Co-Author(s)

Serena Wo, Martha Torujo, Alexis Neuman

Research Mentor(s)

Clint Spiegel

Affiliated Department

Chemistry

Sort Order

46

Start Date

17-5-2017 12:00 PM

End Date

17-5-2017 3:00 PM

Document Type

Event

Abstract

Blood coagulation factor VIII is an essential cofactor in the mammalian blood-clotting cascade. FVIII must bind the phospholipid membrane of activated platelets to function as a cofactor for FIXa. The blood coagulation cascade culminates in the formation of a stable blood clot. In humans, the C1 and C2 domains are implicated in binding phospholipid membranes, however the relative contribution of different residues in the lipid-binding mechanism is unclear. Using site-directed mutagenesis, expression of the isolated C1 and C2 domains in Escherichia coli cells, protein purification with metal affinity chromatography, electrospray ionization mass spectrometry, enzyme-linked immunosorbent assays, liposome sedimentation assays, pull down assays, circular dichroism and intrinsic tryptophan fluorescence, we compare relative binding affinities and stability of five mutations on the C1 and C2 domains. Three of the mutations are on the C2 domain: R2320S, R2320T, and R2215A. The other two mutations are on the C1 domain: R2163H and R2159H. Our results combined with the crystal structure of C1 and C2 soaked with O-phosphatidylserine moieties will help elucidate the roles of these residues in the function and stability of FVIII with regards to Hemophilia A.

Comments

Outstanding Poster Award Recipient

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 documentation for commercial purposes, or for financial gain, shall not be allowed without the author's written permission.

Language

English

Format

application/pdf

Share

COinS
 
May 17th, 12:00 PM May 17th, 3:00 PM

Lipid Binding Studies of Blood Coagulation Factor VIII C1 and C2 Domains

Chemistry

Blood coagulation factor VIII is an essential cofactor in the mammalian blood-clotting cascade. FVIII must bind the phospholipid membrane of activated platelets to function as a cofactor for FIXa. The blood coagulation cascade culminates in the formation of a stable blood clot. In humans, the C1 and C2 domains are implicated in binding phospholipid membranes, however the relative contribution of different residues in the lipid-binding mechanism is unclear. Using site-directed mutagenesis, expression of the isolated C1 and C2 domains in Escherichia coli cells, protein purification with metal affinity chromatography, electrospray ionization mass spectrometry, enzyme-linked immunosorbent assays, liposome sedimentation assays, pull down assays, circular dichroism and intrinsic tryptophan fluorescence, we compare relative binding affinities and stability of five mutations on the C1 and C2 domains. Three of the mutations are on the C2 domain: R2320S, R2320T, and R2215A. The other two mutations are on the C1 domain: R2163H and R2159H. Our results combined with the crystal structure of C1 and C2 soaked with O-phosphatidylserine moieties will help elucidate the roles of these residues in the function and stability of FVIII with regards to Hemophilia A.

 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.