398
PLATELET
RECEPTORS:
ASSAYS AND PURIFICATION
[33]
of H D L support this h y p o t h e s i s . 2°-24 Furthermore, it has been demonstrated in the last few years that LDL causes inhibition of adenylate cyclase, 25 evokes protein phosphorylation and mobilization of thromboxa n e A 2 ,26 and induces the release of inositol phosphates. 27 These effects definitely bring LDL binding in close relationship with platelet reactivity, although they become significant only at high LDL concentrations, far beyond that leading to half-saturation of binding sites. 28 GPIIb-IIIa fulfills a key role in the course of platelet activation. The agonist-induced binding of fibrinogen to GPIIb-IIIa is a necessary prerequisite for aggregation. Consequently, the binding of lipoproteins to this membrane protein complex might have major effects on platelet function in vivo. In fact, we could show that fibrinogen binding to ADP or thrombinstimulated platelets is significantly enhanced in the presence of L D L . 29 2o M. Aviram and J. G. Brook, Atherosclerosis 46, 259 (1983). 21 K. Desai, K. R. Bruckdorfer, R. A. Hutton, and J. S. Owen, J. LipidRes. 30, 831 (1989). 2., E. Koller, Th. Vukovich, W. Doleschel, and W. Auerswald, Atherogenese 4 (Suppl. IV), 53 (1979), 23 D. G. Hassall, J. S. Owen, and K. R. Bruckdorfer, Biochem. J. 216, 43 (1983). 24 R. Farbiszewski, Z. Skrzydlewski, and K. Worowski, Thromb. Diath. Haemostas. 21, 89 (1963). 25 K. R. Bruckdorfer, S. Buckley, and D. G. Hassall, Biochem. J. 223, 189 (1984). 26 H. E. Andrews, J. W. Aitken, D. G. Hassall, V. O. Skinner, and K. R. Bruckdorfer, Biochem. J. 242, 559 (1987). 27 M. Knorr, R. Locher, E. Vogt, W. Vetter, L. H. Block, F. Ferracin, H. Lefkovits, and A. Pletscher, Eur. J. Biochem. 172, 753 (1988). 28 Calculations based on the in vitro interaction between platelets and LDL might, however, be misleading. The presence of additional classes of lipoproteins obviously markedly reduces the strength of this binding. The degree of saturation of sites in vivo may therefore be well below 100%. 29 E. Koller, F. Koller, and B. R. Binder, Thromb. Haemostas. 62, (abstr 830) 261 (1989).
[33] P l a t e l e t I n s u l i n R e c e p t o r By ANTHONY S. HAJEK and J. HEINRICH JOIST Introduction Platelets contain insulin receptors with characteristics that are similar to the insulin receptors found in other types of cells. These include an alkaline pH binding optimum, site-site interactions between receptors (i.e., negative cooperativity), numbers of binding sites per cell surface METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[33]
PLATELET INSULIN RECEPTOR
399
area, and a high-affinity binding constant in the nanomolar range. L2 Insulin binding to platelets can be demonstrated and quantitated by conventional hormone binding techniques, 3'4 including experiments where the association of ~25I-labeled insulin with the platelet insulin receptor is competitively inhibited by native insulin. The resulting data can be used for Scatchard analysis 5 to calculate the number of binding sites on the platelet plasma membrane and to obtain a value for the dissociation constant. Fluorescein isothiocyanate (FITC)-labeled insulin has also been used successfully to study platelet insulin receptors. 2 Presented here is the more traditional method, which utilizes radioiodinated insulin as the ligand.
Methods
Binding Assay Buffer N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; 100 mM), NaCI (120 mM), MgSO4 (1.2 raM), KCI (2.5 mM), glucose (10 mM), EDTA (1 mM), and 10 g/liter bovine serum albumin, pH 8.0. 6 The buffer should be freshly prepared on the day of usage.
