228
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[21]
the slope of the curve and the maximal binding capacity (number of binding sites) from the x intercept. Curve fitting and calculation of the binding constants are improved substantially by using a computer program based on the least squares method. 8 Comments Platelet-activating factor is a phospholipid and therefore requires special handling. Because PAF is insoluble in aqueous solutions, buffers must contain 0. I% (w/v) human serum albumin or 0.25% BSA as carrier proteins. Thus, the binding data obtained in these studies represent an equilibrium between receptor bound- and albumin-bound PAF. Plateletactivating factor is stable for at least 6 months when stored at - 2 0 ° in methanol or 2.5% BSA in PBS under an atmosphere of air. Stock solutions of tritiated PAF and unlabeled PAF may be used repeatedly in binding assays. 8 p. j. Munson and D. Rodbard, Anal Biochem. 107, 220 (1980).
[21] B i n d i n g o f F i b r i n o g e n a n d y o n W i l l e b r a n d F a c t o r to Platelet Glycoprotein IIb-IIIa Complex B y JACEK H A W I G E R a n d SHEILA TIMMONS
The mechanism through which a platelet hemostatic plug is formed involves at least two adhesive macromolecules: von Willebrand factor (vWF) and fibrinogen. ~ von Willebrand factor is thought to provide a molecular anchor between the subendothelium and platelets, because in von Willebrand disease formation of a platelet hemostatic plug is impaired. On the other hand, fibrinogen is considered to provide interplatelet linkages after activation of platelets with ADP and exposure of the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor for fibrinogen. 2 The involvement of fibrinogen in platelet aggregation remained unexplained until it was demonstrated that aggregation of platelets induced by ADP is accompanied by their binding of fibrinogen. Subsequent experiHawiger, in "Hemostasis and Thrombosis: Basic Principles and Clinical Practice" (R. W. Colman, J. Hirsh, V. J. Marder, and E. W. Salzman, eds.), Chapter 12, p. 182. Lippincott, Philadelphia, 1987. 2 j. Hawiger, Ann. N.Y. Acad. Sci. 614, 270 (1991). I j.
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[21]
FIBRINOGEN AND vWF BINDINGTO GPIIb-IIIa COMPLEX
229
ments indicated that the main role of platelet agonists such as ADP, epinephrine, or thrombin is to induce exposure of binding sites for fibrinogen on the platelet membrane. After stimulation with thrombin and inactivation of free thrombin with hirudin, we observed specific binding of tzSI-labeled fibrinogen with remarkably similar characteristics to those after stimulation with ADP. 3 The interaction of fibrinogen with human platelets stimulated with thrombin (0.05 U/ml) fulfills the criteria for specific, saturable, and reversible binding. It is mediated by receptors that bind approximately 44,000 molecules of fibrinogen per platelet with an apparent dissociation constant (KD) of 1.8 × 10 - 7 M. Because the plasma concentration of fibrinogen is approximately 50 times higher, it is apparent that even when its plasma level decreases to 5% of the normal value the fibrinogen level is still sufficient to saturate the available binding sites. The binding of fibrinogen to platelets requires calcium, because it is prevented or reversed by ethylenediaminetetraacetic acid (EDTA). The platelet membrane glycoprotein IIb-IIIa complex that is involved in the binding of fibrinogen belongs to the/33 family of the integrin receptor gene superfamily. 4 About 40,000 molecules of the glycoprotein lib-Ilia complex (integrin Otllb/33) are available on one platelet. 5 This suggests a 1 : 1 stoichiometry between fibrinogen and the glycoprotein IIb-IIIa complex on the membrane of activated platelets. We have shown that this process is either prevented or reversed by cyclic AMP-mediated reactions, inasmuch as an increase in cAMP levels induced by prostacyclin (PGI2) or a nonprostanoid inhibitor, forskolin, correlates with inhibition of binding of fibrinogen to activated platelets, which is paralleled by inhibition of platelet aggregation. 