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ugation at 400 g for 2 min at 4°. The platelets are then pelleted by centrifuging at 3000 g for 15 min at 4° and washed three times in buffer B. The platelet pellet is then resuspended in 3 M KCI (approximately 7 ml/unit used), incubated at 37° for 15 min with occasional swirling, and centrifuged at 3000 g for 15 min at 4 °. The glycocalicin-containing supernatant is removed and recentrifuged twice at 12,000 g for 20 min at 4°. It is then dialyzed overnight at 4° against large volumes of buffer C and then against large volumes of distilled H20 for at least 24 hr with several changes. During dialysis, a glycocalicin-rich precipitate forms that is isolated by centrifugation at 12,000 g for 45 min at 4°. The sediment is resuspended in a solution containing 154 mM NaC1, 0.2 mM EDTA, 15.3 mM NaN 3, pH 7.0 (using approximately 0.5 ml/unit used), stirred at 37° for 10 min, and centrifuged at 12,000 g for 45 min at 4° to remove the undissolved material. The glycocalicin is then purified from contaminants on a 6D1 affinity column as described for GPIb.

[27] F i b r o n e c t i n B i n d i n g to P l a t e l e t s By JANE F O R S Y T H ,


Introduction Fibronectin is a large circulating glycoprotein that has been implicated in cell migration, opsonization, tissue development, and platelet adhesion. 1 Although qualitative studies have suggested the existence of cellular fibronectin receptors,2'3 the first direct demonstration of fibronectin binding to a saturable cellular receptor was performed with platelets. 4 The performance of such assays has permitted the identification of a congenital deficiency of fibronectin receptors, 5 and establishment of two types of fibronectin interaction with platelets. 6 They have also provided important confirmation to the concept 7 that the Arg-Gly-Asp sequence in fibronectin mediates its binding to cell surfaces by the direct demonstration of inhibiI R. O. Hynes and K. M. Yamada, J. Cell Biol. 95, 369 (1982). 2 F. Grinnell, J. Cell Biol. 86, 104 (1980). 3 M. P. Bevilacqua, D. Amrani, M. W. Mosesson, and C. Bianco, J. Exp. Med. 153, 42 (1981).

4 E. F. Plow and M. H. Ginsberg, J. Biol. Chem. 256, 9477 (1981). 5 M. H. Ginsberg, J. Forsyth, A. Lightsey, J. Chediak, and E. F. Plow, J. Clin. Invest. 71, 619 (1983). 6 E. F. Plow, G. A. Marguerie, and M. H. Ginsberg, Blood 66, 26 (1985). : M. D. Pierschbacher and E. Ruoslahti, Nature (London) 309, 30 (1984).


Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.




tion o f fibronectin binding to a cell by such peptides. 8 In addition, assay of fibronectin binding to platelets provided 5 evidence that G P I I b - I I I a (aUb/33 integrin) was a multifunctional receptor, i.e., that fibrinogen and von Willebrand factor shared this receptor with fibronectin.9-12 This multifunctional receptor exists in addition to the fibrinogen receptor belonging to the/31 family ofintegrins (aSfll integrins). 13Thus, the quantitative assessment of fibronectin binding to the platelet surface has proved of value in the general study of cellular adhesion, as well as in the specific analysis of platelet adhesive function. The assays are performed with purified radiolabeled fibronectin and suspensions of washed platelets. The specific details for preparing and radiolabeling of the fibronectin and performance of the binding assays are described below.

Preparation of Gelatin-Sepharose Fibronectin is isolated from human plasma by affinity chromatography on a gelatin-Sepharose column. Gelatin-Sepharose is available commercially, but is costly. I. Wash 750 ml Sepharose 4B (Pharmacia, Piscataway, N J) with distilled water and slowly stir the mixture in an ice bath in a fume hood. 2. Add 1.5 ml of 2 M Na2CO3 and stir on ice until the temperature of the mixture is less than 4 ° . 3. Dissolve 100 g cold C N B r (Sigma, St. Louis, MO) in 50 ml cold acetonitrile; it will take considerable time to dissolve the CNBr, but do not try to rush the process by heating it. 4. Slowly add the C N B r solution to the stirring Sepharose 4B, adding ice crystals occasionally to keep the solution cold. Stir the mixture for 10 min. 5. Filter the beads in a glass sintered funnel, then wash with 10 liters cold 0.2 M NaHCO3, p H 9.5, and add ice crystals to cool. 8 M. Ginsberg, M. D. Pierschbacher, E. Ruoslahti, G. Marguerie, and E. Plow, J. Biol. Chem. 260, 3931 (1985). 9 E. F. Plow, A. H. Srouji, D. Meyer, G. Marguerie, and M. H. Ginsberg, J. Biol. Chem. 259, 5388 (1984). l0 E. F. Plow, R. P. McEver, B. S. Coller, V. L. Woods, Jr., G. A. Marguerie, and M. H. Ginsberg, Blood 66, 724 (1985). " R. Pytela, M. D. Pierschbacher, M.H. Ginsberg, E. F. Plow, and E. Ruoslahti, Science 231, 1559 (1986). 12E. F. Plow, M. D. Pierschbacher, E. R ~oslahti, G. A. Marguerie, and M. H. Ginsberg, Proc. Natl. Acad. Sci. U.S.A. 82, 8057 (1985). 13R. O. Hynes, Cell (Cambridge, Mass.) 48, 549 (1987).