Platelet Isolation Blood is collected by clean venipuncture, using a two-syringe technique, into I/7 vol of acid-citrate-dextrose (ACD) solution, pH 4.5. Platelets are isolated by differential centrifugation according to the method of Mustard et al. 7 Platelets prepared by this method appear to be morphologically (disk shape, normal number and distribution of subcellular organelles) and functionally (normal in vitro aggregation to ADP in the presence of added fibrinogen) intact, i.e., similar to platelets in citrated platelet-rich plasma. All steps in this procedure are carried out at 37° to guarantee maximum effectiveness of apyrase in removing extracellular ADP, and thus prevent platelet activation.~ The ACD blood is centrifuged at 180 g for 15 rain. The platelet-rich plasma is removed and centrifuged at 2000 g t A. S. Hajek, J. H. Joist, R. K. Baker, L. Jarett, and W. H. Daughaday, J. Clin. Invest. 63, 1060 (1979). 2 R. Shimoyama, J. Clin. Endocrinol. Metab. 53, 502 (1981). 3 j. Roth, this series, Vol. 37, p. 66. 4 T. Kono, this series, Vol. 37, p. 193. 5 G. Scatchard, Ann. N . Y . Acad. Sci. 51, 660, 1949. 6 M. M. Rechler and J. M. Podskalny, Diabetes 25, 250 (1976). 7 j. F. Mustard, D. W. Perry, N. G. Ardlie, and M. A. Packham, Br. J. Haematol. 22, 193 (1972).
400
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[33]
for 10 min. The platelet sediment is immediately resuspended in T y r o d e ' s solution (8.0 g/liter NaC1, 0.2 g/liter KC1, 1.0 g/liter N a H C O 3 , 0 . 0 5 g/liter NaH2PO 4 • H20, 0.438 g/liter CaCI 2 • 6H20, pH 7.35) containing 25 units/ ml heparin, 3.5 g/liter bovine serum albumin, and 3 units/ml of potato apyrase prepared as described elsewhere. 8 Approximately 0.5 ml of packed platelets is added to 10 ml of solution. Following centrifugation of the mixture at 1000 g for 10 min, the platelets are resuspended in 10 ml of the above washing solution (except that heparin is omitted), the mixture is again centrifuged at 1000 g for 10 min, and the platelets are resuspended in H E P E S binding assay buffer at p H 8.0. It is important to determine the white blood cell (WBC) concentration in the cell suspension at this point microscopically (hemocytometer) since blood mononuclear cells (monocytes, lymphocytes) also bind insulin. 9'~° White blood cell contamination of 1 WBC/100,000 platelets or less can be readily achieved by this method and appears acceptable. Insulin Iodination
Crystalline, porcine, m o n o c o m p o n e n t insulin may be iodinated by a variety of methods H-~3 or obtained commercially. The preparation should contain 0.4-0.5 iodine atoms per molecule of insulin. Because iodinated insulin deteriorates over time to yield products that are biologically inactive, it is advisable that each preparation be used within 2 weeks after iodination. The insulin preparation can be purified by means of gelfiltration chromatography (Sephadex G-50) 2 or other suitable gel-filtration methods. Insulin Binding A s s a y
All materials (tubes, pipettes) that will come in contact with the labeled insulin should be made of polypropylene. This material does not adsorb insulin as readily as glass or polystyrene so that background radioactivity is minimized. Washed platelets (0.5-2.0 × 106//xl in H E P E S binding assay buffer, pH 8.0) are incubated with a mixture of 125I-labeled insulin and unlabeled insulin (0-10 /zg/ml). The mixture is briefly 8 j. Molnar and L. Lorand, Arch. Biochem. Biophys. 93, 353 (1961).
9 R. A. Schwartz, B. S. Bianco, B. S. Handwerger, and C. R. Kahn, Proc. Natl. Acad. Sci. U.S.A. 72, 474 (1975). 10j. R. Gavin, P. Gorden, J. Roth, J. A. Archer, and D. N. Buell, J. Biol. Chem. 248, 2202 (1973). H j. j. Conahey and F. J. Dixon, this series, Vol. 70, p. 210. 12M. Morrison, this series, Vol. 70, p. 214. t3 j. j. Langone, this series, Vol. 70, p. 221.
