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Regulation of the Adhesive and Signaling Functions of Platelet Integrins

S.J. Shattil, J. Gao, T. Hato, H. Kashiwagi, L. Leng, N. Pampori

Platelets express several members of the integrin family of adhesion receptors, including ₂ß₁, ₅ß₁, ₆ß₁, _vß₃, and _IIbß₃. These transmembrane heterodimers mediate platelet interactions with a variety of ligands in the extracellular matrix that are resident within the walls of normal and diseased blood vessels, and they are required for efficient platelet function during hemostasis and thrombosis. The affinity and avidity of integrins for their adhesive ligands are under dynamic cellular control through a process often referred to as "inside out" signaling. In addition, integrin ligands are multivalent and promote integrin clustering within the plane of the plasma membrane, thus exerting a level of control over adhesion-dependent platelet responses through a process of "outside in" signaling.

A long-term goal of our research program is to understand the mechanisms of integrin signaling in molecular detail. We hypothesize that abnormalities in integrin signaling in platelets can predispose to bleeding or thrombosis, depending on the nature of the defect. In addition, we suggest that pharmacologic interruption of integrin signaling at strategic points in the pathway might provide new approaches to antithrombotic therapy.

One current experimental focus is _IIbß₃, a receptor for von Willebrand factor and fibrinogen. This integrin is expressed only on platelets and megakaryocytes; it mediates platelet aggregation and adhesion, and its biological functions are nonredundant. Recently, we examined the roles of several signaling elements in the functions of _IIbß₃, including the protein tyrosine kinases Syk and Src, the small GTPase RhoA, and the integrin-binding protein ß₃-endonexin.

Syk is essential for the development and function of several hematopoietic cells, and it becomes activated through interaction of its tandem SH2 domains with immune receptor tyrosine activation (ITAM) motifs in immune response receptors. However, Syk is also activated through integrins, which do not contain ITAMs. A model system with CHO cells was used to study the mechanism of Syk activation by _IIbß₃.

As in platelets, in the CHO cells, Syk underwent tyrosine phosphorylation and activation during adhesion of the cells to _IIbß₃ ligands, including fibrinogen. This activation involved Syk autophosphorylation and the tyrosine kinase activity of Src, and it exhibited 2 novel features. First, unlike _IIbß₃-mediated activation of the integrin-dependent tyrosine kinase pp125FAK, activation of Syk could be triggered by the binding of soluble fibrinogen, could be abolished by truncation of the _IIb or the ß₃ cytoplasmic tail, and was resistant to inhibition by cytochalasin D. Second, it did not require phosphorylated ITAMs; it was unaffected by disruption of an ITAM-interaction motif in the SH2(C) domain of Syk or by simultaneous overexpression of the tandem SH2 domains.

These studies indicate that Syk is a proximal component of outside-in signaling through _IIbß₃ and that it is regulated as a consequence of intimate functional relationships with the _IIbß₃ cytoplasmic tails and with Src or a closely related kinase. Furthermore, activation of Syk by _IIbß₃ differs fundamentally from activation by immune response receptors, a finding that suggests a unique role for integrins in Syk function.

RhoA regulates certain actin rearrangements and transcriptional events in cells. Cell adhesion through integrins triggers similar responses, but the precise relationship between these receptors and Rho is not understood. To examine this relationship in the context of _IIbß₃, we incubated platelets with C3 exoenzyme, which specifically ADP-ribosylates and inactivates Rho. Despite inactivation of 90% or more of RhoA, platelets had normal inside-out signaling, as indicated by agonist-induced binding of a fibrinogen-mimetic antibody to _IIbß₃ and by fibrinogen-dependent platelet aggregation. On the other hand, inactivation of RhoA decreased the adhesion of agonist-stimulated platelets to immobilized fibrinogen and the formation of vinculin-rich focal adhesions in adherent platelets. These effects were selective, because retraction of fibrin clots, a response also dependent on _IIbß₃ and actin contractility, was unaffected by C3, as was the content of F-actin in resting or agonist-stimulated platelets. Similar results were obtained in a CHO cell model system of _IIbß₃. The presence of C3 exoenzyme (or overexpression of a dominant-negative form of RhoA) did not influence the activation state of the integrin, but it did block the formation of focal adhesions in fibrinogen-adherent cells. These studies establish that RhoA plays a highly selective role in _IIbß₃ signaling. It does not influence the ligand-binding function of the integrin, but it does regulate a subset of platelet responses to _IIbß₃ ligation that may be dependent on specific actin rearrangements mediated by RhoA.

ß₃-Endonexin is a recently discovered 111 amino acid polypeptide that interacts selectively with the cytoplasmic tail of the ß₃ integrin subunit, both in vitro and within cells. Overexpression of ß₃-endonexin in CHO cells is associated with energy-dependent activation of the fibrinogen-binding function of _IIbß₃. Messenger RNA for this protein is present in platelets but is also found in many other tissues, and the recombinant form of the protein is distributed in both the nucleus and the cytoplasm of cells. To better understand the physiologic functions of ß₃-endonexin, we are characterizing the murine gene for this polypeptide as a prelude to deleting the gene in mice by using homologous recombination.

PUBLICATIONS

Eigenthaler, M., Shattil, S.J. ß₃-Endonexin. In: Guidebook to the Cytoskeletal and Motor Proteins. Kreis, T., Vale, R. (Eds.). Oxford University Press, New York, in press.

Gao, J., Zoller, K.E., Ginsberg, M.H., Brugge, J.S., Shattil, S.J. Regulation of the pp72Syk protein tyrosine kinase by platelet integrin _IIbß₃. EMBO J. 16:6414, 1997.

George, J., Shattil, S.J. Acquired disorders of platelet function. In: Hematology: Basic Principles and Practice, 3rd ed. Hoffman, R., et al. (Eds.). Saunders, Philadelphia, in press.

Hoffman, R., Benz, E., Shattil, S., Furie, B., Cohen, H., Silberstein, L., McGlave, P., (Eds.). Hematology: Basic Principles and Practice, 3rd ed. Saunders, Philadelphia, in press.

Leng. L., Kashiwagi, H., Ren, X.-D., Shattil, S.J. RhoA and integrin signaling in platelets. Blood, in press.

Phillips, D.R., Teng, W., Arfsten, A., Nanizzi-Alaimo, L., White, M.M., Longhurst, C., Shattil, S.J., Randolph, A., Jakubowski, J.A., Jennings, L.K., Scarborough, R.M. Effect of Ca²⁺ on integrilin-GP IIb-IIIa interactions: Enhanced GP IIb-IIIa binding and inhibition of platelet aggregation by reductions in the concentration of ionized calcium in plasma anticoagulated with citrate. Circulation 96:1488, 1997.

Ruggeri, Z.M., FitzGerald, G.A., Shattil, S.J. Platelet thrombus formation and anti-platelet therapy. In: Molecular Basis of Heart Disease. Chien, K.R., et al. (Eds.). Saunders, Philadelphia, in press.

Shattil, S.J., Kashiwagi, H., Pampori, N. Integrin signaling: The platelet paradigm. Blood 91:2645, 1998.

Wang, R., Shattil, S.J., Ambruso, D.R., Newman, P.J. Truncation of the cytoplasmic domain of ß₃ in a variant form of Glanzmann thrombasthenia abrogates signaling through the integrin _IIbß₃ complex. J. Clin. Invest. 100:2393, 1997.