Abstract

The long-term objective of this research program is to seek new information regarding the structure and function of the various proteins involved in blood coagulation, fibrinolysis, and platelet adhesion in order to understand their mechanism of action, their inhibition, their biosynthesis, and their gene structure. In the present grant application, a major focus will be placed on an investigation of the normal and abnormal genes coding for factor XI and the expression of this protein, the isolation and characterization of a human placental coagulant protein that may play an important role in the initiation of blood coagulation, the structure and function of platelet glycoprotein IV, the structure and function of several placental anticoagulant proteins, the structure and function of the a and b subunits of factor XIII and their genes, the gene organization for plasminogen, the structure and function of factor X and the structure of several domains present in plasma protein. The various protein, described above play an important role in the formation of the platelet plug and fibrin generation following vascular damage. One of these proteins (plasminogen) also plays a key role in dissolving the fibrin clot as vascular repair occurs. These proteins are isolated by conventional methods and their amino acid sequence established by amino acid sequence analysis and CDNA cloning. The structure and organization of the normal and abnormal genes coding for these proteins are established by gene cloning and the polymerase chain reaction. The expression of these proteins will be studied in mammalian cell culture after transfection of their cDNAs present in appropriate expression vectors. Various portions of these molecules will also be examined by site-I specific mutagenesis experiments to provide information regarding the role of specific amino acids-and domains in these proteins. It is hoped that this new information will assist in the clinical management of individuals who have vascular damage or injury or suffer from abnormal blood coagulation (such as hemophilia) or abnormal fibrin clot formation leading to coronary thrombosis.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL016919-20
Application #
3335296
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1989-09-01
Project End
1994-08-31
Budget Start
1993-09-01
Budget End
1994-08-31
Support Year
20
Fiscal Year
1993
Total Cost
Indirect Cost

  Institution

Name
University of Washington
Department
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195

  Publications

Black, Peter C; Mize, Gregory J; Karlin, Peter et al. (2007) Overexpression of protease-activated receptors-1,-2, and-4 (PAR-1, -2, and -4) in prostate cancer. Prostate 67:743-56
Kulman, John D; Satake, Masanobu; Harris, Jeff E (2007) A versatile system for site-specific enzymatic biotinylation and regulated expression of proteins in cultured mammalian cells. Protein Expr Purif 52:320-8
Kulman, John D; Harris, Jeff E; Xie, Ling et al. (2007) Proline-rich Gla protein 2 is a cell-surface vitamin K-dependent protein that binds to the transcriptional coactivator Yes-associated protein. Proc Natl Acad Sci U S A 104:8767-72
Davie, Earl W; Kulman, John D (2006) An overview of the structure and function of thrombin. Semin Thromb Hemost 32 Suppl 1:3-15
Kulman, John D; Harris, Jeff E; Nakazawa, Noriko et al. (2006) Vitamin K-dependent proteins in Ciona intestinalis, a basal chordate lacking a blood coagulation cascade. Proc Natl Acad Sci U S A 103:15794-9
Pratt, Kathleen P; Qian, Jiahua; Ellaban, Ekram et al. (2004) Immunodominant T-cell epitopes in the factor VIII C2 domain are located within an inhibitory antibody binding site. Thromb Haemost 92:522-8
Takeshima, Kazuya; Smith, Christina; Tait, Jonathan et al. (2003) The preparation and phospholipid binding property of the C2 domain of human factor VIII. Thromb Haemost 89:788-94
Greenberg, Daniel L; Mize, Gregory J; Takayama, Thomas K (2003) Protease-activated receptor mediated RhoA signaling and cytoskeletal reorganization in LNCaP cells. Biochemistry 42:702-9
Fuentes-Prior, Pablo; Fujikawa, Kazuo; Pratt, Kathleen P (2002) New insights into binding interfaces of coagulation factors V and VIII and their homologues lessons from high resolution crystal structures. Curr Protein Pept Sci 3:313-39
Chung, Dominic W; Fujikawa, Kazuo (2002) Processing of von Willebrand factor by ADAMTS-13. Biochemistry 41:11065-70

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