[33]
PLATELET INSULIN RECEPTOR
401
vortexed and incubated for 3 hr at 24°. Since the platelets tend to settle to the bottom of the tubes with time it is advisable to vortex the tubes gently every 20 min. At the end of the incubation period the mixture is again vortexed and duplicate 0.5-ml aliquots are removed and layered over 0.5 ml of ice-cold HEPES binding buffer in a 1.5-ml microfuge tube and immediately centrifuged in a cold room for 5 min in a microfuge (Beckman Instruments, Inc., Spinco Div., Palo Alto, CA). The supernatant buffer is removed by a two-step method, i.e., the bulk of the supernatant fluid is aspirated, and the remaining traces of buffer are allowed to drain down the inside walls of the microfuge tubes and are removed by a second aspiration. The tips of the tubes containing the platelet pellet are cut off and counted for radioactivity in a 3' counter. Specific binding of 125I-labeled insulin is determined by subtracting the amount of radioactivity (counts per minute, cpm) associated with the platelet pellet in the presence of 10/zg/ml of unlabeled insulin from the total cpm determined in the platelet sediment.
Comments Under the conditions of the assay described here, hormone degradation [as determined by trichloroacetic acid (TCA) precipitation and rebinding experiments] and receptor degradation were not observed.l
Specificity At a platelet concentration of 800,000 ~1 in the incubation mixture, binding reaches a steady state at 2 to 3 hr (half-maximal binding is reached by 30 min), at which time approximately 2% of the labeled hormone is bound to the platelets. Binding of approximately 90% of the labeled insulin is competitively inhibited by 10/xg/ml of native insulin (specific binding). As little as 3 t~g/ml of labeled insulin (a physiological blood concentration) may cause a 40-50% decrease in specific binding of the iodoinsulin. 1 Further evidence for the specificity of the binding of insulin to the platelet surface was obtained from experiments showing that with catfish insulin and porcine proinsulin (which in humans are biologically less potent than native porcine insulin) substantially higher doses are required to produce a decrease in the specific binding of nsIlabeled insulin as compared to porcine insulin. Furthermore, diisopropyl fluorophosphate (DFP)-treated thrombin, human prolactin, human growth hormone, and glucagon did not inhibit the binding of radiolabeled insulin to platelets. 1
402
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[33]
Receptor Site Number and Binding Affinity Scatchard plots 5 of the competitive binding assay data show a curvilinear pattern from which one can derive a high-affinity dissociation constant of approximately 3 x 10 9 m -1. The curvilinear pattern of the plot is consistent with either the presence of two classes of binding sites with different affinities or a single population of insulin receptors that display enhanced dissociation of labeled insulin in the presence of excess unlabeled hormone (negative cooperativity). The number of 500-600 insulin binding sites per platelet, derived from Scatchard plot analysis, is appreciably less than that reported for human lymphocytes (12,500) J° and erythrocytes (2000). Z4 However, when expressed as number of binding sites per platelet surface area, the value of approximately 25//~m 2 is very similar to the insulin receptor density observed in other insulin binding cell types. 1'9'1° It is important to note that the estimation of the density of platelet insulin receptors is based on the assumption of a smooth, continuous outer cell membrane. Since the platelets appear to have an extensive spongelike, internal surface-connecting canalicular system,15 the actual platelet surface area potentially accessible for insulin binding is likely to be greatly in excess of that estimated on the basis of the measurement of platelet size. This means that the value of 25 insulin receptors/tzm 2 for human platelets I is probably an overestimate. The method outlined about is straightforward and yields consistent results. The values for the high-affinity dissociation constant and platelet receptor number obtained with this method are lower than those obtained with FITC-labeled insulin binding experiments, 2 but are in agreement with values reported for 125I-labeled insulin binding in other systems, as well as with alternate methods of analysis (i.e., Lineweaver-Burketype p l o t s ) . 4 The issue of the biologic significance of the platelet insulin receptor remains unsettled. Whereas earlier reports indicated that insulin may increase lactate formation in human platelets 16and inhibit platelet aggregation in vitro, 17 an extensive study in our laboratory 18 failed to show any effect of insulin on in vitro secretion, glucose transport and metabolism, or protein phosphorylation in human platelets in response to stimulation
14 p. DeMeyts, J. Roth, D. M. Neville, Jr., J. R. Gavin, and M. A. Lesniak, Biochem. Biophys. Res. Commun. $5, 154 (1973). 15j. G. White, Am. J. Pathol. 66, 295 (1972). 16 S. Karpatkin, J. Clin. Invest. 46, 409 (1967). 17 A. A. Hassanein, T. A. EI-Garf, and Z. EI-Baz, Thromb. Diath. Haemorrh. 27, 114 (1972). t8 j. H. Joist, L. Babb, and R. K. Baker, unpublished observations.
