Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1996-10-18
2001-04-03
Chan, Christina Y. (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007210, C435S007240, C436S501000, C436S503000, C530S324000, C530S325000
Reexamination Certificate
active
06210913
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the modulation of integrin-mediated signal transduction, particularly signal transduction mediated by GPIIb-IIIa on platelets. The invention relates specifically to the identification of molecules that mediate integrin signaling and to methods of modulating integrin-mediated signaling.
BACKGROUND OF THE INVENTION
Integrins are a family of &agr;&bgr; heterodimers that mediate adhesion of cells to extracellular matrix proteins and to other cells (Clark et al.,
Science
(1995) 268: 233-239). Integrins also participate in signal transduction, as evidenced by either an alteration in adhesive affinity of cell surface integrins in response to cellular activation (termed inside-out signal transduction) or by affecting intracellular signaling pathways following integrin-mediated adhesion (termed outside-in signal transduction). Many biological responses are dependent at least to some extent upon integrin-mediated adhesion and cell migration, including embryonic development, hemostasis, clot retraction, mitosis, angiogenesis, cell migration, inflammation, immune response, leukocyte homing and activation, phagocytosis, bone resorption, tumor growth and metastasis, atherosclerosis, restenosis, wound healing, viral infectivity, amyloid toxicity, programmed cell death and the response of cells to mechanical stress.
The integrin family consists of 15 related known &agr; subunits (&agr;
1
, &agr;2, &agr;3, &agr;4, &agr;5, &agr;6, &agr;7, &agr;8, &agr;9, &agr;E, &agr;V, &agr;IIb, &agr;L, &agr;M, and &agr;X) and 8 related known &bgr; subunits (&bgr;1, &bgr;2, &bgr;3, &bgr;4, &bgr;5, &bgr;6, &bgr;7, and &bgr;8, Luscinskas et al.,
FASEB J
(1994) 8:929-938). Integrin &agr; and &bgr; subunits are known to exist in a variety of pairings as indicated in FIG.
1
. Integrin ligand specificity is determined by the specific pairing of the &agr; and &bgr; subunits, although some redundancy exists as several of the integrins are known to bind the same ligand.
FIG. 2
shows the sequences of the cytoplasmic domains of GPIIb and GPIIIa, including the cytoplasmic domains of other &agr; and &bgr; subunits, respectively, that have homologous cytoplasmic domains. Most integrins containing the &bgr;1, &bgr;2, &bgr;3, &bgr;5, &bgr;6, and &bgr;7 subunits have been found to transduce signals (reviewed by Hynes,
Cell
(1992) 62:11-25). Integrins are involved in both “inside-out” and “outside-in” signaling events.
Various pathologies associated with integrin-related defects are known. For example, inherited deficiencies of GPIIb-IIIa (also termed &agr;II&bgr;3) content or function have been described (termed Glanzmann's thrombasthenia) and are characterized by platelets that do not bind adhesive proteins and therefore fail to aggregate, resulting in a life-long bleeding diathesis. Inhibitors of the binding of fibrinogen and von Willebrand factor to GPIIb-IIIa have been described and have been found to block platelet aggregation in vitro and to inhibit clinical thrombosis in vivo (The EPIC Investigators,
New England Journal of Med
(1994) 330:956-961; Tcheng, J. E. et al.,
Circulation
(1995) 91:2151-2157). Also, leukocyte adhesion deficiency (LAD) results from the absence of a &bgr;2 subunit.
A. Inside-Out Signaling
Inside-out signal transduction has been observed for &bgr;1, &bgr;2, and &bgr;3 integrins. (Hynes, R. O.
Cell
(1992) 69:11-25; Phillips, D. R. et al.,
Cell
(1991) 65:359-362; Smyth, S. S. et al.,
Blood
(1993) 81:2827-2843; Ginsberg, M. H. et al.,
Thromb Haemostasis
(1993) 70:87-93; Juliano, R. L. et al.,
Cell Biol
(1993) 120:577-585; Rouslahti, E.,
J Clin Invest
(1991) 87:1-5.
Perhaps the most widely studied integrin that is involved in inside-out signaling is GPIIb-IIIa, the receptor for the four adhesive proteins, fibrinogen, von Willebrand factor, vitronectin and fibronectin, on stimulated platelets (Phillips, D. R. et al.,
Blood
(1988) 71:83143). The binding of adhesive proteins to GPIIb-IIIa is required for platelet aggregation and normal hemostasis and is also responsible for occlusive thrombosis in high shear arteries.