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6. Transfer the cake to a large beaker containing a chilled solution of 16 g of gelatin in 800 ml of 0.2 M NaHCO3, pH 9.5. Incubate overnight at 4° , stirring slowly. 7. After the overnight incubation, the solution will be gelid and difficult to filter. At room temperature, filter the Sepharose and wash it with 10 liters of phosphate-buffered saline (PBS; 0.01 M Tris base, 0.15 M NaCI), pH 8.6; 10 liters of 0.01 M Tris, 2 M NaC1, pH 8.6; and 10 liters of PBS, pH 8.6. Store at 4 ° until used. 8. Pour the gelatin-Sepharose column at room temperature in a siliconized glass column. Before use, equilibrate the column with PBS (0.01 M sodium phosphate, 0.15 M NaCI) plus 5 mM EDTA, 0.05% (w/v) sodium azide, pH 7.0.

Preparation of Fibronectin Fibronectin is prepared from human plasma drawn into 1/6 vol ACD [0.065 M citric acid, 0.085 M sodium citrate, 2% (w/v) dextrose]. The plasma should be fresh (not more than 2 days old) and kept at room temperature until used; freezing decreases the yield. The entire preparation is done at room temperature. 1. Centrifuge the plasma at 5000 rpm for 30 min, then filter it through Whatman (Clifton, NJ) filter paper (#2) by gravity. Add 0.05% sodium azide, 5 mM EDTA, 1 mM benzamidine to the plasma. 2. Load the plasma onto the preequilibrated column, then wash the column with successive washes of PBS, 1 mM benzamidine, pH 7.0; 1 M urea, 1 mM benzamidine, pH 7.0; and PBS, pH 7.0. The volume of each wash step should be two to three times the column bed volume. 3. Elute the column with 1 M NaBr, 0.02 M acetic acid, pH 5.0. As the fibronectin elutes, it appears as viscous strands. Therefore it is important to collect fractions by time rather than drop counting. 4. Pool the fractions with peak absorption at 280 nm and immediately dialyze the pool against PBS with three changes of buffer. 5. Store the fibronectin at - 7 0 ° in aliquots. Thaw only once. 6. Fibronectin is characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in which it yields a closely spaced doublet at M r 225K reduced and a single Mr 450K band nonreduced. In addition, the concentration of fibronectin in solution can be estimated from a0.1% 1 28 14 its absorption coefficient a = ,11cm =

14 M. Mosesson and C. A. Umfleet, J. Biol. Chem. 245, 5728 (1970).




Radiolabeling of Fibronectin I. To a tube containing 800/zl of fibronectin at approximately 1 mg/ml add 800/zCi ~25I and 20/z[ of chloramine-T at 2 mg/ml in PBS, pH 7.0. 2. Mix and incubate at room temperature for 5 min. 3. Add 20/zl sodium metabisulfite at 2 mg/ml in PBS, pH 7.0, and 20 /zl 1% (w/v) potassium iodide in distilled water. 4. Add 300 /zl of a 1% BSA, 1 mM phenylmethylsulfonyl fluoride (PMSF) solution in PBS. Dialyze to remove free iodine. 5. Before dialysis the label is generally greater than 90% TCA precipitable and after dialysis it is greater than 99%. Binding Assays Suspension of washed platelets can be prepared by gel filtration as described in this series, Volume 169, p. 11. Specific techniques for doing this are described by Ginsberg e t al. ~5 Platelets are adjusted to a final cell concentration of 8 × 108 cells/ml. 1. Binding assays are performed in a 1.5-ml polypropylene Eppendorf centrifuge tube. 2. Precentrifuge a solution containing 10/~M lZSI-labeled fibronectin at 11,750 rpm for 5 min in a Beckman (Palo Alto, CA) microfuge B to remove potential aggregate. 3. Add 110/~l buffer or competing ligand to the Eppendorf tube followed by 50/~l of the ~25I-labeled fibronectin. 4. Add 20/A platelet suspension and equilibrate at 37 ° in a water bath for 10 min. 5. Add 20 /.d of the platelet stimulus (e.g., 10 units/tA human a-thrombin). 6. Incubate at 37 ° for desired time. 7. At selected time points triplicate 50-/A aliquots are layered onto 300 /~l of 20% sucrose (ultrapure; Schwarz/Mann, Orangeburg, NY) in a 400-~1 microfuge tube (West Coast Scientific, Emoryville, CA). 8. Centrifuge at 11,750 g for 5 min at room temperature. 9. The tube tips can then be amputated by slicing them with a razor blade, and counted in a y scintillation spectrometer. Several points are worthy of note. First, it is important to use the platelets within 2 hr of their preparation, as their capacity to respond to thrombin stimulation declines rapidly once the cells are washed. In con15 M. H. Ginsberg, L. Taylor, and R. Painter, Blood 55, 661 (1980).




