Von Willebrand Disease (VWD) is the most common genetic bleeding disorder, with severe health implications in the face of critical injury. The disease impairs effective blood coagulation, specifically in the presence of vessel injury. VWD is caused by a mutation in the gene known as the Von Willebrand Factor (VWF), and causal mutations have been found across the protein sequence [1]. VWF, when acting normally, serves as a critical component of blood vessel maintenance and blood coagulation, as it promotes and regulates the adhesion of platelets and coagulation factors at the site of vessel injury [2]. It is known that VWF’s role is enabled through binding collagen, resulting in proteins attaching to the blood vessel wall and activating a coagulation cascade [3]. A specific domain within the VWF gene, the A1 domain, was discovered to be responsible for the adhesion of platelets in clot formation at the vessel wall [4]. It has been proposed that post-translational modifications play a major role in the proper function of VWF, as the protein undergoes extensive N-linked glycosylation post-translationally [5]. Lastly, it is known that phosphorylation increases the adhesion affinity between platelets and the blood vessel endothelium [6]. However, it is unknown if post-translational phosphorylation of amino acids in the A1 domain plays a role in VWF mediated platelet activation.
The primary goal of this study is to understand how essential phosphorylation of amino acids in the A1 domain is for VWF function and platelet activation. The long-term goal of the project is to discover therapeutic targets, in a either a genetic, genomic, or proteomic sense, that may serve as areas for treatment and rescue of dysfunctional blood coagulation. To achieve such goals, the role of the A1 domain in VWF function will be studied in the zebrafish organism (Danio rerio). Zebrafish are excellent candidates to study this blood disorder due to their transparent bodies and the role of VWF in Danio rerio blood vessel maintenance [7]. Additionally, a microscopic laser can be used to induce a blood vessel injury in zebrafish, and fluorescent tagging of their platelets can allow for observation of consequential platelet activity [8].
Aim 1: Identify conserved phosphorylated amino acids in the A1 domain involved in platelet activation
Approach: Using NCBI-Blast, orthologs of the VWF gene will be identified and their sequences will be comparatively analyzed using Clustal Omega alignment software. From sequence alignment, known A1 domain amino acids, located within AA 1275-1458, will be examined for identified phosphorylation sites. Specifically, serines at positions 1286, 1290, and 1389 will be mutated due to conservation across all model organisms and their known post-translational phosphorylation [9]. Using a CRISPR-Cas9 system, Danio rerio mutants S1285F, S1290F, and S1389F will be created. A laser-induced vessel injury will be performed on control and experimental fish, and their platelets fluorescently tagged and observed for activity following injury.
Rationale: This analysis will allow for the identification of the essential conserved amino acids within the A1 domain that determine VWF function. Understanding the genetic variation within this domain may lead to further indications on what certain mutations mean in terms of VWF function and coagulation efficiency.
Hypothesis: Mutant zebrafish will express lower platelet affinity and VWF function due to mutations in conserved phosphorylation sites in the A1 domain.
Aim 2: Identify differentially expressed genes important for platelet aggregation
Approach: The serine mutants and control zebrafish described in Aim 1 will be used for differential gene expression analysis. Blood from these organisms will be extracted and their endothelial cells isolated for analysis via RNA-sequencing. Sequencing data will identify genes that have increased and decreased expression in both the mutants and the controls. Genes will be sorted by Gene Ontology and their roles in platelet activation validated using a CRISPR-Cas9 system. The genes found to be involved in platelet activation will be knocked-out of WT zebrafish, and their platelet activity will be observed following laser-injury.
Rationale: The results of this aim will identify other genes that play a role in platelet activity and may serve as potential therapeutic targets of VWF dysfunction and the rescue of platelet function.
Hypothesis: Serine mutants will experience down-regulation of genes involved in platelet activation.
Aim 3: Identify proteins that are essential for platelet activation and coagulation
Approach: Proteins from experimental and control zebrafish will be isolated and tagged using 8-plex isobaric tags for relative and absolute quantification (iTRAQ). Following protein tagging, mixture of proteins will be sorted using high-performance liquid chromatography, and proteins identified via Mass Spectrometry. After identification of the novel proteins involved in platelet activation, they will be validated using a CRISPR-Cas9 system. An adeno-associated virus will transfect novel proteins to multiple S1286F mutants, and their platelet activity will be observed following laser-injury.
