Chemical Crosslinking of Factor VIII C Domains Effect on Membrane Binding
Research Mentor(s)
P. Clint Spiegel
Description
Factor VIII is a multi domain glycoprotein that is found in the bloodstream in the form of a heterodimer. Factor VIII is composed of a heavy chain A1-A2 and light chain A3-C1-C2. Factor VIII binds to activated factor IX on the surface of blood platelets during the blood coagulation cascade. Once activated by thrombin cleavage, it dissociates from von Willebrand factor and C1 and C2 domains bind to activated platelet surfaces. Once factor VIII has bound to lipids, it is a cofactor in activating the factor X tenase complex. The membrane dissociation conformation of the C1 and C2 domains is not fully understood. Previous x ray crystallography data has shown that the C2 domain is highly flexible. An alternative model from previous electron microscopy suggests that C2 domain is in extended conformation when binds membranes. In this study we hypothesize that anchoring FIII C domains together in canonical conformations via a disulfide bond will not affect membrane binding. C1 and C2 domain mutants were expressed and purified in E. coli. C2 expression plasmids were cloned into pET32a vectors via cloning. C1 and C2 domains membrane binding will be investigated via sedimentation assays and BLI.
Document Type
Event
Start Date
May 2022
End Date
May 2022
Location
Carver Gym (Bellingham, Wash.)
Department
CSE - Chemistry
Genre/Form
student projects; posters
Type
Image
Rights
Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.
Language
English
Format
application/pdf
Chemical Crosslinking of Factor VIII C Domains Effect on Membrane Binding
Carver Gym (Bellingham, Wash.)
Factor VIII is a multi domain glycoprotein that is found in the bloodstream in the form of a heterodimer. Factor VIII is composed of a heavy chain A1-A2 and light chain A3-C1-C2. Factor VIII binds to activated factor IX on the surface of blood platelets during the blood coagulation cascade. Once activated by thrombin cleavage, it dissociates from von Willebrand factor and C1 and C2 domains bind to activated platelet surfaces. Once factor VIII has bound to lipids, it is a cofactor in activating the factor X tenase complex. The membrane dissociation conformation of the C1 and C2 domains is not fully understood. Previous x ray crystallography data has shown that the C2 domain is highly flexible. An alternative model from previous electron microscopy suggests that C2 domain is in extended conformation when binds membranes. In this study we hypothesize that anchoring FIII C domains together in canonical conformations via a disulfide bond will not affect membrane binding. C1 and C2 domain mutants were expressed and purified in E. coli. C2 expression plasmids were cloned into pET32a vectors via cloning. C1 and C2 domains membrane binding will be investigated via sedimentation assays and BLI.