[34]
CROSS-LINKING OF PLATELET MEMBRANE GLYCOPROTEINS
403
by different agonists. Others reported a lack of correlation between ADPinduced platelet aggregation and serum insulin levels in patients with type I and type II diabetes 19 and a lack of effect of continuous subcutaneous insulin infusion on platelet aggregation to ADP and epinephrine or thromboxane B 2 generation. 2° t9 D. B. Jones, T. M. Davis, E. Brown, R. D. Carter, J. I. Mann, and R. J. Prescott, Diabetologia 29, 291 (1986). 2o L. H. Monnier, M. Rodier, A. Gancel, P. Crastes De Paulet, C. Colette, M. Piperno, and J. Crastes DePaulet, Diabete Metab. 13, 210 (1987).
[34] M e m b r a n e - I m p e r m e a n t Cross-Linking Reagents for S t r u c t u r a l a n d F u n c t i o n a l A n a l y s e s o f P l a t e l e t Membrane Glycoproteins
By JAMES V.
STAROS,
NICOLAS J. KOT1TE, and
LEON W. CUNNINGHAM
The interactions among platelet membrane glycoproteins and between these molecules and macromolecular components of the subendothelial matrix and of the plasma have been areas of great and growing interest. Chemical cross-linking is a technique that has proven useful in many studies of protein-protein interactions. 1-4 Indeed, cross-linking has provided useful information concerning platelet supramolecular structure 5-s and the interactions of specific macromolecules 7,9-J4 or synthetic pepF. Wold, this series, Vol. 25, p. 623. 2 K. Peters and F. M. Richards, Annu. Rev. Biochem. 46, 523 (1977). 3 T. H. Ji, this series, Vol. 91, p. 580. 4 j. V. Staros and P. S. R. Anjaneyulu, this series, Vol. 172, p. 609. 5 G. E. Davies and J. Palek, Blood 59, 502 (1982). 6 S. M. Jung and M. Moroi, Biochim. Biophys. Acta 761, 152 (1983). v N. J. Kotite, J. V. Staros, and L. W. Cunningham, Biochemistry 23, 3099 (1984). s A. Sonnenberg, H. Janssen, F. Hogervorst, J. Calafat, and J. Hilgers, J. Biol. Chem. 262, 10376 (1987). 9 N. E. Larsen and E. R. Simons, Biochemistry 20, 4141 (1981). l0 j. Lahav, M. A. Schwartz, and R. O. Hynes, Cell (Cambridge, Mass.) 31, 253 (1982). II j. Takamatsu, M. K. Horne, Ill, and H. R. Gralnick, J. Clin. Invest. 77, 362 (1986). t2 F. C. Molinas, J. Wietzerbin, and E. Falcoff, J. lmmunol. 138, 802 (1987). t~ M. Jandrot-Perrus, D. Didry, M.-C. Guillin, and A. T. Nurden, Eur. J. Biochem. 174, 359 (1988). ~4 R. K. Andrews, J. J. Gorman, W. J. Booth, G. L. Corino, P. A. Castaldi, and M. C. Berndt, Biochemistt3' 28, 8326 (1989).
METHODS IN ENZYMOLOGY.VOL. 215
Copyright © 1992by AcademicPress, Inc. All rights of reproduction in any form reserved.
