The Journal of Experimental Medicine
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© The Rockefeller University Press, 0022-1007/1998/2/329/ $5.00
The Journal of Experimental Medicine, Volume 187, Number 3, February 2, 1998 329-339

Articles

Adhesion of Activated Platelets to Endothelial Cells: Evidence for a GPIIbIIIa-dependent Bridging Mechanism and Novel Roles for Endothelial Intercellular Adhesion Molecule 1 (ICAM-1), {alpha}vβ3 Integrin, and GPIb{alpha}

Thomas Bombeli, Barbara R. Schwartz, and John M. Harlan

From the Division of Hematology, University of Washington, Seattle, Washington 98195-7710

Although it has been reported that activated platelets can adhere to intact endothelium, the receptors involved have not been fully characterized. Also, it is not clear whether activated platelets bind primarily to matrix proteins at sites of endothelial cell denudation or directly to endothelial cells. Thus, this study was designed to further clarify the mechanisms of activated platelet adhesion to endothelium. Unstimulated human umbilical vein endothelial cell (HUVEC) monolayers were incubated with washed, stained, and thrombin-activated human platelets. To exclude matrix involvement, HUVEC were harvested mechanically and platelet binding was measured by flow cytometry. Before the adhesion assay, platelets or HUVEC were treated with different receptor antagonists. Whereas blockade of platelet β1 integrins, GPIb{alpha}, GPIV, P-selectin, and platelet-endothelial cell adhesion molecule (PECAM)-1 did not reduce platelet adhesion to HUVEC, blockade of platelet GPIIbIIIa by antibodies or Arg-Gly-Asp (RGD) peptides markedly decreased adhesion. Moreover, when platelets were treated with blocking antibodies to GPIIbIIIa-binding adhesive proteins, including fibrinogen and fibronectin, and von Willebrand factor (vWF), platelet binding was also reduced markedly. Addition of fibrinogen, fibronectin, or vWF further increased platelet adhesion, indicating that both endogenous platelet-exposed and exogenous adhesive proteins can participate in the binding process. Evaluation of the HUVEC receptors revealed predominant involvement of intercellular adhesion molecule (ICAM)-1 and {alpha}vβ3 integrin. Blockade of these two receptors by antibodies decreased platelet binding significantly. Also, there was evidence that a component of platelet adhesion was mediated by endothelial GPIb{alpha}. Blockade of β1 integrins, E-selectin, P-selectin, PECAM-1, vascular cell adhesion molecule (VCAM)-1 and different matrix proteins on HUVEC did not affect platelet adhesion. In conclusion, we show that activated platelet binding to HUVEC monolayers is mediated by a GPIIbIIIa-dependent bridging mechanism involving platelet-bound adhesive proteins and the endothelial cell receptors ICAM-1, {alpha}vβ3 integrin, and, to a lesser extent, GPIb{alpha}.

Address correspondence to John M. Harlan, MD., Division of Hematology, Box 357710, 1959 Pacific Street NE, University of Washington, Seattle, WA, 98195-7710. Phone: 206-685-7866; Fax: 206-685-3062; E-mail: jharlan{at}u.washington.edu

1 Abbreviations used in this paper: HUVEC, human umbilical vein endothelial cells; ICAM, intercellular adhesion molecule; PECAM, platelet endothelial cell adhesion molecule; RGD, Arg-Gly-Asp; TEM, transmission electron microscopy; TSP, thrombospondin; VCAM, vascular cell adhesion molecule; vWF, von Willebrand factor.

The authors gratefully acknowledge Dr. R. Rothlein, Dr. D.C. Altieri, Dr. M.R. Zocchi, and Dr. G.J. Roth for providing antibodies. We also thank Dr. J.F. Tait for the generous gift of recombinant annexin V and Dr. S. Lana for performing electron microscopy.


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