[30]
PLATELET FACTOR XIa RECEPTOR
[30] Binding
of Coagulation Human
Factor
XIa to Receptor
361
on
Platelets
B y DIPALI SINHA and PETER N. WALSH
General Methods Principle
H u m a n platelets h a v e b e e n s h o w n to p r o m o t e the p r o t e o l y t i c activation o f c o a g u l a t i o n f a c t o r s X I I and XI. 1 A n a p p a r e n t r e q u i r e m e n t for this c o n t r i b u t i o n o f platelets to c o n t a c t activation is that first the protein cofactor n o n c o v a l e n t l y associates with f a c t o r XI in plasma, and then f a c t o r X I binds to a c t i v a t e d platelets in the p r e s e n c e o f high m o l e c u l a r weight kininogenfl T h e f a c t o r X I a f o r m e d as a c o n s e q u e n c e binds specifically to a site on platelets distinct f r o m that for f a c t o r XI,3 and retains its structural and functional properties. 3'4 Materials P r o t e i n s . F a c t o r X I (specific activity = 270 U / m g ) 5"6 and high m o l e c u lar weight kininogen 7 (15 U / m g specific activity) are purified as p r e v i o u s l y described. F a c t o r X I a is p r e p a r e d b y incubation with f a c t o r X I I a . 6 Prekallikrein (133 U / m g specific activity), 8 kallikrein, 9 h u m a n a - t h r o m b i n , 3 and p r o t h r o m b i n 6 are p r e p a r e d as described. All proteins should be > 9 8 % pure as j u d g e d b y s o d i u m d o d e c y l sulfate (SDS) p o l y a c r y l a m i d e slab gel electrophoresis. 10 O t h e r R e a g e n t s . M e t h y l silicone oil (1.0. DC200) and Hi P h e n y l silic o n e oil (125 DC550) are o b t a i n e d f r o m William F. N y e , Inc. (Fairhaven, MA); carrier-free Na125I-labeled B o l t o n - H u n t e r reagent and s o d i u m
i p. N. Walsh and J. H. Griffin, Blood 57, 106 (1981). ' J. S. Greengard, M. J. Heeb, E. Ersdal, P. N. Walsh, and J. H. Griffin, Biochemistry 25, 3884 (1986). 3 D. Sinha, F. S. Seaman, A. Koshy, L. C. Knight, and P. N. Walsh, J. Clin. Invest. 73, 1550 (1984). 4 p. N. Walsh, D. Sinha, F. Kueppers, F. S. Seaman, and K. B. Blanstein, J. Clin. Invest. 80, 1578 (1987). 5 B. N. Bouma and J. H. Griffin, J. Biol. Chem. 252, 6432 (1977). 6 p. N. Walsh, H. Bradford, D. Sinha, J. R. Piperno, and G. P. Tuszynski, J. Clin. Invest. 73, 1392 (1984). 7 D. M. Kerbiriou and J. H. Griffin, J. Biol. Chem. 254, 12020 (1979). 8 D. M. Kerbiriou, B. N. Bouma, and J. H. Griffin, J. Biol. Chem. 255, 3952 (1980). 9 B. N. Bouma, L. A. Miles, G. Berena, and J. H. Griffin, Biochemistry 19, 1151 (1980). to U. K. Laemmli, Nature (London) 227, 680 (1970).
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
362
PLATELET RECEPTORS; ASSAYS AND PURIFICATION
[30]
[51Cr]chromate are from New England Nuclear (Boston, MA); Factor XIdeficient plasma is from George King Biomedical (Overland Park, KS); Ultro] HEPES is from Calbiochem Corp. (San Diego, CA); and the chromogenic substrate Pyr-Glu-Pro-Arg-p-nitroanilide hydrochloride (S-2366) is from A. B. Kabi Peptide Research (Stockholm, Sweden).
Radiolabeling For binding experiments, factor XI is radiolabeled with 1251either by the procedure of Bolton and Hunter 1~ or by the Iodogen method ~z as previously described) Free ~zsIis then separated from the labeled protein by passage through a 1-ml Sephadex G-25 column, ~3 and the protein is then dialyzed in the presence of ovalbumin (1 mg/ml). ~25I-Labeled factor XIa is then prepared by incubating the zymogen with factor XIIa?
Assays Coagulation proteins are assayed as previously described, 3 utilizing appropriate congenitally deficient substrate plasmas. Factor XIa is measured by amidolytic assay TM and by radioimmunoassay. TM Protein assays are performed by the method of Lowry et al.~5
Preparation of Cell Suspensions Platelet-rich plasma is obtained from citrated human blood and gel filtered on a Sepharose 2B column into calcium-free HEPES-buffered Tyrode's solution, pH 7.4, containing bovine serum albumin (1 mg/ml), 138 mM NaCI, 2.7 mM KCI, 1.0 mM MgC12 • 6H20, 3.3 mM NaHzPO4 • H20, 15 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 5.5 mM dextrose as previously described? Erythrocyte suspensions are prepared from fresh human blood washed five times in Hanks' balanced salt solution to remove plasma and other cells as previously described. 3 The red cells are finally suspended in calcium-free HEPES-buffered Tyrode's solution, pH 7.4.