trast, the platelets are stable in platelet-rich plasma, prior to washing, for at least 12 hr. In addition, if there is variability in replicate determinations, several potential errors should be explored. First, it is possible that platelets may be aggregating in the tubes. This problem can be readily addressed by reducing the platelet concentration, or avoiding agitation during incubations. A second potential problem is failure to preclear all the aggregates from the radiolabeled fibronectin solution. This should become apparent if platelet-free controls are included in the binding assay. Another source of variability may be a problem with amputating the tube tips. To avoid this, fresh razor blades should be used, and frequently replaced. In addition, an effort should be made to slice the tube tip just above the visible platelet pellet. In addition to the platelet-free control alluded to above, other controls routinely included are those in which no stimulus is added, and those in which a 100-fold molar excess of unlabeled fibronectin is added as competing ligand. Data Analysis. Binding data can be expressed as counts per minute bound, or picograms bound per specified number of platelets. This is readily calculated from the known specific activity of the ligand. Another popular method of expressing such data is in molecules per platelet, which is readily calculated from the approximate molecular weight for fibronectin of 4.5 × 105. In each case we routinely subtract the nonsaturable binding estimated by addition of greater than 100-fold molar excess of unlabeled fibronectin to the reaction mixture. Analysis of the binding parameters generated from binding isotherms can utilize any of the popular means of expressing this data. It is important to recognize that fibronectin binding to platelets has not been established to obey simple mass action and, therefore, estimated parameters such as Kd and number of sites are only descriptive approximations. In the past, we have employed Scatchard plots to analyze such isotherms. In this case, it is advisable to manually subtract the nonsaturable binding from each data point. An alternative to this is to analyze the data utilizing nonlinear least-squares curve fitting (LIGAND program of Munson and Rodbard).16 This program assomes simple mass action, binding to a finite number of independent binding sites, and that the nonsaturable binding component is a constant fraction of the free ligand. Fibronectin binding isotherms readily fit a single-site binding model, and estimates of Kd and numbers of sites confirming our published values with Scatchard plots have been obtained. This particular method is advantageous in the analysis of treatments thought to affect affinity or number of binding sites, because statisti16 p. j. M u n s o n and D. Rodbard, Anal. Biochem. 107, 220 (1980).






cal estimation of the likelihood that a particular manuever affects either parameter can readily be obtained. Whatever the method of analysis of such binding isotherms, it is essential to present the raw binding data in addition to the transformed data. Acknowledgment Supported by Grants #HL28235, HL16411, and AM27214 from the NIH. This is publication #4376-IMM from the Research Institute of Scripps Clinic.

[28] M a t h e m a t i c a l S i m u l a t i o n o f P r o t h r o m b i n a s e B y MICHAEL E . NESHEIM, RUSSELL P. TRACY, PAULA B. TRACY, DANILO S. BOSKOVIC, a n d KENNETH G. MANN

Introduction Prothrombinase is a multicomponent enzyme complex involved in blood coagulation. It comprises a serine protease (factor Xa), a cofactor (factor Va), Ca 2+, and a catalytic surface. 1,2 In model systems in vitro the surface consists of negatively charged phospholipid vesicles, whereas the presumed surface in vivo consists of a component or components of the platelet. In model systems, as well as on platelets, the complex consists of a noncovalent but tightly bound complex of factor Xa and factor Va in which factor Va provides the equivalent of a surface receptor for factor Xa. These components together efficiently catalyze the proteolytic activation of the zymogen prothrombin to the blood-clotting enzyme thrombin. Because prothrombin is also a surface (phospholipid) binding protein, the reaction can be best described as a surface-dependent event, even though factor Xa alone will slowly catalyze prothrombin activation in solution in the absence of either factor Va, Ca 2÷ , or the catalytic surface. However, it does so at a rate about five orders of magnitude slower than the complete complex. 3-5 K. G. Mann, B. H. Odegaard, S. Krishnaswamy, P. B. Tracy, and M. E. Nesheim, in "Proteases in Biological Control and Biotechnology," p. 235. Alan R. Liss, New York, 1987. 2 M. E. Nesheim, Surv. Synth. Pathol. Res. 31, 219 (1984). 3 M. E. Nesheim, J. B. Taswell, and K. G. Mann, J. Biol. Chem. 254, 10952 (1979). 4 N. H. Kane, M. J. Lindhout, C. M. Jackson, and P. W. Majerus, J. Biol. Chem. 255, 1170 (1980).


Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Fibronectin binding to platelets.

[2 7] FIBRONECTIN BINDING 31 1 ugation at 400 g for 2 min at 4°. The platelets are then pelleted by centrifuging at 3000 g for 15 min at 4° and was...
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