Differentiation of Patients with Symptomatic Low von Willebrand Factor from Those with Asymptomatic Low von Willebrand FactorFunding This project has been supported by an Investigator-Initiated Grant from CSL Behring and, for equipment maintenance, by the National Institutes of Health (grant No. R01 DK 11 7313).
22 August 2019
05 March 2020
05 May 2020 (online)
Background Accurate diagnosis of symptomatic low von Willebrand factor (VWF) remains a major challenge in von Willebrand disease (VWD). However, present tests do not adequately take into account flow forces that, at very high shear rates, reveal a weakness in the VWF-platelet glycoprotein glycoprotein Ib bond in normal subjects. The degree of this weakness is greater in symptomatic, but not asymptomatic, low VWF.
Objective The aim of this study is to distinguish patients with symptomatic low VWF (levels in the 30–50 IU/dL range) from those with asymptomatic low VWF and normal subjects.
Methods We measured platelet adhesion (PA)/aggregation in our novel microfluidic flow system that permits real-time assessment of PA (surface coverage) and PA/aggregation (V, aggregate volume) using epifluorescence digital videomicroscopy in flowing noncitrated whole blood at 4,000 second−1. Blood samples from 24 low VWF patients and 15 normal subjects were collected into plastic tubes containing 4 U/mL enoxaparin. MetaMorph software was used to quantify rates of PA and V increase.
Results Rates of PA increase showed a bimodal distribution, with values for 16/24 patients (Group I) all below the 2.5th percentile of normal, and values for 8/24 patients (Group II) similar to controls. Bleeding scores (mean ± standard error) were 5.50 ± 0.45 versus 2.75 ± 0.45 (p = 0.00077), and 10 clinically significant bleeding events were observed in seven versus zero (p = 0.0295) Group I and Group II subjects, respectively.
Conclusion The present approach may offer a definitive means to distinguish symptomatic low VWF from either asymptomatic low VWF or normal controls.
Keywordsvon Willebrand disease - platelet aggregation - microfluidics - platelet function tests - von Willebrand factor
E.F.G. developed the flow system, conceived of the study, performed many of the experiments, wrote the manuscript and enrolled his pediatric patients. L.B. enrolled her adult patients and critically reviewed the manuscript. E.M.V.C. advised on the comparisons with standard VWD assays, revised the discussion text, and critically reviewed the manuscript. R.L.S. collected the information regarding clinically significant bleeding events. D.C.B. assisted with the experiments, digital image analysis, and figures.
- 1 Grabowski EF, Curran MA, Van Cott EM. Assessment of a cohort of primarily pediatric patients with a presumptive diagnosis of type 1 von Willebrand disease with a novel high shear rate, noncitrated blood flow device. Thromb Res 2012; 129 (04) e18-e24
- 2 Yago T, Lou J, Wu T. , et al. Platelet glycoprotein Ibalpha forms catch bonds with human WT vWF but not with type 2B von Willebrand disease vWF. J Clin Invest 2008; 118 (09) 3195-3207
- 3 Bowie EJW, Owen Jr CA, Thompson JH, Didisheim P. Platelet adhesiveness in von Willebrand's disease. Am J Clin Pathol 1969; 52 (01) 69-77
- 4 Weiss HJ, Turitto VT, Baumgartner HR. Effect of shear rate on platelet interaction with subendothelium in citrated and native blood. I. Shear rate--dependent decrease of adhesion in von Willebrand's disease and the Bernard-Soulier syndrome. J Lab Clin Med 1978; 92 (05) 750-764