tl A. E. Bolton and W. M. Hunter, Biochem. J. 133, 529 (1973). 12 p. j. Fraker and J. C. Speck, Biochem. Biophys. Res. Commun. 80, 849 (1978). 13 G. P. Tuszynski, L. Knight, J. R. Piperno, and P. N. Walsh, Anal. Biochem. 106, 118 (1980). 14 C. F. Scott, D. Sinha, F. S. Seaman, P. N. Walsh, and R. W. Colman, Blood63, 42 0984). i50. H. Lowry, H. J. Rosebrough, A. L. Farr, and F. J. Randall, J. Biol. Chem. 193, 365 (1951).
[30]
PLATELET FACTORXIa RECEPTOR
363
Binding Measurements To measure the binding of factor XIa to platelets over varying incubation times, at variable concentrations of added factor XIa, or under differing conditions, gel-filtered platelets (2-4 × 108/ml) or control cells (e.g., erythrocytes) are incubated at 37 ° in 1.5-ml polypropylene centrifuge tubes (Sarstedt, Inc., Princeton, N J) with 125I-labeled factor XIa, in the presence or absence of high molecular weight kininogen or thrombin (or other platelet agonists). Determinations of specificity of binding and measurements of nonspecific binding are made in the presence of 50- to 100-fold molar excesses of unlabeled factor XIa or other proteins. At specified incubation times, 100-/zl aliquots are layered over a mixture of silicone oils, (I vol of DC200 to 5 vol DC550) contained in microsediment tubes with narrow-bore extended tips (Sarstedt, Inc.). After centrifugation for 2 min in a microfuge (model B; Beckman Instruments, Inc., Cedar Grove, N J), the tips containing the sediments are cut off with wire cutters and the sediments and supernatants are counted separately in a y counter.
Validation of Binding Assay To validate the binding assay it is necessary to ascertain that it separates bound from free ligand. Therefore, it should initially be demonstrated that the fraction of cells sedimented is acceptably high and that the fraction of protein not specifically bound (i.e., the "trapped volume") is acceptably low. In experiments with platelets labeled with 51Cr, >94% of the 51Cr is recovered in the pellet, thus confirming that any ligand bound to the platelets would be expected to appear in the sediment. When platelets are incubated with 125I-labeled bovine serum albumin, the fraction of radioactivity appearing in the pellet is routinely
PLATELET FACTOR XIa RECEPTOR
[30] Binding
of Coagulation Human
Factor
XIa to Receptor
361
on
Platelets
B y DIPALI SINHA and PETER N. WALSH
General Methods Principle
H u m a n platelets h a v e b e e n s h o w n to p r o m o t e the p r o t e o l y t i c activation o f c o a g u l a t i o n f a c t o r s X I I and XI. 1 A n a p p a r e n t r e q u i r e m e n t for this c o n t r i b u t i o n o f platelets to c o n t a c t activation is that first the protein cofactor n o n c o v a l e n t l y associates with f a c t o r XI in plasma, and then f a c t o r X I binds to a c t i v a t e d platelets in the p r e s e n c e o f high m o l e c u l a r weight kininogenfl T h e f a c t o r X I a f o r m e d as a c o n s e q u e n c e binds specifically to a site on platelets distinct f r o m that for f a c t o r XI,3 and retains its structural and functional properties. 3'4 Materials P r o t e i n s . F a c t o r X I (specific activity = 270 U / m g ) 5"6 and high m o l e c u lar weight kininogen 7 (15 U / m g specific activity) are purified as p r e v i o u s l y described. F a c t o r X I a is p r e p a r e d b y incubation with f a c t o r X I I a . 6 Prekallikrein (133 U / m g specific activity), 8 kallikrein, 9 h u m a n a - t h r o m b i n , 3 and p r o t h r o m b i n 6 are p r e p a r e d as described. All proteins should be > 9 8 % pure as j u d g e d b y s o d i u m d o d e c y l sulfate (SDS) p o l y a c r y l a m i d e slab gel electrophoresis. 10 O t h e r R e a g e n t s . M e t h y l silicone oil (1.0. DC200) and Hi P h e n y l silic o n e oil (125 DC550) are o b t a i n e d f r o m William F. N y e , Inc. (Fairhaven, MA); carrier-free Na125I-labeled B o l t o n - H u n t e r reagent and s o d i u m
i p. N. Walsh and J. H. Griffin, Blood 57, 106 (1981). ' J. S. Greengard, M. J. Heeb, E. Ersdal, P. N. Walsh, and J. H. Griffin, Biochemistry 25, 3884 (1986). 3 D. Sinha, F. S. Seaman, A. Koshy, L. C. Knight, and P. N. Walsh, J. Clin. Invest. 73, 1550 (1984). 4 p. N. Walsh, D. Sinha, F. Kueppers, F. S. Seaman, and K. B. Blanstein, J. Clin. Invest. 80, 1578 (1987). 5 B. N. Bouma and J. H. Griffin, J. Biol. Chem. 252, 6432 (1977). 6 p. N. Walsh, H. Bradford, D. Sinha, J. R. Piperno, and G. P. Tuszynski, J. Clin. Invest. 73, 1392 (1984). 7 D. M. Kerbiriou and J. H. Griffin, J. Biol. Chem. 254, 12020 (1979). 8 D. M. Kerbiriou, B. N. Bouma, and J. H. Griffin, J. Biol. Chem. 255, 3952 (1980). 9 B. N. Bouma, L. A. Miles, G. Berena, and J. H. Griffin, Biochemistry 19, 1151 (1980). to U. K. Laemmli, Nature (London) 227, 680 (1970).
