The aims of this proposal are the identification and characterization of the different regions on the light chain (LC) and heavy chain (HC), of high molecular weight kininogen (HK) responsible for the novel in- teraction with platelet thrombospondin (TSP), to define the primary structure and its functional role. Peptide fragments from the HK-LC and HK-HC will be generated by the use of a thiol protease enzyme from B. gingivalis, which cleaves efficiently both chains into small fragments: The proteolytic fragments will the be applied to a C-18 or C-4 column and purified by size exclusion or reverse phase HPLC. Peaks from HPLC will then be dot blotted onto a PVDF membrane. Radiolabeled TSP will serve as a probe to identify peaks containing binding domains, and a polyclonal antibody directed against either HK-LC or HK-HC will determine their chain of origin. The fraction(s) containing fragments reacting against both probes(labeled TSP and each of the two different antibodies) will be analyzed by SDS-PAGE electrophoresis to confirm its size and purity. The N-terminal sequence of each purified polypeptide will be determined, which will place them in the known amino acid sequence. The isolated peptides will be tested for their ability to compete for the binding of radiolabeled TSP to microtiter plates precoated with HK. To confirm the minimum sequence needed for the binding to TSP, a molecular biological approach will also be used to provide independent information. The HK-LC will be expressed in E. coli. After assessing the functional biological activity of the rHK-LC, its structure will be altered ( based on the results from the protein chemistry experiments) by site specific mutagenesis using polymerase chain reaction (PCR). The long-term goals are related to the functional role of the HK binding to TSP. TSP and HK are two multifunctional proteins playing an important role on the platelet function although their mechanism of action remains obscure. The correlation of structure-function will provide a better understanding of the interaction of TSP-HK during pathophysiological conditions.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
1K08HL002681-01
Application #
3083093
Study Section
Research Manpower Review Committee (MR)
Project Start
1991-09-30
Project End
1996-09-29
Budget Start
1991-09-30
Budget End
1992-09-29
Support Year
1
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Temple University
Department
Type
Schools of Medicine
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Selim, T E; Ghoneim, H R; Abdel Ghaffar, H A et al. (2001) High molecular mass kininogen inhibits cathepsin G-induced platelet activation by forming a complex with cathepsin G. Hematol J 2:371-7
Majluf-Cruz, A; Manns, J M; Uknis, A B et al. (2000) Residues F16-G33 and A784-N823 within platelet thrombospondin-1 play a major role in binding human neutrophils: evaluation by two novel binding assays. J Lab Clin Med 136:292-302
DeLa Cadena, R A; Majluf-Cruz, A; Stadnicki, A et al. (1998) Recombinant tumor necrosis factor receptor p75 fusion protein (TNFR:Fc) alters endotoxin-induced activation of the kinin, fibrinolytic, and coagulation systems in normal humans. Thromb Haemost 80:114-8
Kaplan, R; DeLa Cadena, R A (1998) Mechanism of the coagulopathy associated with acute promyelocytic leukemia -clinical conference-. Am J Hematol 59:234-7
Khan, M M; Kunapuli, S P; Lin, Y et al. (1998) Three noncontiguous peptides comprise binding sites on high-molecular-weight kininogen to neutrophils. Am J Physiol 275:H145-50
DeLa Cadena, R A; Kunapuli, S P; Walz, D A et al. (1998) Expression of thrombospondin 1 on the surface of activated platelets mediates their interaction with the heavy chains of human kininogens through Lys 244-Pro 254. Thromb Haemost 79:186-94
Selim, T E; Ghoneim, H R; Uknis, A B et al. (1997) High-molecular-mass and low-molecular-mass kininogens block plasmin-induced platelet aggregation by forming a complex with kringle 5 of plasminogen/plasmin. Eur J Biochem 250:532-8
Bradford, H N; Dela Cadena, R A; Kunapuli, S P et al. (1997) Human kininogens regulate thrombin binding to platelets through the glycoprotein Ib-IX-V complex. Blood 90:1508-15
Chouhan, V D; De La Cadena, R A; Nagaswami, C et al. (1997) Simultaneous occurrence of human antibodies directed against fibrinogen, thrombin, and factor V following exposure to bovine thrombin: effects on blood coagulation, protein C activation and platelet function. Thromb Haemost 77:343-9
Dela Cadena, R A; Stadnicki, A; Uknis, A B et al. (1995) Inhibition of plasma kallikrein prevents peptidoglycan-induced arthritis in the Lewis rat. FASEB J 9:446-52

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