von Willebrand disease Giancarlo Castaman Center for Bleeding - PowerPoint PPT Presentation

11 th Hemostasis Seminar 1-2 October 2019, Bucharest, Rumenia Clinical and Laboratory aspects of von Willebrand disease Giancarlo Castaman Center for Bleeding Disorders and Coagulation, Department of Oncology, Careggi University Hospital, Florence, Italy

SVEZIA FINLANDIA

Family S, Föglö Island Klas Oskar Augusta 78 y 74 y † † † † † † † Dagny Harald Sylvia Runar Hjördis Greta Gerda Anna Lars Dagny Thomas 2 y 44 y 41 y 40 y 13 y 5 y 34 y 4 y 29 y 2y 31 y Helga Viking Robert Birgitta Roger Cecilia Tage Jan Uluf Anders Monika Lars-Uwe Börje 17 y 10 y 17 y 15 y 13 y 10 y 9 y 1 y 8 y 4 y 1 y 6 y Severe bleeding Mild or dubious bleeding symptoms

Von Willebrand disease (VWD) is an inherited bleeding disorder due to a quantitative and/or qualitative deficiency of von Willebrand factor, first identified by E. von Willebrand in 1926 Erik Adolf von Willebrand (1870-1949)

VON WILLEBRAND FACTOR (VWF): The role of ADAMTS-13-dependent proteolysis Cleavage site of ADAMTS-13 S-S (Y1605-M1606) Propeptide Multimers S-S Signal peptide Dimer D2 D’ D2 D3 D3 D1 A1 A2 H 2 N A3 A3 D4 B2 B3 B1 C1 C2 CK COOH 22 763 FVIII GP Ib  Collagen I & III 2813 Collagen VI GP IIb/IIIa Heparin

SHEAR STRESS FORCES OF THE BLOOD min max Globular Extended VWF VWF When shear stress is high enough to stretch VWF exposing the buried A2 domain, proteolysis is rapid (Dong et al, 2002) Siedecki et al Blood 1996

Synthesis Proteolysis Steady state (ADAMTS-13) (Normal Plasma) Σ = EC → Plasma

VWF locus (chrom 12) Dimerization Multimerization Allele Allele B1 D’ D1 D2 D3 A1 A2 A3 D4 C1 C2 CK B3 Endothelial cell PP PP C N C NH 2 COOH FVIII GPIb Collagen GpIIb/IIIa Dimerization Heparin Collagen Heparin PP PP N N PP PP Propeptide cleavage Multimerization - - - - Weibel-Palade bodies Proteolysis by ADAMTS-13 Plasma Plasma VWF Constitutive Regulated release of secretion ultralarge multimers Clearance

The he mu mult ltimeric imeric com omposi position tion of of VWF

VWF Journey: From Inactive Globular Form to Activation of Platelets Leebeek FWG, Eikenboom JCJ. N Engl J Med 2016;375:2067-80.

Von n Wi Wille llebrand brand Fa Factor tor • Multimeric, adhesive protein, composed of a series of dimers of mature subunits up to 20,000 Kd (multiplicative effect of binding activities) • Carrier er of FVIII III: localization and prevention of inactivation by the Protein C system • Platel elet et adhesion on to the subendothelium at high shear stress flow (via Gp Ib,  2  1 ) • Platelet elet-to to-plat platelet elet cohesi sion n and aggreg egat ation in cooperation with fibrinogen (via Gp IIb/IIIa,  IIb  3 )

Von Willebrand disease • Bleeding disorder due to a quantitative or qualitative defect of VWF • Depending on the particular defect the disease may be inherited either in a dominant or recessive manner • The spectrum of clinical symptoms is greatly influenced by a wide variation in expressivity and penetrance

VWD is an inherited bleeding disorder due to a quantitative or qualitative defect of VWF Bleeding risk in Bleeding - propositus disorder - family members Quantity VWF:Ag (Type 1, 3) Quality VWF:RCo/Ag (Type 2) VWF deficiency FVIII/VWF:Ag Multimeric composition Inheritance Proven or inferred

GROUP A: Autosomal dominant inheritance, high penetrance and expressivity (C1130F; Castaman et al, BJH 2000) Family A Family B Family C ND Rsa I ND - + - + + - - + ND Rsa I - + - - - - + - - + + + Hph I - - + + + + - - + - + - VNTR I 10 11 7 7 7 7 7 12 7 7 10 7 VNTR II 4 4 3 3 3 3 4 4 3 2 Rsa I - + - + - - - - Rsa I - - - + - + - + Hph I + - + - + - + - VNTR I 7 10 7 7 7 7 7 7 VNTR II 3 4 3 4 3 2 3 2 Family D Family E Rsa I + + - - + - - + + + Rsa I + + - + + + - - - - Hph I - - + - + - + + + - VNTR I 7 7 7 8 6 8 7 7 11 7 VNTR II 3 3 3 3 2 3 3 3 4 4 Rsa I - + - + - + + + Rsa I - + - + - - - - Hph I + - + - + - - + • Single VWF haplotype VNTR I 7 7 7 7 7 7 7 7 VNTR II 3 3 3 3 3 4 4 3 • VWF ~ 10 U/dL • BS > 5