- 5 Tschopp TB, Weiss HJ, Baumgartner HR. Decreased adhesion of platelets to subendothelium in von Willebrand's disease. J Lab Clin Med 1974; 83 (02) 296-300
- 6 Fressinaud E, Sakariassen KS, Rothschild C, Baumgartner HR, Meyer D. Shear rate-dependent impairment of thrombus growth on collagen in nonanticoagulated blood from patients with von Willebrand disease and hemophilia A. Blood 1992; 80 (04) 988-994
- 7 Schlammadinger A, Kerenyi A, Muszbek L, Boda Z, Trossaert M. Comparison of the O'Brien filter test and the PFA-100 platelet analyzer in the laboratory diagnosis of von Willebrand's disease. Thromb Haemost 2000; 84 (01) 88-92
- 8 Hayward CP, Harrison P, Cattaneo M, Ortel TL, Rao AK. ; Platelet Physiology Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. Platelet function analyzer (PFA)-100 closure time in the evaluation of platelet disorders and platelet function. J Thromb Haemost 2006; 4 (02) 312-319
- 9 Nichols WL, Hultin MB, James AH. , et al. von Willebrand disease (VWD): evidence-based diagnosis and management guidelines, the National Heart, Lung, and Blood Institute (NHLBI) Expert Panel report (USA). Haemophilia 2008; 14 (02) 171-232
- 10 Smits-Engelsman B, Klerks M, Kirby A. Beighton score: a valid measure for generalized hypermobility in children. J Pediatr 2011; 158 (01) 119-123
- 11 Hickey SE, Varga EA, Kerlin B. Epidemiology of bleeding symptoms and hypermobile Ehlers-Danlos syndrome in paediatrics. Haemophilia 2016; 22 (05) e490-e493
- 12 McEver RP, Bennett EM, Martin MN. Identification of two structurally and functionally distinct sites on human platelet membrane glycoprotein IIb-IIIa using monoclonal antibodies. J Biol Chem 1983; 258 (08) 5269-5275
- 13 McEver RP, Baenziger NL, Majerus PW. Isolation and quantitation of the platelet membrane glycoprotein deficient in thrombasthenia using a monoclonal hybridoma antibody. J Clin Invest 1980; 66 (06) 1311-1318
- 14 Grabowski EF. Platelet aggregation in flowing blood at a site of injury to an endothelial cell monolayer: quantitation and real-time imaging with the TAB monoclonal antibody. Blood 1990; 75 (02) 390-398
- 15 Grabowski EF, Yam K, Gerace M. Evaluation of hemostasis in flowing blood. Am J Hematol 2012; 87 (Suppl. 01) S51-S55
- 16 Grabowski EF, Van Cott EM, Bornikova L, Boyle DC. Differentiation of patients with type 1 von willebrand disease (VWD) and with symptomatic low normal von willebrand factor (VWF) from those with asymptomatic low VWF, using a very high shear rate, high image resolution blood flow chamber. Blood 2017; 130: 2338 (presented as a poster at the 2017 Annual Meeting of the American Society of Hematology, Atlanta, GA, December 10)
- 17 Silverberg A. Thickness of adsorbed protein layers on glass: Adsorption of proteins in multilayers. In: Polymer Adsorption and Dispersion Stability. Am. Chem. Soc 1984; 161-167
- 18 Rodeghiero F, Tosetto A, Abshire T. , et al; ISTH/SSC joint VWF and Perinatal/Pediatric Hemostasis Subcommittees Working Group. ISTH/SSC bleeding assessment tool: a standardized questionnaire and a proposal for a new bleeding score for inherited bleeding disorders. J Thromb Haemost 2010; 8 (09) 2063-2065
- 19 Gill JC, Cristopherson PA, Flood VH, Friedman KD, Montgomery RR. Bleeding scores in Von Willebrand disease re-visited: analysis of the TS Zimmerman program for the molecular and clinical biology of VWD. Blood 2008; 112: 162a
