Haematologica 2004;89: supplement no. 8 - Supplements ...

90Oral CommunicationsOral CommunicationsVON WILLEBRAND FACTOR AND DISEASECO-134IN VITRO EXPRESSION STUDY OF A NEW MUTATION (R1308L)FOUND IN A FAMILY WITH TYPE 2B VON WILLEBRAND DISEASECHARACTERIZED BY ALL SET OF MULTIMERS IN PLASMA ANDNO THROMBOCYTOPENIA AFTER DESMOPRESSINBaronciani L, Federici AB, Cozzi G, Beretta M,Canciani MT, Mannucci PMAngelo Bianchi Bonomi Hemophilia and ThrombosisCenter, IRCCS Maggiore Hospital and University ofMilan, ItalyType 2B von Willebrand disease (VWD) is characterizedby enhanced ristocetin-induced plateletagglutination (RIPA) and by loss of high molecularweigh multimers (HMWM) in plasma. A novel 2B vonWillebrand factor (VWF) variant (4173G>T, R1308L)was found in four patients with RIPA (0.3-0.4mg/mL), VWF:Ag (27-51 U/dL), VWF:RCo (23-49U/dL), bleeding time (6-11 min), all set of multimersin plasma and normal platelet count. Interestingly, acommon VWD type 2B mutation, R1308C is characterizedby loss of HMWM, low platelet count andRIPA (0.7-0.8 mg/mL), VWF:Ag (26-48 U/dL),VWF:RCo (13-39 U/dL), bleeding time (10-27 min).Mutated rVWF-R1308L and rVWF-R1308C, weretransiently expressed in Cos 7 cells, and analyzed fortheir ability to bind GpIb receptor along with rVWF-WT. Binding of rVWFs to the GpIb platelet receptorwas tested by an ELISA method (Federici et al.Haematologica 89:77, 2004), at increasing concentrationsof ristocetin (0, 0.125, 0.25 and 0.5 mg/mL),and the rVWF bound to GpIb; was revealed by anti-VWF Antibody-HRP reading O.D. 492 nm. The threerVWFs have a similar VWF:Ag and a full set of multimers.Our data demonstrated that the new mutation(R1308L) can enhance the VWF capacity to bindto GpIb receptor, but with lower efficiency comparedto the R1308C variant. This might explain why thesepatients have normal VWF multimers in plasma. Theincreased binding capacity of VWF-R1308L for GpIbdoes not cause its spontaneous interaction withplatelets, or at least, not as efficiently as VWF-R1308C does. The enhanced RIPA of 0.4 mg/mL,observed in patients with VWF-R1308L, versus 0.7mg/mL of VWF-R1308C is probably due to the presenceof all set of multimers, that strongly compensatefor the lower binding capacity to GpIb of thisnew variant.CO-135BIOCHEMICAL CHARACTERIZATION OF RECOMBINANT VONWILLEBRAND FACTOR (VWF) WITH A TYPE 2B MUTATION(P1337L) COEXPRESSED WITH A TYPE 1 VWF DEFECT (C275R):A COMPLEX MOLECULAR DEFECT IN A PATIENT PREVIOUSLYDIAGNOSED WITH TYPE 2A VON WILLEBRAND DISEASEBaronciani L, Federici AB, Beretta M, Cozzi G,Canciani MT, Mannucci PMAngelo Bianchi Bonomi Hemophilia and ThrombosisCenter, IRCCS Maggiore Hospital and University ofMilan, ItalyIn a patient with apparently type 2A VWD (meanvalues: FVIII:C = 32 U/dL, VWF:Ag = 7 U/dL, VWF:RCo= < 6 U/dL, VWF:CB = 2.0 mg/mL, loss ofhigh and intermediate molecular weight multimersin plasma and low platelet VWF) a transient thrombocytopeniaoccurred after an infusion test withdesmopressin. The propositus' brother showed a lesssevere laboratory data (mean values: FVIII:C = 36U/dL, VWF:Ag = 17 U/dL, VWF:RCo = 6 U/dL,VWF:CB=1, RIPA = 1.2 mg/mL). The two brothersresulted to be compound heterozygous for mutationP1337L and a novel candidate defect C275R. In theirchildren, mutations were associated with type 2B(P1337L) and type 1 (C275R) VWD. rVWF-P1337Land rVWF-C275R, were transiently expressed in Cos7 cells, on their own, together and with the rWVF-WT. VWFs. rVWFs were tested for VWF:Ag and multimeranalysis. Binding of rVWFs to the GpIb plateletreceptor was evaluated by an ELISA method (Federiciet al. Haematologica 89:77, 2004), at increasingconcentrations of ristocetin (0, 0.125, 0.25, 0.5, 0.8and 1 mg/mL), and the rVWF bound to GpIb wasrevealed by anti-VWF Antibody-HRP reading O.D.492 nm. Only the expression of rVWF-C275R aloneshowed a strongly reduced VWF:Ag in cells mediumand a complete absence of multimers. The remainingrVWFs (P1337L, P1337L/WT and C275R/WT)showed only a slightly reduce VWF:Ag, in comparisonto rVWF-WT, and a full set of multimers. GpIbbinding assay (Table) showed that rVWF obtained byco-expression of mutation P1337L with C275Rbehave very similarly to rVWF-P1337L. It seems thatrVWF-C275R molecules do not contribute with therVWF-P1337L to form multimers.rVWFs Ristocetin Ristocetin Ristocetin Ristocetin Ristocetin Ristocetin0 mg/ml 0.125 mg/ml 0.25 mg/ml 0.5 mg/ml 0.8 mg/ml 1 mg/mlWT 0.066 O.D. 0.071 O.D. 0.128 O.D. 0.154 O.D. 0.691 O.D. 0.924 O.D.P1337L 0.117 O.D. 0.310 O.D. 0.689 O.D. 0.984 O.D. 0.984 O.D. 1.039 O.D.P1337L/WT 0.063 O.D. 0.166 O.D. 0.409 O.D. 0.740 O.D. 0.894 O.D. 1.008 O.D.P1337L/C275R 0.084 O.D. 0.372 O.D. 0.635 O.D. 0.925 O.D. 1.105 O.D. 1.117 O.D.haematologica vol. 89(suppl. n. 8):september 2004