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
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Abstract

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

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