- 20 Marcus PD, Nire KG, Grooms L, Klima J, O'Brien SH. The power of a standardized bleeding score in diagnosing paediatric type 1 von Willebrand's disease and platelet function defects. Haemophilia 2011; 17 (02) 223-227
- 21 Tosetto A, Rodeghiero F, Castaman G. , et al. A comparison between two semi-quantitative bleeding scales for the diagnosis and assessment of bleeding severity in type 1 von Willebrand disease. Haemophilia 2011; 17 (01) 165-166
- 22 Lethagen S, Hillarp A, Ekholm C, Mattson E, Halldén C, Friberg B. Distribution of von Willebrand factor levels in young women with and without bleeding symptoms: influence of ABO blood group and promoter haplotypes. Thromb Haemost 2008; 99 (06) 1013-1018
- 23 Zhang W, Deng W, Zhou L. , et al. Identification of a juxtamembrane mechanosensitive domain in the platelet mechanosensor glycoprotein Ib-IX complex. Blood 2015; 125 (03) 562-569
- 24 Shankaran H, Alexandridis P, Neelamegham S. Aspects of hydrodynamic shear regulating shear-induced platelet activation and self-association of von Willebrand factor in suspension. Blood 2003; 101 (07) 2637-2645
- 25 Dunne E, Qi QM, Shaqfeh ES. , et al. Blood group alters platelet binding kinetics to von Willebrand factor and consequently platelet function. Blood 2019; 133 (12) 1371-1377
- 26 Gill JC, Endres-Brooks J, Bauer PJ, Marks Jr WJ, Montgomery RR. The effect of ABO blood group on the diagnosis of von Willebrand disease. Blood 1987; 69 (06) 1691-1695
- 27 Ju L, Dong JF, Cruz MA, Zhu C. The N-terminal flanking region of the A1 domain regulates the force-dependent binding of von Willebrand factor to platelet glycoprotein Ibα. J Biol Chem 2013; 288 (45) 32289-32301
- 28 Moroi M, Jung SM, Shinmyozu K, Tomiyama Y, Ordinas A, Diaz-Ricart M. Analysis of platelet adhesion to a collagen-coated surface under flow conditions: the involvement of glycoprotein VI in the platelet adhesion. Blood 1996; 88 (06) 2081-2092
- 29 Polanowska-Grabowska R, Gibbins JM, Gear ARI. Platelet adhesion to collagen and collagen-related peptide under flow: roles of the α2β1 integrin, GPVI, and Src tyrosine kinases. Arterioscler Thromb Vasc Biol 2003; 23 (10) 1934-1940
- 30 Polanowska-Grabowska R, Gear ARI. High-speed platelet adhesion under conditions of rapid flow. Proc Natl Acad Sci U S A 1992; 89 (13) 5754-5758
- 31 Reininger AJ. VWF attributes--impact on thrombus formation. Thromb Res 2008; 122 (Suppl. 04) S9-S13
- 32 Reininger AJ. Function of von Willebrand factor in haemostasis and thrombosis. Haemophilia 2008; 14 (Suppl. 05) 11-26
- 33 Fressinaud E, Veyradier A, Truchaud F. , et al. Screening for von Willebrand disease with a new analyzer using high shear stress: a study of 60 cases. Blood 1998; 91 (04) 1325-1331
- 34 Lehmann M, Ashworth K, Manco-Johnson M, Di Paola J, Neeves KB, Ng CJ. Evaluation of a microfluidic flow assay to screen for von Willebrand disease and low von Willebrand factor levels. J Thromb Haemost 2018; 16 (01) 104-115
- 35 Hubbell JA, McIntire LV. Platelet active concentration profiles near growing thrombi. A mathematical consideration. Biophys J 1986; 50 (05) 937-945
- 36 Gudmundsdottir BR, Marder VJ, Onundarson PT. Risk of excessive bleeding associated with marginally low von Willebrand factor and mild platelet dysfunction. J Thromb Haemost 2007; 5 (02) 274-281
- 37 Flood VH, Cox JC, Christopherson PA. , et al. Comparison of Type I, Type III, and Type VI collagen binding assays in diagnosis of VWD. J Thromb Haemost 2012; 10: 1537-1554