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
362
PLATELET RECEPTORS; ASSAYS AND PURIFICATION
[30]
[51Cr]chromate are from New England Nuclear (Boston, MA); Factor XIdeficient plasma is from George King Biomedical (Overland Park, KS); Ultro] HEPES is from Calbiochem Corp. (San Diego, CA); and the chromogenic substrate Pyr-Glu-Pro-Arg-p-nitroanilide hydrochloride (S-2366) is from A. B. Kabi Peptide Research (Stockholm, Sweden).
Radiolabeling For binding experiments, factor XI is radiolabeled with 1251either by the procedure of Bolton and Hunter 1~ or by the Iodogen method ~z as previously described) Free ~zsIis then separated from the labeled protein by passage through a 1-ml Sephadex G-25 column, ~3 and the protein is then dialyzed in the presence of ovalbumin (1 mg/ml). ~25I-Labeled factor XIa is then prepared by incubating the zymogen with factor XIIa?
Assays Coagulation proteins are assayed as previously described, 3 utilizing appropriate congenitally deficient substrate plasmas. Factor XIa is measured by amidolytic assay TM and by radioimmunoassay. TM Protein assays are performed by the method of Lowry et al.~5
Preparation of Cell Suspensions Platelet-rich plasma is obtained from citrated human blood and gel filtered on a Sepharose 2B column into calcium-free HEPES-buffered Tyrode's solution, pH 7.4, containing bovine serum albumin (1 mg/ml), 138 mM NaCI, 2.7 mM KCI, 1.0 mM MgC12 • 6H20, 3.3 mM NaHzPO4 • H20, 15 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 5.5 mM dextrose as previously described? Erythrocyte suspensions are prepared from fresh human blood washed five times in Hanks' balanced salt solution to remove plasma and other cells as previously described. 3 The red cells are finally suspended in calcium-free HEPES-buffered Tyrode's solution, pH 7.4.
tl A. E. Bolton and W. M. Hunter, Biochem. J. 133, 529 (1973). 12 p. j. Fraker and J. C. Speck, Biochem. Biophys. Res. Commun. 80, 849 (1978). 13 G. P. Tuszynski, L. Knight, J. R. Piperno, and P. N. Walsh, Anal. Biochem. 106, 118 (1980). 14 C. F. Scott, D. Sinha, F. S. Seaman, P. N. Walsh, and R. W. Colman, Blood63, 42 0984). i50. H. Lowry, H. J. Rosebrough, A. L. Farr, and F. J. Randall, J. Biol. Chem. 193, 365 (1951).
[30]
PLATELET FACTORXIa RECEPTOR
363
Binding Measurements To measure the binding of factor XIa to platelets over varying incubation times, at variable concentrations of added factor XIa, or under differing conditions, gel-filtered platelets (2-4 × 108/ml) or control cells (e.g., erythrocytes) are incubated at 37 ° in 1.5-ml polypropylene centrifuge tubes (Sarstedt, Inc., Princeton, N J) with 125I-labeled factor XIa, in the presence or absence of high molecular weight kininogen or thrombin (or other platelet agonists). Determinations of specificity of binding and measurements of nonspecific binding are made in the presence of 50- to 100-fold molar excesses of unlabeled factor XIa or other proteins. At specified incubation times, 100-/zl aliquots are layered over a mixture of silicone oils, (I vol of DC200 to 5 vol DC550) contained in microsediment tubes with narrow-bore extended tips (Sarstedt, Inc.). After centrifugation for 2 min in a microfuge (model B; Beckman Instruments, Inc., Cedar Grove, N J), the tips containing the sediments are cut off with wire cutters and the sediments and supernatants are counted separately in a y counter.
Validation of Binding Assay To validate the binding assay it is necessary to ascertain that it separates bound from free ligand. Therefore, it should initially be demonstrated that the fraction of cells sedimented is acceptably high and that the fraction of protein not specifically bound (i.e., the "trapped volume") is acceptably low. In experiments with platelets labeled with 51Cr, >94% of the 51Cr is recovered in the pellet, thus confirming that any ligand bound to the platelets would be expected to appear in the sediment. When platelets are incubated with 125I-labeled bovine serum albumin, the fraction of radioactivity appearing in the pellet is routinely
