The objectives of this proposal are to characterize the mechanisms whereby platelet high molecular weight kininogen (HMWK) can be an extracellular regulator of contact phase zymogen activation, a possible intracellular inhibitor of platelet calpains, and an indirect regulator of endothelial function.
The specific aims of this investigation are as follows: (1) Platelet HMWK present on the external membrane of activated platelets will be quantified and characterized by 125I-monoclonal anti HMWK Fab binding studies; (2) Platelet HMWK will be characterized in unstimulated and activated platelets as to its location and distribution by immunocytochemical staining using colloidal gold; (3) Purified HMWK subunit binding to platelets will be done to determine the portion of the HMWK molecule that binds to platelets and to determine if the affinity of 125I-HMWK-platelet binding is altered when platelets are activated; (4) Coordinate factor XI/XIa-HMWK platelet binding will be performed to determine if HMWK serves as their platelet receptor; (5) The molecular structure of platelet HMWK will be characterized by immunoblot to determine if it is different from plasma HMWK or if it is cleaved by platelet calpain; (6) The effect of purified platelet calpain on purified HMWK will be studied to determine if platelet calpain activates HMWK and produces an altered HMWK with a structure similar to the HMWK seen in platelets, and to determine if HMWk itself can be an inhibitor of platelet calpain; (7) The availability and function of bradykinin derived from platelet HMWK will be ascertained to determine whether it is directly secreted from platelets and to determine whether a sufficient amount can be made available from platelet HMWK to stimulate endothelial cell secretion of prostacyclin. These studies are intended to characterize the platelet surface as a site for contact phase zymogen activation through the expression of HMWK on the platelet external membrane; to demonstrate that HMWK is activated by and is an inhibitor of platelet calpain; and to determine that bradykinin from secreted platelet HMWK is sufficient to stimulate endothelial cell prostacyclin secretion. These objectives are consistent with the hypothesis that platelet HMWK is a mediator and regulator of the surface-activated defense reactions of the proteins of the Hageman factor pathways which participate in the initiation of coagulation, the inflammatory response, and local injury-site blood pressure control.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL035553-02
Application #
3349536
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1986-07-01
Project End
1989-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Temple University
Department
Type
Schools of Medicine
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Schmaier, A H (2000) Plasma kallikrein/kinin system: a revised hypothesis for its activation and its physiologic contributions. Curr Opin Hematol 7:261-5
Schmaier, A H; Rojkjaer, R; Shariat-Madar, Z (1999) Activation of the plasma kallikrein/kinin system on cells: a revised hypothesis. Thromb Haemost 82:226-33
Hasan, A A; Rebello, S S; Smith, E et al. (1999) Thrombostatin inhibits induced canine coronary thrombosis. Thromb Haemost 82:1182-7
Rojkjaer, R; Schmaier, A H (1999) Activation of the plasma kallikrein/kinin system on endothelial cells. Proc Assoc Am Physicians 111:220-7
Rojkjaer, R; Schmaier, A H (1999) Activation of the plasma kallikrein/kinin system on endothelial cell membranes. Immunopharmacology 43:109-14
Hasan, A A; Zisman, T; Schmaier, A H (1998) Identification of cytokeratin 1 as a binding protein and presentation receptor for kininogens on endothelial cells. Proc Natl Acad Sci U S A 95:3615-20
Musial, J; Gluszko, P; Undas, A et al. (1998) Gamma interferon administration to patients with atopic dermatitis inhibits fibrinolysis and elevates C1 inhibitor. Thromb Res 89:253-61
Motta, G; Rojkjaer, R; Hasan, A A et al. (1998) High molecular weight kininogen regulates prekallikrein assembly and activation on endothelial cells: a novel mechanism for contact activation. Blood 91:516-28
Spalding, A; Vaitkevicius, H; Dill, S et al. (1998) Mechanism of epinephrine-induced platelet aggregation. Hypertension 31:603-7
Schmaier, A H; Wahl, R; Fisher, S J et al. (1998) The pharmacokinetics of the kininogens. Thromb Res 92:293-7

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