PLATELET
RECEPTORS:
ASSAYS AND PURIFICATION
[33]
of H D L support this h y p o t h e s i s . 2°-24 Furthermore, it has been demonstrated in the last few years that LDL causes inhibition of adenylate cyclase, 25 evokes protein phosphorylation and mobilization of thromboxa n e A 2 ,26 and induces the release of inositol phosphates. 27 These effects definitely bring LDL binding in close relationship with platelet reactivity, although they become significant only at high LDL concentrations, far beyond that leading to half-saturation of binding sites. 28 GPIIb-IIIa fulfills a key role in the course of platelet activation. The agonist-induced binding of fibrinogen to GPIIb-IIIa is a necessary prerequisite for aggregation. Consequently, the binding of lipoproteins to this membrane protein complex might have major effects on platelet function in vivo. In fact, we could show that fibrinogen binding to ADP or thrombinstimulated platelets is significantly enhanced in the presence of L D L . 29 2o M. Aviram and J. G. Brook, Atherosclerosis 46, 259 (1983). 21 K. Desai, K. R. Bruckdorfer, R. A. Hutton, and J. S. Owen, J. LipidRes. 30, 831 (1989). 2., E. Koller, Th. Vukovich, W. Doleschel, and W. Auerswald, Atherogenese 4 (Suppl. IV), 53 (1979), 23 D. G. Hassall, J. S. Owen, and K. R. Bruckdorfer, Biochem. J. 216, 43 (1983). 24 R. Farbiszewski, Z. Skrzydlewski, and K. Worowski, Thromb. Diath. Haemostas. 21, 89 (1963). 25 K. R. Bruckdorfer, S. Buckley, and D. G. Hassall, Biochem. J. 223, 189 (1984). 26 H. E. Andrews, J. W. Aitken, D. G. Hassall, V. O. Skinner, and K. R. Bruckdorfer, Biochem. J. 242, 559 (1987). 27 M. Knorr, R. Locher, E. Vogt, W. Vetter, L. H. Block, F. Ferracin, H. Lefkovits, and A. Pletscher, Eur. J. Biochem. 172, 753 (1988). 28 Calculations based on the in vitro interaction between platelets and LDL might, however, be misleading. The presence of additional classes of lipoproteins obviously markedly reduces the strength of this binding. The degree of saturation of sites in vivo may therefore be well below 100%. 29 E. Koller, F. Koller, and B. R. Binder, Thromb. Haemostas. 62, (abstr 830) 261 (1989).
[33] P l a t e l e t I n s u l i n R e c e p t o r By ANTHONY S. HAJEK and J. HEINRICH JOIST Introduction Platelets contain insulin receptors with characteristics that are similar to the insulin receptors found in other types of cells. These include an alkaline pH binding optimum, site-site interactions between receptors (i.e., negative cooperativity), numbers of binding sites per cell surface METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[33]
PLATELET INSULIN RECEPTOR
399
area, and a high-affinity binding constant in the nanomolar range. L2 Insulin binding to platelets can be demonstrated and quantitated by conventional hormone binding techniques, 3'4 including experiments where the association of ~25I-labeled insulin with the platelet insulin receptor is competitively inhibited by native insulin. The resulting data can be used for Scatchard analysis 5 to calculate the number of binding sites on the platelet plasma membrane and to obtain a value for the dissociation constant. Fluorescein isothiocyanate (FITC)-labeled insulin has also been used successfully to study platelet insulin receptors. 2 Presented here is the more traditional method, which utilizes radioiodinated insulin as the ligand.
Methods
Binding Assay Buffer N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; 100 mM), NaCI (120 mM), MgSO4 (1.2 raM), KCI (2.5 mM), glucose (10 mM), EDTA (1 mM), and 10 g/liter bovine serum albumin, pH 8.0. 6 The buffer should be freshly prepared on the day of usage.
Platelet Isolation Blood is collected by clean venipuncture, using a two-syringe technique, into I/7 vol of acid-citrate-dextrose (ACD) solution, pH 4.5. Platelets are isolated by differential centrifugation according to the method of Mustard et al. 7 Platelets prepared by this method appear to be morphologically (disk shape, normal number and distribution of subcellular organelles) and functionally (normal in vitro aggregation to ADP in the presence of added fibrinogen) intact, i.e., similar to platelets in citrated platelet-rich plasma. All steps in this procedure are carried out at 37° to guarantee maximum effectiveness of apyrase in removing extracellular ADP, and thus prevent platelet activation.~ The ACD blood is centrifuged at 180 g for 15 rain. The platelet-rich plasma is removed and centrifuged at 2000 g t A. S. Hajek, J. H. Joist, R. K. Baker, L. Jarett, and W. H. Daughaday, J. Clin. Invest. 63, 1060 (1979). 2 R. Shimoyama, J. Clin. Endocrinol. Metab. 53, 502 (1981). 3 j. Roth, this series, Vol. 37, p. 66. 4 T. Kono, this series, Vol. 37, p. 193. 5 G. Scatchard, Ann. N . Y . Acad. Sci. 51, 660, 1949. 6 M. M. Rechler and J. M. Podskalny, Diabetes 25, 250 (1976). 7 j. F. Mustard, D. W. Perry, N. G. Ardlie, and M. A. Packham, Br. J. Haematol. 22, 193 (1972).