3.6Isolated glycoproteins l i b - I l i a in the form of a calcium-held heterodimer bind fibrinogen attached to solid support. 7 Isolated GPIIb-IIIa complexes can be incorporated into phospholipid vesicles (liposomes) and used for binding studies. Their binding capacity, however, is much lower. 8 The primary structure of GPIIb and GPIIIa was elucidated by cDNA cloning and sequencing. 9'1° 3 j. Hawiger, S. Parkinson, and S. Timmons, Nature (London) 283, 195 (1980). 4 E. Ruoslahti and M. D. Pierschbacher, Science 238, 491 (1987). 5 B. S. Coller, E. I. Peerschke, L. E. Scuder, and C. A. Sullivan, J. Clin. Invest. 72, 325 (1983). 6 S. Graber and J. Hawiger, J. Biol. Chem. 257, 14606 (1982). 7 R. L. Nachman, L. L. K. Leung, M. Kloczewiak, and J. Hawiger, J. Biol. Chem. 259, 8584 (1984). 8 L. Parise and D. R. Phillips, J. Biol. Chem. 261, 14011 (1986). 9 M. Poncz, R. Eisman, R. Heidenreich, S. M. Silver, G. Vilaire, S. Surrey, E. Schwartz, and J. S. Bennett, J. Biol. Chem. 262, 8467 (1987). l0 L. A. Fitzgerald, B. Steiner, S. C. Rail, Jr., S. S. Lo, and D. R. Phillips, J. Biol. Chem. 262, 3936 (1987).
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Human fibrinogen interacts with binding sites exposed on GPIIb-IIIa of stimulated platelets through the tentacles present on ,/and a chains.ll The 12-residue carboxyl-terminal segment of the y chain, encompassing residues 400-411, was pinpointed by us as the platelet receptor recognition domain. 12-14 Following our findings that isolated a chains from human fibrinogen were reactive with ADP-activated platelets, 11 we showed that the sequences RGDF (a95-98) and RGDS (o
PLATELET
RECEPTORS; ASSAYS AND PURIFICATION
[21]
the slope of the curve and the maximal binding capacity (number of binding sites) from the x intercept. Curve fitting and calculation of the binding constants are improved substantially by using a computer program based on the least squares method. 8 Comments Platelet-activating factor is a phospholipid and therefore requires special handling. Because PAF is insoluble in aqueous solutions, buffers must contain 0. I% (w/v) human serum albumin or 0.25% BSA as carrier proteins. Thus, the binding data obtained in these studies represent an equilibrium between receptor bound- and albumin-bound PAF. Plateletactivating factor is stable for at least 6 months when stored at - 2 0 ° in methanol or 2.5% BSA in PBS under an atmosphere of air. Stock solutions of tritiated PAF and unlabeled PAF may be used repeatedly in binding assays. 8 p. j. Munson and D. Rodbard, Anal Biochem. 107, 220 (1980).
[21] B i n d i n g o f F i b r i n o g e n a n d y o n W i l l e b r a n d F a c t o r to Platelet Glycoprotein IIb-IIIa Complex B y JACEK H A W I G E R a n d SHEILA TIMMONS
The mechanism through which a platelet hemostatic plug is formed involves at least two adhesive macromolecules: von Willebrand factor (vWF) and fibrinogen. ~ von Willebrand factor is thought to provide a molecular anchor between the subendothelium and platelets, because in von Willebrand disease formation of a platelet hemostatic plug is impaired. On the other hand, fibrinogen is considered to provide interplatelet linkages after activation of platelets with ADP and exposure of the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor for fibrinogen. 2 The involvement of fibrinogen in platelet aggregation remained unexplained until it was demonstrated that aggregation of platelets induced by ADP is accompanied by their binding of fibrinogen. Subsequent experiHawiger, in "Hemostasis and Thrombosis: Basic Principles and Clinical Practice" (R. W. Colman, J. Hirsh, V. J. Marder, and E. W. Salzman, eds.), Chapter 12, p. 182. Lippincott, Philadelphia, 1987. 2 j. Hawiger, Ann. N.Y. Acad. Sci. 614, 270 (1991). I j.
