Arteriosclerosis and Thrombosis, Vol 11, 704-718, Copyright © 1991 by American Heart Association

ARTICLES

Thrombin-induced platelet aggregates have a dynamic structure. Time- dependent redistribution of glycoprotein IIb-IIIa complexes and secreted adhesive proteins

E Heilmann, P Hourdille, A Pruvost, A Paponneau and AT Nurden
URA 1464 CNRS, Pathologie Cellulaire de l'Hemostase, Hopital Cardiologique, Pessac, France.

The role of glycoprotein (GP) IIb-IIIa complexes and of adhesive proteins in mediating platelet aggregation is now well defined. However, less is known of the changes that occur once aggregation has begun. We report immunogold staining of thin sections of platelets or platelet aggregates, embedded in Lowicryl K4M, after the use of polyclonal antibodies to GP IIb or GP IIIa, fibrinogen (Fg), von Willebrand factor (vWF), and thrombospondin (TSP). Bound immunoglobulin G (IgG) was located by species-specific anti-IgG coupled to 5-nm gold particles and by electron microscopy. Initial experiments with platelet- rich plasma confirmed the feasibility of visualizing adhesive proteins between platelets in aggregates. Experiments then continued, using stirred suspensions of washed platelets incubated with alpha-thrombin. After 20 seconds, platelets were in contact without detectable release, although giant secretory vesicles containing adhesive proteins were seen. Internal pools of GP IIb-IIIa were progressively externalized within the aggregate. Secreted Fg was readily detected between platelets at 40 seconds. After 3 minutes, when most of the secretion had occurred, Fg had a patchwork-like distribution within the aggregate. After 6 minutes, zones with closely interspaced surface membranes, usually representing pseudopods, were dominant and Fg free. Results for vWF and TSP were similar to those for Fg. Nonetheless, GP IIb-IIIa complexes continued to be located between adjacent surface membranes throughout the aggregate. Thrombin-induced platelet aggregates were isolated, and sodium dodecyl sulfate-soluble extracts were obtained. Western blot experiments showed that, although fibrinopeptide A had been cleaved, degradation of adhesive proteins by platelet proteases had not occurred. These results emphasize that a platelet aggregate is a dynamic structure and suggest that not all surface-contact interactions are mediated by Fg or the other adhesive proteins tested in this study.

This article has been cited by other articles:

				A. Ono, E. Westein, S. Hsiao, W. S. Nesbitt, J. R. Hamilton, S. M. Schoenwaelder, and S. P. Jackson Identification of a fibrin-independent platelet contractile mechanism regulating primary hemostasis and thrombus growth Blood, July 1, 2008; 112(1): 90 - 99. [Abstract] [Full Text] [PDF]

				P. Nurden, C. Poujol, C. Durrieu-Jais, J. Winckler, R. Combrie, L. Macchi, C. Bihour, C. Wagner, R. Jordan, and A. T. Nurden Labeling of the Internal Pool of GP IIb-IIIa in Platelets by c7E3 Fab Fragments (abciximab): Flow and Endocytic Mechanisms Contribute to the Transport Blood, March 1, 1999; 93(5): 1622 - 1633. [Abstract] [Full Text] [PDF]

				C. Bihour, C. Durrieu-Jais, L. Macchi, C. Poujol, P. Coste, P. Besse, P. Nurden, and A. T. Nurden Expression of Markers of Platelet Activation and the Interpatient Variation in Response to Abciximab Arterioscler Thromb Vasc Biol, February 1, 1999; 19(2): 212 - 219. [Abstract] [Full Text] [PDF]

				O. Peyruchaud, A.T. Nurden, S. Milet, L. Macchi, A. Pannochia, P.F. Bray, N. Kieffer, and F. Bourre R to Q Amino Acid Substitution in the GFFKR Sequence of the Cytoplasmic Domain of the Integrin alpha IIb Subunit in a Patient With a Glanzmann's Thrombasthenia-Like Syndrome Blood, December 1, 1998; 92(11): 4178 - 4187. [Abstract] [Full Text] [PDF]

				V. Dubernard, B. B. Arbeille, M. B. Lemesle, and C. Legrand Evidence for an {alpha}-Granular Pool of the Cytoskeletal Protein {alpha}-Actinin in Human Platelets That Redistributes With the Adhesive Glycoprotein Thrombospondin-1 During the Exocytotic Process Arterioscler Thromb Vasc Biol, October 1, 1997; 17(10): 2293 - 2305. [Abstract] [Full Text]

				M. Humbert, P. Nurden, C. Bihour, J.-M. Pasquet, J. Winckler, E. Heilmann, P. Savi, J.-M. Herbert, T. J. Kunicki, and A. T. Nurden Ultrastructural Studies of Platelet Aggregates From Human Subjects Receiving Clopidogrel and From a Patient With an Inherited Defect of an ADP-Dependent Pathway of Platelet Activation Arterioscler Thromb Vasc Biol, December 1, 1996; 16(12): 1532 - 1543. [Abstract] [Full Text]

				M. M. Rooney, L. V. Parise, and S. T. Lord Dissecting Clot Retraction and Platelet Aggregation J. Biol. Chem., April 12, 1996; 271(15): 8553 - 8555. [Abstract] [Full Text] [PDF]

				A. A. Lopes, N. Y. Maeda, A. Almeida, R. Jaeger, M. Ebaid, and D. Fischer Chamone Circulating Platelet Aggregates Indicative of in Vivo Platelet Activation in Pulmonary Hypertension Angiology, September 1, 1993; 44(9): 701 - 706. [Abstract] [PDF]