400
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[33]
for 10 min. The platelet sediment is immediately resuspended in T y r o d e ' s solution (8.0 g/liter NaC1, 0.2 g/liter KC1, 1.0 g/liter N a H C O 3 , 0 . 0 5 g/liter NaH2PO 4 • H20, 0.438 g/liter CaCI 2 • 6H20, pH 7.35) containing 25 units/ ml heparin, 3.5 g/liter bovine serum albumin, and 3 units/ml of potato apyrase prepared as described elsewhere. 8 Approximately 0.5 ml of packed platelets is added to 10 ml of solution. Following centrifugation of the mixture at 1000 g for 10 min, the platelets are resuspended in 10 ml of the above washing solution (except that heparin is omitted), the mixture is again centrifuged at 1000 g for 10 min, and the platelets are resuspended in H E P E S binding assay buffer at p H 8.0. It is important to determine the white blood cell (WBC) concentration in the cell suspension at this point microscopically (hemocytometer) since blood mononuclear cells (monocytes, lymphocytes) also bind insulin. 9'~° White blood cell contamination of 1 WBC/100,000 platelets or less can be readily achieved by this method and appears acceptable. Insulin Iodination
Crystalline, porcine, m o n o c o m p o n e n t insulin may be iodinated by a variety of methods H-~3 or obtained commercially. The preparation should contain 0.4-0.5 iodine atoms per molecule of insulin. Because iodinated insulin deteriorates over time to yield products that are biologically inactive, it is advisable that each preparation be used within 2 weeks after iodination. The insulin preparation can be purified by means of gelfiltration chromatography (Sephadex G-50) 2 or other suitable gel-filtration methods. Insulin Binding A s s a y
All materials (tubes, pipettes) that will come in contact with the labeled insulin should be made of polypropylene. This material does not adsorb insulin as readily as glass or polystyrene so that background radioactivity is minimized. Washed platelets (0.5-2.0 × 106//xl in H E P E S binding assay buffer, pH 8.0) are incubated with a mixture of 125I-labeled insulin and unlabeled insulin (0-10 /zg/ml). The mixture is briefly 8 j. Molnar and L. Lorand, Arch. Biochem. Biophys. 93, 353 (1961).
9 R. A. Schwartz, B. S. Bianco, B. S. Handwerger, and C. R. Kahn, Proc. Natl. Acad. Sci. U.S.A. 72, 474 (1975). 10j. R. Gavin, P. Gorden, J. Roth, J. A. Archer, and D. N. Buell, J. Biol. Chem. 248, 2202 (1973). H j. j. Conahey and F. J. Dixon, this series, Vol. 70, p. 210. 12M. Morrison, this series, Vol. 70, p. 214. t3 j. j. Langone, this series, Vol. 70, p. 221.
[33]
PLATELET INSULIN RECEPTOR
401
vortexed and incubated for 3 hr at 24°. Since the platelets tend to settle to the bottom of the tubes with time it is advisable to vortex the tubes gently every 20 min. At the end of the incubation period the mixture is again vortexed and duplicate 0.5-ml aliquots are removed and layered over 0.5 ml of ice-cold HEPES binding buffer in a 1.5-ml microfuge tube and immediately centrifuged in a cold room for 5 min in a microfuge (Beckman Instruments, Inc., Spinco Div., Palo Alto, CA). The supernatant buffer is removed by a two-step method, i.e., the bulk of the supernatant fluid is aspirated, and the remaining traces of buffer are allowed to drain down the inside walls of the microfuge tubes and are removed by a second aspiration. The tips of the tubes containing the platelet pellet are cut off and counted for radioactivity in a 3' counter. Specific binding of 125I-labeled insulin is determined by subtracting the amount of radioactivity (counts per minute, cpm) associated with the platelet pellet in the presence of 10/zg/ml of unlabeled insulin from the total cpm determined in the platelet sediment.