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[21]
FIBRINOGEN AND vWF BINDINGTO GPIIb-IIIa COMPLEX
229
ments indicated that the main role of platelet agonists such as ADP, epinephrine, or thrombin is to induce exposure of binding sites for fibrinogen on the platelet membrane. After stimulation with thrombin and inactivation of free thrombin with hirudin, we observed specific binding of tzSI-labeled fibrinogen with remarkably similar characteristics to those after stimulation with ADP. 3 The interaction of fibrinogen with human platelets stimulated with thrombin (0.05 U/ml) fulfills the criteria for specific, saturable, and reversible binding. It is mediated by receptors that bind approximately 44,000 molecules of fibrinogen per platelet with an apparent dissociation constant (KD) of 1.8 × 10 - 7 M. Because the plasma concentration of fibrinogen is approximately 50 times higher, it is apparent that even when its plasma level decreases to 5% of the normal value the fibrinogen level is still sufficient to saturate the available binding sites. The binding of fibrinogen to platelets requires calcium, because it is prevented or reversed by ethylenediaminetetraacetic acid (EDTA). The platelet membrane glycoprotein IIb-IIIa complex that is involved in the binding of fibrinogen belongs to the/33 family of the integrin receptor gene superfamily. 4 About 40,000 molecules of the glycoprotein lib-Ilia complex (integrin Otllb/33) are available on one platelet. 5 This suggests a 1 : 1 stoichiometry between fibrinogen and the glycoprotein IIb-IIIa complex on the membrane of activated platelets. We have shown that this process is either prevented or reversed by cyclic AMP-mediated reactions, inasmuch as an increase in cAMP levels induced by prostacyclin (PGI2) or a nonprostanoid inhibitor, forskolin, correlates with inhibition of binding of fibrinogen to activated platelets, which is paralleled by inhibition of platelet aggregation. 3.6Isolated glycoproteins l i b - I l i a in the form of a calcium-held heterodimer bind fibrinogen attached to solid support. 7 Isolated GPIIb-IIIa complexes can be incorporated into phospholipid vesicles (liposomes) and used for binding studies. Their binding capacity, however, is much lower. 8 The primary structure of GPIIb and GPIIIa was elucidated by cDNA cloning and sequencing. 9'1° 3 j. Hawiger, S. Parkinson, and S. Timmons, Nature (London) 283, 195 (1980). 4 E. Ruoslahti and M. D. Pierschbacher, Science 238, 491 (1987). 5 B. S. Coller, E. I. Peerschke, L. E. Scuder, and C. A. Sullivan, J. Clin. Invest. 72, 325 (1983). 6 S. Graber and J. Hawiger, J. Biol. Chem. 257, 14606 (1982). 7 R. L. Nachman, L. L. K. Leung, M. Kloczewiak, and J. Hawiger, J. Biol. Chem. 259, 8584 (1984). 8 L. Parise and D. R. Phillips, J. Biol. Chem. 261, 14011 (1986). 9 M. Poncz, R. Eisman, R. Heidenreich, S. M. Silver, G. Vilaire, S. Surrey, E. Schwartz, and J. S. Bennett, J. Biol. Chem. 262, 8467 (1987). l0 L. A. Fitzgerald, B. Steiner, S. C. Rail, Jr., S. S. Lo, and D. R. Phillips, J. Biol. Chem. 262, 3936 (1987).
230
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[21]
Human fibrinogen interacts with binding sites exposed on GPIIb-IIIa of stimulated platelets through the tentacles present on ,/and a chains.ll The 12-residue carboxyl-terminal segment of the y chain, encompassing residues 400-411, was pinpointed by us as the platelet receptor recognition domain. 12-14 Following our findings that isolated a chains from human fibrinogen were reactive with ADP-activated platelets, 11 we showed that the sequences RGDF (a95-98) and RGDS (o
