The biochemistry, cell and molecular biology of high (HK) and low (LK) molecular weight kininogens will be studied to learn about structure-function correlates as they relate to binding to and effects on blood and vascular cells. D5 has been identified as the region that interacts with anionic surfaces and as a binding site for HKA to neutrophils. Deletion mutants as well as synthesized peptides should allow mapping of the sequence(s) required for binding to neutrophils and endothelial cells. Physical studies of D5 with and without Zn plus plus will be performed. A 31 amino acid sequence in D6 contains sufficient information to bind to two noncontinuous sites on prekallikrein. Using peptides and deletion mutagenesis of D6, the minimal sequences for binding and the topology of HK binding to prekallikrein will be determined. We use these peptides and cognate peptides of prekallikrein to down-regulate fibrinolysis on endothelial cells. D3 has previously been identified as one cell binding region for platelets, endothelial cells and neutrophils. Exons 7, 8, and 9 coding for D3 will be expressed to test the hypothesis that one of the exon products contains all of the information for neutrophil binding on the heavy chain, while another is responsible for inhibiting the binding of thrombin to platelets and endothelial cells. Synthesized, conformationally restrained peptides will be used for fine mapping based on surface-accessible regions of a molecular model of D3 of HK. HK binds to neutrophils on Mac-1, and we will further map the ligand and receptor to better define the interaction. We have recently shown that fibrinogen does not compete with HK binding to endothelial cells, and an antibody to alpha nu beta 3 does not inhibit HK binding, ruling out alpha nu beta 3 as the receptor for HK. However, we found that vitronectin and soluble urokinase receptor inhibit HK binding, suggesting that the UK receptor is the binding site for HK. We will further test this hypothesis by mapping the HK binding site on the UK receptor and map the binding site on HK to the receptor. The mechanism by which HK blocks thrombin binding to platelets by interaction with the thrombin receptor or GPIb will be explored. Kininogen-deficient rats will be used to verify whether HK or LK is an important anti-thrombotic protein. Peptides from kininogen domains will be tested in rats for their antiadhesive and antiplatelet potential. Such polypeptides could serve as templates for peptidomimetic compounds, which should be therapeutic in sepsis, arthritis, hereditary angioedema, and gingival disease in addition to reocclusion after thrombolytic therapy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
1P01HL056914-02
Application #
6273208
Study Section
Project Start
1998-07-01
Project End
1999-06-30
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Temple University
Department
Type
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Guo, Yan-Lin; Wang, Shujie; Cao, Dian J et al. (2003) Apoptotic effect of cleaved high molecular weight kininogen is regulated by extracellular matrix proteins. J Cell Biochem 89:622-32
Wang, Shujie; Hasham, Muneer G; Isordia-Salas, Irma et al. (2003) Upregulation of Cdc2 and cyclin A during apoptosis of endothelial cells induced by cleaved high-molecular-weight kininogen. Am J Physiol Heart Circ Physiol 284:H1917-23
Ahmad, S S; London, F S; Walsh, P N (2003) The assembly of the factor X-activating complex on activated human platelets. J Thromb Haemost 1:48-59
Zhang, H; Colman, R W; Sheng, N (2003) Regulation of CD11b/CD18 (Mac-1) adhesion to fibrinogen by urokinase receptor (uPAR). Inflamm Res 52:86-93
Colman, R W; Pixley, R A; Sainz, I M et al. (2003) Inhibition of angiogenesis by antibody blocking the action of proangiogenic high-molecular-weight kininogen. J Thromb Haemost 1:164-70
Baglia, Frank A; Shrimpton, Corie N; Lopez, Jose A et al. (2003) The glycoprotein Ib-IX-V complex mediates localization of factor XI to lipid rafts on the platelet membrane. J Biol Chem 278:21744-50
Chavakis, Triantafyllos; Santoso, Sentot; Clemetson, Kenneth J et al. (2003) High molecular weight kininogen regulates platelet-leukocyte interactions by bridging Mac-1 and glycoprotein Ib. J Biol Chem 278:45375-81
Baird, T Regan; Walsh, Peter N (2003) Factor XI, but not prekallikrein, blocks high molecular weight kininogen binding to human umbilical vein endothelial cells. J Biol Chem 278:20618-23
Pixley, R A; Lin, Y; Isordia-Salas, I et al. (2003) Fine mapping of the sequences in domain 5 of high molecular weight kininogen (HK) interacting with heparin and zinc. J Thromb Haemost 1:1791-8
Mastellos, Dimitrios; Morikis, Dimitrios; Isaacs, Stuart N et al. (2003) Complement: structure, functions, evolution, and viral molecular mimicry. Immunol Res 27:367-86

Showing the most recent 10 out of 49 publications