Comments Under the conditions of the assay described here, hormone degradation [as determined by trichloroacetic acid (TCA) precipitation and rebinding experiments] and receptor degradation were not observed.l
Specificity At a platelet concentration of 800,000 ~1 in the incubation mixture, binding reaches a steady state at 2 to 3 hr (half-maximal binding is reached by 30 min), at which time approximately 2% of the labeled hormone is bound to the platelets. Binding of approximately 90% of the labeled insulin is competitively inhibited by 10/xg/ml of native insulin (specific binding). As little as 3 t~g/ml of labeled insulin (a physiological blood concentration) may cause a 40-50% decrease in specific binding of the iodoinsulin. 1 Further evidence for the specificity of the binding of insulin to the platelet surface was obtained from experiments showing that with catfish insulin and porcine proinsulin (which in humans are biologically less potent than native porcine insulin) substantially higher doses are required to produce a decrease in the specific binding of nsIlabeled insulin as compared to porcine insulin. Furthermore, diisopropyl fluorophosphate (DFP)-treated thrombin, human prolactin, human growth hormone, and glucagon did not inhibit the binding of radiolabeled insulin to platelets. 1
402
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[33]
Receptor Site Number and Binding Affinity Scatchard plots 5 of the competitive binding assay data show a curvilinear pattern from which one can derive a high-affinity dissociation constant of approximately 3 x 10 9 m -1. The curvilinear pattern of the plot is consistent with either the presence of two classes of binding sites with different affinities or a single population of insulin receptors that display enhanced dissociation of labeled insulin in the presence of excess unlabeled hormone (negative cooperativity). The number of 500-600 insulin binding sites per platelet, derived from Scatchard plot analysis, is appreciably less than that reported for human lymphocytes (12,500) J° and erythrocytes (2000). Z4 However, when expressed as number of binding sites per platelet surface area, the value of approximately 25//~m 2 is very similar to the insulin receptor density observed in other insulin binding cell types. 1'9'1° It is important to note that the estimation of the density of platelet insulin receptors is based on the assumption of a smooth, continuous outer cell membrane. Since the platelets appear to have an extensive spongelike, internal surface-connecting canalicular system,15 the actual platelet surface area potentially accessible for insulin binding is likely to be greatly in excess of that estimated on the basis of the measurement of platelet size. This means that the value of 25 insulin receptors/tzm 2 for human platelets I is probably an overestimate. The method outlined about is straightforward and yields consistent results. The values for the high-affinity dissociation constant and platelet receptor number obtained with this method are lower than those obtained with FITC-labeled insulin binding experiments, 2 but are in agreement with values reported for 125I-labeled insulin binding in other systems, as well as with alternate methods of analysis (i.e., Lineweaver-Burketype p l o t s ) . 4 The issue of the biologic significance of the platelet insulin receptor remains unsettled. Whereas earlier reports indicated that insulin may increase lactate formation in human platelets 16and inhibit platelet aggregation in vitro, 17 an extensive study in our laboratory 18 failed to show any effect of insulin on in vitro secretion, glucose transport and metabolism, or protein phosphorylation in human platelets in response to stimulation
14 p. DeMeyts, J. Roth, D. M. Neville, Jr., J. R. Gavin, and M. A. Lesniak, Biochem. Biophys. Res. Commun. $5, 154 (1973). 15j. G. White, Am. J. Pathol. 66, 295 (1972). 16 S. Karpatkin, J. Clin. Invest. 46, 409 (1967). 17 A. A. Hassanein, T. A. EI-Garf, and Z. EI-Baz, Thromb. Diath. Haemorrh. 27, 114 (1972). t8 j. H. Joist, L. Babb, and R. K. Baker, unpublished observations.
[34]
CROSS-LINKING OF PLATELET MEMBRANE GLYCOPROTEINS
403
by different agonists. Others reported a lack of correlation between ADPinduced platelet aggregation and serum insulin levels in patients with type I and type II diabetes 19 and a lack of effect of continuous subcutaneous insulin infusion on platelet aggregation to ADP and epinephrine or thromboxane B 2 generation. 2° t9 D. B. Jones, T. M. Davis, E. Brown, R. D. Carter, J. I. Mann, and R. J. Prescott, Diabetologia 29, 291 (1986). 2o L. H. Monnier, M. Rodier, A. Gancel, P. Crastes De Paulet, C. Colette, M. Piperno, and J. Crastes DePaulet, Diabete Metab. 13, 210 (1987).
[34] M e m b r a n e - I m p e r m e a n t Cross-Linking Reagents for S t r u c t u r a l a n d F u n c t i o n a l A n a l y s e s o f P l a t e l e t Membrane Glycoproteins
By JAMES V.
STAROS,
NICOLAS J. KOT1TE, and
LEON W. CUNNINGHAM
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METHODS IN ENZYMOLOGY.VOL. 215
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