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Protease
Signaling 
Platelets are critical effectors of hemostasis and thrombosis, and the
coagulation protease thrombin is arguably the best activator of platelets
ex vivo. Protease signaling in other cell types is likely important in
inflammation, angiogenesis, and embryonic development. How does a protease
behave like a hormone to regulate cellular behavior? We have characterized
a family of protease-activated G protein-coupled receptors (PARs) that
provide one answer. Thrombin cleaves PAR1's amino terminal exodomain to
unmask a new amino terminus that then serves as a tethered peptide ligand,
binding intramolecularly to the body of the receptor to cause transmembrane
signaling. PAR1 is the prototype for a small family of PARs. PAR1, PAR3,
and PAR4 can be activated by thrombin. PAR2 can be activated by trypsin,
mast cell tryptase, and coagulation factors upstream of thrombin (see
below) but not by thrombin itself. Current work focuses on:
Mechanisms of PAR activation. We are examining PAR-PAR interactions
and novel cofactor mechanisms by which protease and receptor are brought
together. For example, unlike other PARs, mouse PAR3 does not itself mediate
transmembrane signaling but instead binds thrombin and promotes cleavage
and activation of mouse PAR4. The role of receptor oligomerization and/or
localization to specific plasma membrane "rafts" in this interesting paradigm
for receptor interaction is being explored. We are also examining PAR
activation by proteases other than thrombin and the role of known protease
cofactors in this process. We recently showed that tissue factor/VIIa
complex and factor Xa can activate PAR2. Thus, while not activated by
thrombin, PAR2 may still sense activation of the coagulation cascade like
PAR1 and PAR4, and proteases upstream of thrombin may have effector functions
other than triggering thrombin generation.
PAR signaling. PAR1 couples to Gq, Gi, and G12/13. The G12/13 signaling
pathway is incompletely characterized. Efforts to identify the effectors
of the G12/13 pathway and the importance of this pathway in platelet,
endothelial, and leukocyte biology are ongoing. Approaches to the latter
include cell type-specific transgene rescue of G13-/- mouse embryos (which
die at ~E9.0) and cell type-specific knockout of G13.
PARs in physiology and disease. Together with the coagulation cascade,
PARs provide a mechanism for cells to sense tissue injury and vascular
leak. Accordingly, PARs may orchestrate cellular responses involved in
hemostasis and thrombosis and inflammation, and perhaps in angiogenesis.
To define the roles of PARs in vivo, we have generated mice deficient
in PAR1, PAR2, PAR3, and PAR4 as well as mice with combined PAR deficiencies.
We are exploring the importance of PAR signaling in platelets and endothelial
cells in mouse models of hemostasis and thrombosis, inflammation, tumor
invasion and angiogenesis.
PARs in embryonic development. Approximately half of PAR1-deficient
mouse embryos die at ~E9.5, apparently from bleeding. This cannot be attributed
to defective platelet function, and strikingly, trangenic expression of
PAR1 in endothelial cells prevented death of PAR1-deficient embryos. Thus
PAR1 signaling in endothelial cells is important for normal vascular development
and hemostasis in the mouse embryo. Efforts to identify the specific endothelial
responses involved and the other targets of the coagulation cascade that
are important for embryonic development are in progress. We expect that
this line of research will identify novel roles for coagulation protease
and their receptors in embryonic development.
Selected Publications:
Kahn, M.L., Zheng, Y.-W., Huang, W., Bigornia, V., Zeng, D., Moff, S.,
Farese, R.V., Jr., Tam, C., and Coughlin, S.R. (1998) A two thrombin receptor
system for platelet activation. Nature 394: 690-694.
Kahn, M.L., Nakanishi-Matsui, M., Shapiro, M.J., Ishihara, H., and Coughlin,
S.R. (1999). Protease-activated receptors 1 and 4 mediate activation of
human platelets by thrombin. J. Clin. Invest. 103: 879-887.
Nakanishi-Matsui, M., Zheng, Y.-W., Sulciner, D., Weiss, E.J., Ludeman,
M.J. and Coughlin, S.R. (2000) PAR3 is a cofactor for PAR4 activation
by thrombin. Nature 404: 609-613.
Camerer, E., Huang, W., and Coughlin, S.R. (2000) Tissue factor- and Factor
X-dependent activation of PAR2 by Factor VIIa. Proc. Natl. Acad. Sci.
USA 97(10): 5255-5260.
Sambrano, G., Weiss, E., Zheng, Y.W., Huang, W., and Coughlin, S.R. (2001)
Role of thrombin signaling in platelets in hemostasis and thrombosis.
Nature (In Press).
Griffin, C.T., Srinivasan, Y., Zheng, Y.W., Huang, W., and Coughlin, S.R.
(2001) A role for thrombin receptor signaling in endothelial cells during
embryonic development. Science (In Press).
Contact Information:
Email: coughlin@cvrimail.ucsf.edu
Phone: 415/476-6174
Address: Box 0130, Room HSW 1300
The University of California, San Francisco, CA 94143, (415) 476-9000
Copyright 2003, The Regents of the University of California.
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