Effect of Stress Hormones - American Chemical Society

Biomacromolecules 2003, 4, 1506-1513

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Fibrinogen Conformation and Platelet Reactivity in Relation to Material-Blood Interaction: Effect of Stress Hormones Rolando Barbucci,* Stefania Lamponi, and Agnese Magnani C.R.I.S.M.A. and Department of Chemical and Biosystem Sciences and Technologies, University of Siena, Via Aldo Moro 2, Siena, Italy 53100 Received February 4, 2003; Revised Manuscript Received July 16, 2003

The performance of many biomaterials in hemocompatibility tests is altered when blood is drawn from stressed subjects. A salient physiological response during stress is one in which hormones are released into plasma by the hypothalamo-pituitary-adrenal axis. We investigated the influence of basal and stress levels of epinephrine and β-endorphin on the conformation of fibrinogen (Fbg), both in saline solution (under physiological conditions) and after its adsorption to polyethylene (PE), by FT-IR spectroscopy. Moreover, as Fbg is one of the major mediators of platelet adhesion, the behavior of platelets in contact with PE was also evaluated as a function of the two different hormone concentrations. Epinephrine was found to affect Fbg conformation and to increase platelet adhesion to PE at stress level. Basal and stress levels of β-endorphin did not significantly affect the Fbg conformation and only induced adhesion of isolated platelets to the PE surface. A direct relationship was therefore found between Fbg conformation and platelet behavior. The response of platelets was affected by the stress status of donors through the influence of epinephrine on Fbg conformation. Introduction The interaction of plasma proteins with solid surfaces is the first event which takes place when a material is exposed to the blood stream. This interaction seems to involve adsorption, sometimes followed by unfolding, and sequential replacement of various proteins.1 Platelets can be used to attain insight into how and why adsorbed proteins affect cell-biomaterial interactions and why their adhesion and aggregation are tightly correlated to the type and conformation of adsorbed plasma proteins.2-5 In particular, fibrinogen (Fbg) is one of the major mediators of platelet adhesion and aggregation, binding to the platelet GP IIb/IIIa receptor [via a few small peptide segments].6,7 The regions implicated are the two RGD sequences in the A-R chain (RGDF at 95-98 and RGDS at 572-575) and the C-terminal dodecapeptide of the γ chain.8 We have recently demonstrated that, together with protein adsorption, the stress condition of blood donors affects the response of platelets to materials as well. In fact, we demonstrated that stress induced high platelet aggregation and activation on various commercial materials.9-11 This behavior may be due to changes in the hormonal environment that acute stress causes, inducing activation of the hypothalamic pituitary adrenal axis and the subsequent release of some hormones, such as epinephrine12 and β-endorphin.13 These substances modify the activity of different systems and, in particular, the increase in epinephrine (E) levels during stress, which is particularly important because this substance is a potent stimulant for platelets in vitro,14-16 acting through * To whom correspondence should be addressed. Phone: +39 0577 234382. Fax: +39 0577 234383. E-mail: [email protected].

alpha-adrenergic receptors on the platelet membrane (R2adrenoreceptor of R2A subtype).14,17 Fbg conformation plays an important role in plateletbiomaterial interactions, mainly under stress conditions. In fact, higher platelet adhesion during acute stress cannot be due to higher levels of Fbg because its plasma concentration does not change with stress18,19 but may be attributed to a change in protein conformation.10 Platelets aggregate and adhere to a material9-11 if the change in Fbg conformation upon adsorption leads to the exposure of the “receptor induced binding site” (RIBS), a sequence9 for interaction with the ligand induced binding site (LIBS) on the platelet membrane.9 This paper deals with the study of the interaction of Fbg with two stress hormones in relation to the response of a synthetic surface to platelet adhesion and aggregation. The interaction of epinephrine and β-endorphin with Fbg was thus analyzed by infrared spectroscopy in a physiological solution at two different hormone concentrations and through those corresponding to their basal and stress levels in human blood10 to assess the influence of these molecules on Fbg conformation. The same study was then performed when Fbg was capable of being adsorbed onto a low-density polyethylene (PE) surface. The results were correlated to the platelet adhesion process which took place at the surface, to understand the role of the acute stress on the haemocompatibility and performance of biomaterials. Materials and Methods Samples. Low-density polyethylene (PE) was obtained from Vygon (Ecouen, France). Human Fbg (Fbg, 41000

10.1021/bm0340366 CCC: $25.00 © 2003 American Chemical Society Published on Web 09/05/2003

Effect of Stress Hormones

Biomacromolecules, Vol. 4, No. 6, 2003 1507

Figure 1. Deconvoluted IR difference spectra of (a) Fbg in normal saline; (b) Fbg and epinephrine (60 pg/mL) in normal saline; (c) Fbg and epinephrine (80 pg/mL) in normal saline.