Rationale: Understanding the proteins that VWF interacts with to mediate platelet adhesion can help us confirm the role of the A1 domain, and its phosphorylation sites, in this coagulation complex. It can also identify novel proteins for therapeutic advances.
Hypothesis: The serine mutants will exhibit decreased expression of proteins involved in platelet activation and blood coagulation compared to control zebrafish.
Through these aims, I hope to establish the role of phosphorylation in the A1 domain and its necessity for VWF function.
The primary goal of this study is to understand how essential phosphorylation of amino acids in the A1 domain is for VWF function and platelet activation. The long-term goal of the project is to discover therapeutic targets, in a either a genetic, genomic, or proteomic sense, that may serve as areas for treatment and rescue of dysfunctional blood coagulation. To achieve such goals, the role of the A1 domain in VWF function will be studied in the zebrafish organism (Danio rerio). Zebrafish are excellent candidates to study this blood disorder due to their transparent bodies and the role of VWF in Danio rerio blood vessel maintenance [7]. Additionally, a microscopic laser can be used to induce a blood vessel injury in zebrafish, and fluorescent tagging of their platelets can allow for observation of consequential platelet activity [8].
Aim 1: Identify conserved phosphorylated amino acids in the A1 domain involved in platelet activation
Approach: Using NCBI-Blast, orthologs of the VWF gene will be identified and their sequences will be comparatively analyzed using Clustal Omega alignment software. From sequence alignment, known A1 domain amino acids, located within AA 1275-1458, will be examined for identified phosphorylation sites. Specifically, serines at positions 1286, 1290, and 1389 will be mutated due to conservation across all model organisms and their known post-translational phosphorylation [9]. Using a CRISPR-Cas9 system, Danio rerio mutants S1285F, S1290F, and S1389F will be created. A laser-induced vessel injury will be performed on control and experimental fish, and their platelets fluorescently tagged and observed for activity following injury.
Rationale: This analysis will allow for the identification of the essential conserved amino acids within the A1 domain that determine VWF function. Understanding the genetic variation within this domain may lead to further indications on what certain mutations mean in terms of VWF function and coagulation efficiency.
Hypothesis: Mutant zebrafish will express lower platelet affinity and VWF function due to mutations in conserved phosphorylation sites in the A1 domain.
Aim 2: Identify differentially expressed genes important for platelet aggregation
Approach: The serine mutants and control zebrafish described in Aim 1 will be used for differential gene expression analysis. Blood from these organisms will be extracted and their endothelial cells isolated for analysis via RNA-sequencing. Sequencing data will identify genes that have increased and decreased expression in both the mutants and the controls. Genes will be sorted by Gene Ontology and their roles in platelet activation validated using a CRISPR-Cas9 system. The genes found to be involved in platelet activation will be knocked-out of WT zebrafish, and their platelet activity will be observed following laser-injury.
Rationale: The results of this aim will identify other genes that play a role in platelet activity and may serve as potential therapeutic targets of VWF dysfunction and the rescue of platelet function.
Hypothesis: Serine mutants will experience down-regulation of genes involved in platelet activation.
Aim 3: Identify proteins that are essential for platelet activation and coagulation
Approach: Proteins from experimental and control zebrafish will be isolated and tagged using 8-plex isobaric tags for relative and absolute quantification (iTRAQ). Following protein tagging, mixture of proteins will be sorted using high-performance liquid chromatography, and proteins identified via Mass Spectrometry. After identification of the novel proteins involved in platelet activation, they will be validated using a CRISPR-Cas9 system. An adeno-associated virus will transfect novel proteins to multiple S1286F mutants, and their platelet activity will be observed following laser-injury.
Rationale: Understanding the proteins that VWF interacts with to mediate platelet adhesion can help us confirm the role of the A1 domain, and its phosphorylation sites, in this coagulation complex. It can also identify novel proteins for therapeutic advances.
Hypothesis: The serine mutants will exhibit decreased expression of proteins involved in platelet activation and blood coagulation compared to control zebrafish.
Through these aims, I hope to establish the role of phosphorylation in the A1 domain and its necessity for VWF function.