TYPE 3 VWD (IVS46 +1, G>T n.7770+1) FVIII:C 160 IU/dL FVIII:C 131 IU/dL VWF:Ag 98 IU/dL VWF:Ag 140 IU/dL FVIII:C 121 IU/dL FVIII:C 90 IU/dL FVIII:C 86 IU/dL FVIII:C 72 IU/dL VWF:Ag 99 IU/dL VWF:Ag 45 IU/dL VWF:Ag 30 IU/dL VWF:Ag 82 IU/dL FVIII:C 57 IU/dL FVIII:C 61 IU/dL FVIII:C 1.2 IU/dL VWF:Ag 63 IU/dL VWF:Ag 35 IU/dL VWF:Ag 1.8 IU/dL Homozygous IVS 46 +1 G>T

HOW TO DIAGNOSE VON WILLEBRAND DISEASE

The pleiotropic effects of von Willebrand factor PFA-100 FVIII:C Bleeding time VWF:RCo VWF:Ag VWF:CB VWF:FVIIIB No single test reflects the whole spectrum of VWF activities

PHENOTYPIC DIAGNOSIS OF VWD Tests in Use • Basic Tests • Advanced tests – Platelet count – VWF/FVIII binding – BT (PFA-100) – Platelet VWF assessment – RIPA – Multimer profile – VWF:Ag – VWF:RCo – VWF:CB – FVIII:C

ADHESION ACTIVITIES OF VWF PLATELET Collagen GPIb  VWF:RCo Heparin Sulphatide A1 A2 C C C C VWF:CB A3 SUBENDOTHELIUM COLLAGEN

Why VWF:RCo as screening test for von Willebrand disease ? • Time-honored surrogate test to explore interaction with platelet GpIb • Greater diagnostic sensitivity compared to classic tests for diagnosis of VWD BUT It does not reflect a true physiologic VWF function

Platelet-dependent VWF Activity: Nomenclature Abbreviation Description Principle Ristocetin cofactor activity: “traditional” assays that use VWF:RCo ristocetin to induce binding Platelet + ristocetin + VWF to platelets Assays based on ristocetin- induced binding of VWF to VWF:GPIbR OR recombinant wild-type GPIb fragment rWT-GPIb + ristocetin + VWF Assays based on spontaneous binding of VWF VWF:GPIbM OR to gain-of-function mutant GPIb fragment Gain-of-function rGPIb +VWF Assays based on binding of a VWF:Ab monoclonal antibody to a VWF A1 domain epitope Α nti-A1 MoAb + VWF

VON WILLEBRAND FACTOR: RIPA Ristocetin induced platelet agglutination Normal VWD 2A VWD 2B Ristocetin mg/ml Transmission 1 min. 1 min. 1 min. Platelet Rich Plasma from Patients + RISTOCETIN [0.2-2.0 mg/ml]

Test Pathophysiologic Diagnostic significance significance Interaction of normal FVIII Binding of VIII:C to Allows the identification of type 2 with patient plasma VWF N, characterized by low binding VWF values and suspected in case of reduced VIII:C/VWF:Ag Simulates primary More sensitive than BT in Closure time PFA-100 hemostasis after injury to a screening for VWD; not tested in small vessel bleeding subjects without specific diagnosis; specificity unknown; poor sensitivity to mildly reduced VWF levels Measures the amount of Increased VWFpp/VWF:Ag ratio Propeptide assay VWFpp released in plasma identifies patients with shortened VWF survival after desmopressin; still for research purposes

INCREASED VWF CLEARANCE: A SINGLE LABORATORY PHENOTYPE ? 14 VWFpp/VWF:Ag Ratio 12 10 8 6 4 2 0 0 10 20 30 40 50 60 70 VWF:Ag (IU/dL) R1205H C1130F W1144G S2179F Modified from Haberichter, 2008; Castaman, 2009

FLOW CHART FOR THE DIAGNOSIS OF A PATIENT WITH VWD: FLOW CHART FOR THE DIAGNOSIS OF A PATIENT WITH V Type 3 Absent Absent a) plasma vWF:Ag plasma VWF:Ag Absent a) a) plasma vWF:Ag f) platelet vWF f) platelet VWF f) platelet vWF Present Present Present Proportionate Proportionate Proportionate Type 1 Proportionate Proportionate Proportionate (0.7 - 1.2) (0.7 - 1.2) (0.7 - 1.2) b) plasma vWF:RCo vs vWF:Ag plasma VWF:RCo vs VWF:Ag b) b) plasma vWF:RCo vs vWF:Ag c) plasma Factor VIII:C vs vWF:Ag plasma Factor VIII:C vs VWF:Ag c) c) plasma Factor VIII:C vs vWF:Ag vWF:(RCo/Ag) VWF:(RCo/Ag) vWF:(RCo/Ag) Discrepant Discrepant Discrepant Discrepant Discrepant Discrepant Type 2 N Type 2 (< 0.6) (< 0.7) (< 0.7) Increased Increased Increased d) Ristocetin Induced platelet aggiutination Ristocetin Induced platelet agglutination g) FVIII binding assay FVIII binding assay d) g) d) Ristocetin Induced platelet aggiutination g) FVIII binding assay (0.2-0.8) (0.2-0.8) (0.2-0.8) R.I.P.A. (mg/ml) R.I.P.A. (mg/ml) R.I.P.A. (mg/ml) Type 2 A Absent Absent Absent Type 2 B Decreased Decreased Decreased e) plasma High Multimers e) plasma High Multimers e) plasma High Multimers (>1.2) (>1.2) Type 2 M (>1.2) Present Present Present