Figure 2. Deconvoluted IR difference spectra of (a) Fbg in normal saline, (b) Fbg and β-endorphin (6pg/mL) in normal saline; (c) Fbg and β-endorphin (50 pg/mL) in normal saline.

MW) was obtained from Calbiochem (CA). Human E (183 MW) was obtained from Laboratorio Chimico Farmaceutico (Brescia, Italy), and β-endorphin were obtained from SigmaAldrich (Germany). All chemical reagents were commercially available and used without further purification. Human Blood Sampling and Processing. Ten selected donors were healthy young adult males and females who had fasted for more than 8 h and had not taken any medication for at least 14 days. Unstressed and stressed donors

were selected on the basis of plasma levels of E: unstressed, e 60 pg/mL; stressed, g 80 pg/mL. Venous blood from individuals was drawn into plastic tubes containing sodium citrate (9:1, v/v). Platelet rich plasma (PRP) was obtained by spinning the blood samples at 150 g for 15 min at room temperature. Preparation of Washed Platelets. The standard non protein method was used to wash platelets.10 Briefly, equal volumes of Krebs-Ringer solution (4 mM KCl, 107 mM

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Barbucci et al.

Table 1. Procedure for Platelet Adhesion in Presence of Basal and Stress Concentrations of Epinephrine or β-Endorphina

a

human platelets suspension medium

aim

Pw-KR with basal concentration of epinephrine (60 pg/mL) Pw-KR with basal concentration of epinephrine (60 pg/mL), and Fbg (250 mg/dL) Pw-KR with basal concentration of β-endorphin (6 pg/mL) Pw-KR with basal concentration of β-endorphin (6 pg/mL), and Fbg (250 mg/dL) Pw-KR with stress concentration of epinephrine (80 pg/mL) Pw-KR with stress concentration of epinephrine (80 pg/mL), and Fbg (250 mg/dL) Pw-KR with stress concentration of β-endorphin (50 pg/mL) Pw-KR with stress concentration of β-endorphin (50 pg/mL), and Fbg (250 mg/dL)

To assess the role of epinephrine under normal conditions To assess the role of epinephrine and Fbg under normal conditions To assess the role of β-endorphin under normal conditions To assess the role of β-endorphin and Fbg under normal conditions To assess the role of epinephrine under conditions of stress To assess the role of epinephrine and Fbg under conditions of stress To assess the role of β-endorphin under conditions of stress To assess the role of β-endorphin e and Fbg under conditions of stress

Pw-KR ) platelets washed (Pw) and suspended in Krebs-Ringer solution (KR).

Figure 3. Deconvoluted IR difference spectra of (a) Fbg in normal saline; (b) Fbg adsorbed on PE.

NaCl, 20 mM NaHCO3, 2 mM Na2SO4, 19 mM tri-sodium citrate, 0.5% (w/v) glucose in H2O, pH 6.1) and PRP were centrifuged at 500 g for 10 min. The supernatant was removed, and the pelleted platelets were resuspended in 2 mL of Krebs-Ringer solution (the vessel was rotated gently to prevent platelet aggregates breaking off the pellet) and centrifuged at 500 g for 10 min. This process was repeated twice, the final suspension being made up to the desired platelet concentration (350 000 platelets/µL) with KrebsRinger solution (Pw-KR). Epinephrine-Fbg and β-Endorphin-Fbg Mediated Platelet Adhesion. Basal and stress concentrations of epinephrine (e60 pg/mL and g80 pg/mL) or β-endorphin (≈6 pg/mL and g50 pg/mL) and/or basal concentration of Fbg (250 mg/ dl) were added to human washed platelets (Pw) suspended in Krebs-Ringer (KR) solution. The procedure followed for

the study of platelet adhesion is reported in Table 1. PE samples were then placed on the bottom of Petri dishes and incubated at room temperature for 1 h with the different media. The experiment was repeated three times for each hormone concentration. SEM Analysis. At the end of the incubation time, the media were removed from each Petri dish, and the samples were rinsed with PBS to remove any non adhering platelets and incubated in 2.5% (v/v) glutaraldehyde in 100 mM cacodylate sodium for 30 min. The samples were washed in 100 mM cacodylate buffer for 30 s, then rinsed with distilled water, and dehydrated in a series of ethanol solutions. Finally, the PE samples were dried overnight under vacuum and coated with gold in an automatic sputter coater (BAL-TEC SCD 050, Balzers, Germany). Platelet adhesion was observed by SEM (Philips XL 20, The Netherlands) at 15 kV.


