A Novel Diagnostic Assay for Platelet-Type von Willebrand Disease Open Access

Su, Ally (Spring 2022)

Permanent URL: https://etd.library.emory.edu/concern/etds/tb09j703s?locale=en


Platelet-type von Willebrand disease (PT-VWD) is rare and often misdiagnosed due to its phenotypically similar symptoms with type 2B VWD. Both subtypes possess gain-of-function mutations that increase the affinity between the von Willebrand factor (VWF) and the glycoprotein Ib⍺ of the platelets. For PT-VWD, this mutation occurs on GPIb⍺. For type 2B VWD, it develops on VWF. The current diagnosis utilizes ristocetin-induced platelet aggregation mixed studies and genetic sequencing, which are unstandardized and expensive. This study established a cell-based model for PT-VWD to circumvent obtaining patient plasma samples and developed a flow cytometry assay to identify PT-VWD. A stable cell line expressing mutated GPIb⍺ (W230L) was generated through transfection and maintained with hygromycin. GPIb⍺(W230L) bound to full-length VWF and monomeric A1 at a higher affinity than GPIb⍺ WT, as observed in flow cytometry. The binding difference between GPIb⍺ (W230L) and GPIb⍺ WT was amplified in a dose-dependent manner when incubated with activators such as botrocetin and 1D12. Utilizing truncated A1 (NAIM-A1) or diluting plasma at a 1:1 ratio also heightened the binding difference between GPIb⍺ (W230L) and GPIb⍺ WT. By employing these parameters to maximize the binding of GPIb⍺ (W230L), it becomes possible to identify PT-VWD and diagnose patients via a rapid and accessible assay.

Table of Contents

Table of Contents

Chapter 1. An Introduction to von Willebrand Disease and von Willebrand Factor       1

1.1.  An Overview                                                                                                              2

1.2.  von Willebrand Disease                                                                                           2

1.3.  Glycoprotein Ib-IX                                                                                                    3

1.4.  Platelet-Type von Willebrand Disease                                                                  3

1.4.1.     Ristocetin-Induced Platelet Aggregation Mixed Studies                           4

1.4.2.     Genetic Testing                                                                                                   5

1.4.3.     Issues                                                                                                                   5

2.          kk

Chapter 2. Materials and Methods                                                                  6

2.1.  Experimental Overview                                                                                        7

2.2.  Establishment of a Stable Cell Line                                                                    7

2.2.1.     The Plasmid                                                                                                      7

2.2.2.     Restriction Digestion                                                                                       7

2.2.3.     Cell Culture                                                                                                        8

2.2.4.     Transfection                                                                                                      9

2.2.5.     Cell Lysis                                                                                                            9

2.2.6.     Western Blot                                                                                                      9

2.2.7.     Cell Sorting                                                                                                       10

2.3.  Flow Cytometry                                                                                                      10

2.3.1.     Expression of GPIbβ and GPIX                                                                     11

2.3.2.     GPIb⍺ Binding to Full-length VWF                                                              11

2.4.  Activators                                                                                                                 11

2.5.  Data analysis on FlowJo                                                                                        12

3.     Results 

Chapter 3. Results                                                                                                 13

3.1.  Establishment of a Stable Cell Line                                                                        14

3.1.1.     Confirming the plasmid with Restriction Digestion                                     14

3.1.2.     Visualizing GFP in CHO cells                                                                          14

3.1.3.     Checking the Success of the Transient transfection of GPIb⍺                   15

3.1.4.     Flow cytometry                                                                                                  16

3.2.  Higher Binding affinity of GPIb⍺                                                                             17

3.3.  Optimizing the conditions                                                                                        17

3.3.1.     GPIb⍺ (W230L) Responds Dose-Dependently to Botrocetin                       18

3.3.2.     GPIb⍺ (W230L) Exhibits Higher Binding to NAIM-A1 (1238-1461)           19

3.3.3.     Determining the Ideal Plasma Concentration at a 1:1 Dilution                  20

3.3.4.     1D12 is a Great Activator 

4.         Discussion

Chapter 4. Discussion                                                                                             21

4.1.  Optimizing the Use of Streptavidin-Conjugated Violet Fluorophore Beads   22

4.2.  Further Studies with Ristocetin                                                                                24

4.3.  The Declining Homogenity of the CHO cell line                                                  24

4.4.  Conclusion                                                                                                                   25

References                                                                                                                            26

Figure 1                                                                                                                                     8

Figure 2                                                                                                                                   12

Figure 3                                                                                                                                   15

Figure 4                                                                                                                                   17

Figure 5                                                                                                                                   18

Figure 6                                                                                                                                   19

Figure 7                                                                                                                                   23

About this Honors Thesis

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
  • English
Research Field
Committee Chair / Thesis Advisor
Committee Members
Last modified

Primary PDF

Supplemental Files