References:
[1] Centers for Disease Control and Prevention. (2023b, July 7). What is von willebrand disease?. Centers for Disease Control and Prevention. https://www.cdc.gov/ncbddd/vwd/facts.html
[2] Sadler, J. E. (1998). Biochemistry and genetics of von Willebrand factor. Annual Review of Biochemistry, 67(1), 395–424. https://doi.org/10.1146/annurev.biochem.67.1.395
[3] Manon-Jensen, T., Kjeld, N. G., & Karsdal, M. A. (2016). Collagen-mediated hemostasis. Journal of thrombosis and haemostasis: JTH, 14(3), 438–448. https://doi.org/10.1111/jth.13249
[4] Goodeve A. C. (2010). The genetic basis of von Willebrand disease. Blood reviews, 24(3), 123–134. https://doi.org/10.1016/j.blre.2010.03.003
[5] Simon F. De Meyer, Hans Deckmyn, Karen Vanhoorelbeke; von Willebrand factor to the rescue. Blood 2009; 113 (21): 5049–5057. doi: https://doi.org/10.1182/blood-2008-10-165621
[6] Karlaftis, V., Perera, S., Monagle, P., & Ignjatovic, V. (2016). Importance of post-translational modifications on the function of key haemostatic proteins. Blood coagulation & fibrinolysis: an international journal in haemostasis and thrombosis, 27(1), 1–4. https://doi.org/10.1097/MBC.0000000000000301
[7] Carrillo, M., Kim, S., Rajpurohit, S. K., Kulkarni, V., & Jagadeeswaran, P. (2010). Zebrafish von Willebrand factor. Blood cells, molecules & diseases, 45(4), 326–333. https://doi.org/10.1016/j.bcmd.2010.10.002
[8] Fish, R. J., Freire, C., Di Sanza, C., & Neerman-Arbez, M. (2021). Venous Thrombosis and Thrombocyte Activity in Zebrafish Models of Quantitative and Qualitative Fibrinogen Disorders. International journal of molecular sciences, 22(2), 655. https://doi.org/10.3390/ijms22020655
[9] Department of Health Technology . (n.d.). NetPhos. Bioinformatic tools and services - DTU health tech. https://services.healthtech.dtu.dk/cgi-bin/webface2.cgi?jobid=661F167C0000788D2D0380AD&wait=20
[2] Sadler, J. E. (1998). Biochemistry and genetics of von Willebrand factor. Annual Review of Biochemistry, 67(1), 395–424. https://doi.org/10.1146/annurev.biochem.67.1.395
[3] Manon-Jensen, T., Kjeld, N. G., & Karsdal, M. A. (2016). Collagen-mediated hemostasis. Journal of thrombosis and haemostasis: JTH, 14(3), 438–448. https://doi.org/10.1111/jth.13249
[4] Goodeve A. C. (2010). The genetic basis of von Willebrand disease. Blood reviews, 24(3), 123–134. https://doi.org/10.1016/j.blre.2010.03.003
[5] Simon F. De Meyer, Hans Deckmyn, Karen Vanhoorelbeke; von Willebrand factor to the rescue. Blood 2009; 113 (21): 5049–5057. doi: https://doi.org/10.1182/blood-2008-10-165621
[6] Karlaftis, V., Perera, S., Monagle, P., & Ignjatovic, V. (2016). Importance of post-translational modifications on the function of key haemostatic proteins. Blood coagulation & fibrinolysis: an international journal in haemostasis and thrombosis, 27(1), 1–4. https://doi.org/10.1097/MBC.0000000000000301
[7] Carrillo, M., Kim, S., Rajpurohit, S. K., Kulkarni, V., & Jagadeeswaran, P. (2010). Zebrafish von Willebrand factor. Blood cells, molecules & diseases, 45(4), 326–333. https://doi.org/10.1016/j.bcmd.2010.10.002
[8] Fish, R. J., Freire, C., Di Sanza, C., & Neerman-Arbez, M. (2021). Venous Thrombosis and Thrombocyte Activity in Zebrafish Models of Quantitative and Qualitative Fibrinogen Disorders. International journal of molecular sciences, 22(2), 655. https://doi.org/10.3390/ijms22020655
[9] Department of Health Technology . (n.d.). NetPhos. Bioinformatic tools and services - DTU health tech. https://services.healthtech.dtu.dk/cgi-bin/webface2.cgi?jobid=661F167C0000788D2D0380AD&wait=20
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Specific Aims Draft #2
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