GPIIb-IIIa is known to be involved in inside-out signal transduction because GPIIb-IIIa on the surface of unstimulated platelets is capable of recognizing only immobilized fibrinogen. In response to platelet stimulation by agents such as thrombin, collagen and ADP, GPIIb-IIIa becomes a receptor for the four adhesive proteins in the previous paragraph, and the binding of fibrinogen and von Willebrand factor causes platelets to aggregate. A monoclonal antibody has been described which detects the activated, receptor competent state of GPIIb-IIIa, suggesting that the conformation of the receptor competent form of GPIIb-IIIa differs from that of GPIIb-IIIa which does not bind soluble fibrinogen or von Willebrand factor (Shattil, S. J. et al.,
J Biol Chem
(1985) 260:11107-11114). It has been postulated that inside-out GPIIb-IIIa signal transduction is dependent on cellular proteins that act to repress or stimulate GPIIb-IIIa activation (Ginsberg, M. H. et al.,
Curr Opin Cell Biol
(1992) 4:766-771).
&bgr;2 integrins on leukocytes also respond to inside-out signal transduction which accounts, for example, for the increased binding activity of LFA-1 on stimulated lymphocytes and the increased binding activity of MAC-1 on stimulated neutrophils (reviewed by Springer, T.,
Curr Biol
(1994) 4:506-517).
B. Outside-In Signaling
Most integrins can be involved in outside-in signal transduction as evidenced by observations showing that binding of adhesive proteins or antibodies to integrins affects the activities of many cells, for example cellular differentiation, various markers of cell activation, gene expression, and cell proliferation (Hynes, R. O.
Cell
(1992) 69:11-25). The involvement of GPIIb-IIIa in outside-in signaling is apparent because the binding of unstimulated platelets to immobilized fibrinogen, a process mediated by GPIIb-IIIa, leads to platelet activation and platelet spreading (Kieffer, N. et al.,
J Cell Biol
(1991) 113:451-461).
Outside-in signaling through GPIIb-IIIa also occurs during platelet aggregation. Signaling occurs because fibrinogen or von Willebrand factor bound to the activated form of GPIIb-IIIa on the surface of stimulated platelets, coupled with the formation of platelet-platelet contacts, causes further platelet stimulation through GPIIb-IIIa signal transduction. In this manner, binding of adhesive proteins to GPIIb-IIIa can both initiate platelet stimulation or can augment stimulation induced by the other platelet agonists such as ADP, thrombin and collagen. The binding of soluble fibrinogen to GPIIb-IIIa on unstimulated platelets can also be induced by selected GPIIb-IIIa antibodies such as LIBS6 (Huang, M -M. et al.,
J Cell Biol
(1993) 122:473-483): although platelets with fibrinogen bound in this manner are not believed to be stimulated, such platelets will aggregate if agitated and will become stimulated following aggregation through GPIIb-IIIa signal transduction.
Outside-in integrin signal transduction results in the activation of one or more cascades within cells. For GPIIb-IIIa, effects caused by integrin ligation include enhanced actin polymerization, increased Na
+
/H
+
exchange, activation of phospholipases, increased phosphatidyl turnover, increased cytoplasmic Ca
++
, and activation of kinases. Kinases known to be activated include PKC, myosin light chain kinase, src, syk and pp125FAK. Kinase substrates identified include pleckstrin, myosin light chain, src, syk, pp125FAK, and numerous proteins yet to be identified (reviewed in Clark, E. A. et al.,
Science
(1995) 268:233-239). Many of these signaling events, including phosphorylations, also occur in response to ligation of other integrins (reviewed in Hynes, R. O.
Cell
(1992) 69:11-25). Although these other integrins have distinct sequences and distinct &agr;-&bgr;3 parings that allow for ligand specificity, the highly conserved nature of the relatively small cytoplasmic domains, bot
Alaimo Lisa Nannizzi
Law Deborah Ann
Phillips David R.
Chan Christina Y.
COR Therapeutics Inc.
Ewoldt Gerald R.
Morgan & Lewis & Bockius, LLP
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