MPV was found in 12 cases but giant platelet and aggregates in only 1 case. Activated VWF as tested by AuVWFa11 was positive in all but 3 (R1308L) cases, with values ranging from 2 to 6 times higher than normal controls: values &gt; 3 correlate with loss of large VWF multimers and mild-moderate thrombocytopenia.
Conclusions: The AVWF11a can show activated VWF in most type 2B VWD patients, especially when 2B VWF mutants induce significant loss of large multimers and thrombocytopenia. Therefore AuVWF11a can be a useful additional tool in the diagnosis of type 2B VWD.
Table 1 Mutation (n) RIPA (mg/ml) VWF.Ag (U/dL) Plat Count (×10^9/L) MPV (micron^3) Loss of HMW Mult AuVWFa11 (ratioNPP) R1306W (5) 0.65 40±9 165±39 10.3±2.3 YES 3.7±1.5 R1308C (3) 0.72 53±16 163±61 11.5±1.9 YES 3.3±2.3 R1308L (3) 0.50 48±13 341±104 8.1±3.1 NO 0.5±0.2 I1309V (1) 0.40 115 222 11.8 PARTIAL 2.1 V1316M (2) 0.50 32±7 119±30 9.2±2.4 YES 4.4±0.1 P1337L (1) 0.50 48 222 9.5 PARTIAL 1.3 R1341Q (1) 0.67 43 422 9.9 PARTIAL 2.9