Figure 4. SEM micrographs of platelet adhesion to PE surface (a) platelet adhesion for Pw-KR; (b) platelet adhesion for Pw-KR + Fbg. PE surface does not affect the protein conformation as demonstrated by the low degree of platelet adhesion with the two different media. Moreover, both with and without Fbg, the adhered platelets maintain their round shape, confirming the low activating power of the surface.


FT-IR Analysis. The IR spectra of Fbg were obtained with a Bio-Rad FTS-6000 Fourier transform infrared spectrometer operating between 4000 and 750 cm-1. An ATR cell for liquid and solid samples, equipped with a germanium crystal, was used to record the spectra in solution and on the polyethylene substrate. All spectra were recorded in single beam mode. FT-IR/ATR spectra were first recorded both in a solution containing only Fbg and in a Fbg solution containing basal or stress concentrations of E or β-endorphin. A second series of experiments were carried out allowing these protein-hormone interactions to occur in contact with a standard material: low-density polyethylene (PE). The conformation of Fbg on the PE surface was thus investigated together with that of the adsorbing protein in the presence of basal and stress concentrations of epinephrine and β-endorphin. In the case of systems in solution, the spectra of both phosphate buffered solution (PBS) and Fbg or Fbg-hormone PBS solution were acquired first. Then the spectra of both the protein and protein-hormone system were obtained subtracting the spectrum of PBS from that of the sample solution and correcting the difference spectrum for the constitution of the protein and protein-hormone system adsorbed at the Ge crystal surface. The scale factor for subtraction of the PBS spectrum was chosen so that the spectral region between 2000 and 1700 cm-1 was flat. In the case of protein adsorption on PE, the spectrum of PE was acquired first. Then the PE sample was immersed in a PBS solution containing Fbg or the Fbg-hormone system for 2 h. After the sample was washed with fresh PBS and dried in air, the IR spectrum was collected again. The spectrum of the adsorbed Fbg or Fbg-hormone system was obtained by subtracting the

Figure 5. Deconvoluted IR difference spectra of (a) Fbg adsorbed on PE; (b) Fbg + basal concentration (60 pg/mL) of epinephrine adsorbed on PE.

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Figure 6. SEM micrographs of platelet adhesion to PE surface (a) platelet adhesion in PRP from unstressed donors; (b) platelet adhesion for Pw-KR + Fbg + basal epinephrine concentration. The degree of platelet adhesion using nonstressed PRP is the same obtained with basal level of epinephrine. With both the media, a few adhered and aggregated platelets are observed on the PE surface.

spectrum of PE from the spectrum of PE with the adsorbed system. The subtraction criterion were adopted to minimize the pure band of the component to be subtracted (PE) and, once again, to obtain a flat baseline between 2000 and 1700 cm-1. The absorption bands of E and β-endorphin did not appear in the IR spectra of the analyzed systems because concentrations of 60-80 pg/mL E and 6-50 pg/mL β-endorphin were below the detection limit of infrared spectroscopy. Therefore, the difference IR spectra of the Fbg-E and Fbg-β-endorphin systems were those of Fbg as affected by basal or stress levels of hormones. To distinguish over-lapping bands, mathematical resolution enhancement was performed by spectral deconvolution. The deconvolution process moves intensity from the outer wings of a band into the center of the band, reducing its effective half-width and improving its observability. The quality of the deconvolution procedure is controlled by two variables: the half-bandwidth of the Lorentzian line used for deconvolution and the resolution enhancement achieved. All of the experiments, both in solution and on the PE surface, were repeated three times for each hormone concentration.

Barbucci et al.

