A Novel Diagnostic Assay for Platelet-Type von Willebrand Disease Öffentlichkeit
Su, Ally (Spring 2022)
Published
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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