The specific aims of this project are to solve the three- dimensional structures of recombinant fragments of human blood clotting factors. The ultimate goals of these structural studies are to attain a better understanding of the molecular events that occur in the blood coagulation cascade and to aid the design of new anticoagulant drugs. 1. We will determine the structure of the C2 domain of human factor VIII, a central regulatory factor of the coagulation cascade. The C2 domain is essential for association of the protein with von Willebrand factor (vWF), and for the localization of factor VIII to sites of blood vessel injury, where it binds tightly and specifically to phosphatidyl serine (PS) head groups. An 18 kDa protein corresponding to the C2 domain of factor VIII has been crystallized in a space group that diffracts to 2.0 A resolution. An excellent heavy-metal derivative, made through incorporation of a free surface cysteine and binding of a mercurial reagent, has been used to generate phases. Model building is now underway. An additional data set has been collected after soaking crystals of the C2 domain with the PS head group analog glycerophosphoserine. 24 mutations in the C2 domain have been associated with type A hemophilia, an often devastating bleeding disorder that affects about 17,000 people in the United States. Ten of these mutations have been introduced into recombinant C2 molecules, and crystallographic studies of several will be carried out, upon completion of the native structure. 2. We will determine the structures of two fibrinogen gamma-chain mutants associated with dysfibringenemia using molecular replacement. These data sets have been collected to 2.3 A resolution and initial MR searches are complete. Crystal structures of a 30 kDa fragment from the c-terminal end of the fibrin(ogen) gamma chain have been determined recently, and the primary fibrin polymerization site has been identified and characterized. 3. We will begin the next stage of these studies by purifying milligram quantities of the N-terminal domain from von Willebrand factor (residues 1 to 272), which is the site of association with factor VIII. We will attempt to crystallize this domain alone and in complex with several factor VIII constructs: recombinant C2 domain, recombinant acidic region (14 kDa) from the factor VIII light chain, and a synthetic acidic peptide from the same region. These factor VIII constructs are known to constitute the high affinity binding site for vWF, and independently display affinity constants as low as 72 nM.
| Spiegel, P Clint; Kaiser, Shari M; Simon, Julian A et al. (2004) Disruption of protein-membrane binding and identification of small-molecule inhibitors of coagulation factor VIII. Chem Biol 11:1413-22 |
| Spiegel Jr, P Clint; Stoddard, Barry L (2002) Optimization of factor VIII replacement therapy: can structural studies help in evading antibody inhibitors? Br J Haematol 119:310-22 |
| Liu, M L; Shen, B W; Nakaya, S et al. (2000) Hemophilic factor VIII C1- and C2-domain missense mutations and their modeling to the 1.5-angstrom human C2-domain crystal structure. Blood 96:979-87 |