Results and Discussion Infrared study of Fbg-epinephrine and Fbg-β-endorphin interaction in physiological solution as a function of hormone level. The infrared spectrum of native Fbg in saline solution together with those of the two protein-hormone systems are reported in Figures 1 and 2, respectively. In both cases, the hormone concentrations used in the experiments were too low to allow the vibrational absorptions of epinephrine and β-endorphin to be detected by IR. In this way, the variations observed in the Fbg spectrum can only be assigned to the hormone-induced conformational changes of the protein. (a) Fbg-Epinephrine Systems. The comparison of the spectrum of native Fbg with those of the protein-epinephrine system suggested a protein conformational change induced by the hormone. The presence of the basal level of epinephrine caused a drop in the maximum of the amide I band. This band, which was centered at 1650 cm-1 in the spectrum of the native protein (spectrum a in Figure 1), shifted to 1630 cm-1 in the spectrum of the Fbg-(≈60 pg/ mL) epinephrine system, suggesting an increase of the β-sheet component in the secondary structure of Fbg accompanied by a decrease of its R-helix content.20 This finding made the hypothesis of a Fbg-epinephrine interaction reasonable. When the concentration of epinephrine in the protein solution was increased up to the stress level (≈80 pg/mL), the variations observed in the Fbg-epinephrine spectrum (spectrum c in Figure 1) increased as well, supporting the hypothesis of an interaction between Fbg and epinephrine, which depends on hormone concentration. In spectrum c of Figure 1, the absorptions in the 16801660 (random coil component) and 1630-1600 cm-1 (βsheet and β-turns components) spectral ranges increased greatly with respect to that at 1650 cm-1 (R-helix component), which showed the higher intensity in the spectrum of native Fbg.20 In this case, the protein-epinephrine interaction thus led to a relevant increase of the random-coil and β-sheet and β-turn components to the prejudice of the R-helix one, suggesting a radical conformational change of the protein. (b) Fbg-β-Endorphin Systems. The infrared spectra of Fbg in the Fbg-β-endorphin systems did not differ as much as that of the native protein, in the sense that the amide I band remained centered at around 1650 cm-1, even if the hormone concentration was increased up to the stress level (Figure 2). When the β-endorphin concentration corresponded to the basal level (6 pg/mL), a small shift to lower wavenumbers of the amide I band was observed together with an increase of the bandwidth, suggesting the presence of a greater number of local microstructures, possibly because of the interaction with the hormone. However, as in the case of basal level of epinephrine, the band-shape did not change significantly. Unlike E, when the concentration of β-endorphin was increased up to the stress level (≈50 pg/mL), the infrared spectrum of the Fbg-hormone did not undergo further changes.



Figure 7. Deconvoluted IR difference spectra of (a) Fbg adsorbed on PE; (b) Fbg + stress level (80 pg/mL) of epinephrine adsorbed on PE.

This finding demonstrated that the increase of the concentration of β-endorphin in solution was not reflected in a drastic conformational change of the Fbg, suggesting that the two molecules interact weakly, so that the conformation of the protein in solution is substantially preserved. The different behavior of the two hormones in affecting the protein conformation may be explained in terms of their different molecular structure. Epinephrine is a small molecule with the following structure:

In aqueous media the molecole is protonated.

The protonated epinephrine can bind to the different domains of the protein through electrostatic, polar, and hydrophobic interactions. Because of its small size, a greater number of E molecules can interact with fibrinogen, and such interactions can be strong enough to cause the protein unfolding. The unfolded protein may in turn expose the RIBS (receptor-induced-binding sites) required for platelet interaction. It has been demonstrated that one domain recognized by resting GPIIb-IIIa on surface-bound fibrinogen corresponds to the dodecapeptide sequence of the fibrinogen

γ-chain (γ401-411).21 As this domain is not recognized by resting GPIIb-IIIa on soluble fibrinogen, it is tempting to speculate that it becomes exposed and thus more accessible for resting GPIIb-IIIa, when a protein conformational change does occur. β-endorphin is a oligopeptide, so that its interaction with fibrinogen, if any, may be less strong than that of epinephrine, preserving the protein from significant conformational changes. When the protein native conformation is retained, Fbg is not recognized by resting GPIIb-IIIa, and thus, the Fbg-platelets interactions may be inhibited. Fbg Adsorption on PE and Platelet Adhesion in the Presence of Epinephrine. The spectrum of Fbg adsorbed on the PE surface is reported in Figure 3 and compared to that of Fbg in saline solution. As it can be seen from the figure, the conformation of Fbg upon adsorption on PE was almost the same as in the saline solution; only a slight shift of the maximum of the amide I band to higher wavenumbers is observed. Thus, we may assume that PE surface did not significantly affect the Fbg conformation. That trend was confirmed by platelet adhesion as shown in Figure 4. After contact with Pw-KR without Fbg, as well as with the protein, a few platelets adhered to the PE surface maintaining their round shape. In fact, it is well-known that platelet adhesion to polymeric materials is due primarily to the adsorption of Fbg and not only to the amount of adsorbed protein.3,22,23 In particular, Fbg binds the GPIIb-IIIa glycoprotein present on the platelet membrane only if the change in the protein conformation inhibits a particular sequence: the so-called RIBS (receptor-inducing binding site) able to interact specifically with the LIBS sequence (ligand-inducing binding site) on the GPIIb-IIIa platelet receptors. If the amount of adsorbed Fbg is small and the protein does not

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Figure 9. SEM micrographs of platelet adhesion to PE surface (a) platelet adhesion for Pw-KR + epinephrine (60 pg/mL); (b) platelet adhesion for Pw-KR + stress levels of epinephrine (80 pg/mL). Figure 8. SEM micrographs of platelet adhesion to PE surface (a) platelet adhesion in PRP from stressed donor (the donor had plasma levels of 89 pg/mL epinephrine and 271 ng/mL cortisol); (b) platelet adhesion for Pw-KR + Fbg + stress levels of epinephrine (80 pg/ mL). Using stressed PRP, as well as stress level of epinephrine, a high degree of platelet adhesion and aggregation is obtained on the PE surface. A carpet of platelets covers the entire material surface. Platelets form numerous pseudopodia and fuse their membranes. The change from round to spread-dendridic and spread shape underlines the high platelet activating power of the surface.

undergo a conformational change, a low degree of platelet adhesion is observed on the material surface.22 The spectrum of PE adsorbed Fbg in the presence of the basal level concentration of E slightly changed in the amide I region (Figure 5). The wideness of the amide I band reflected the presence of a higher number of microstructures in the secondary structure of the protein: in particular a small increase of the random coil and β-sheet components was observed accompanied by a small decrease of the R-helix content.20 This finding was associated with a low degree of platelet adhesion and aggregation. A few single spread platelets and small aggregates were in fact observed on PE in PRP from unstressed donors (Figure 6a) and with (Pw-KR+ basal E + Fbg) (Figure 6b). When E in the adsorbing Fbg solution was increased up to the stress level, a dramatic change in the conformation of the protein adsorbed on PE was observed with respect to

the native one (Figure 7, parts a and b): the R-helix component largely decreased, and the random coil, β-sheet, and β-turns components increased sharply. This system closely resembles the Fbg-E (at the stress level concentration) in saline solution (Figure 1), once again emphasising the influence of E in inducing conformational changes on native and adsorbed Fbg. The platelets adhered on PE were fully adhered and aggregated with PRP from stressed donors (Figure 8a) and with (Pw-KR + stress E + Fbg) (Figure 8b), showing many pseudopodia and fused membranes. They formed a carpet over the whole PE surface. The level of epinephrine affects only platelet reactivity. In fact, platelets aggregate with both normal and stressed level of the hormone but, lacking the adhesive protein Fbg, the aggregates cannot adhere to the PE surface. When the direct influence of E on platelet reactivity was assessed adding Pw-KR with basal (Figure 9a) and stress levels (Figure 9b) of E, but without Fbg, clumping of platelets was observed, confirming the potent stimulant effect of the hormone for platelets in vitro through alpha-adrenergic receptors on the platelet membrane.14 At basal levels of E, the aggregates were much smaller than at stress concentrations of E, and SEM micrographs showed that in both cases the aggregates did not adhere to the PE. Thus, we may conclude that epinephrine stimulated the increase of platelet aggregation degree, but in the absence



In this respect, the influence of β-endorphin was meaningless. A close direct relationship between changes in Fbg conformation and platelet response was also found. Fbg played a determinant role as conveyer of surface chemical features to platelets in material-platelet interactions. Without this protein, platelets could have changed morphology but did not adhere and/or aggregate to the surface. We can thus conclude that the influence of epinephrine, both on Fbg conformation and platelet “state”, must be taken into account in material haemocompatibility assessments. Stress is a common condition during which high plasma levels of E occur. Epinephrine affects the blood-compatibility of biomaterials by changing at the same time both the conformation of Fbg, which may cause in turn platelets adhesion, aggregation, and/or activation, and the physiological state of platelets from quiescent to stimulated. Acknowledgment. The authors thank the Ministry of the University and Technological and Scientific Research for financial support (ex-60%). References and Notes

Figure 10. SEM micrographs of platelet adhesion to PE surface (a) platelet adhesion for Pw-KR + β-endorphin (6 pg/mL); b) platelet adhesion for Pw-Kr + stress level of β-endorphin (50 pg/mL).

of the adhesive protein (Fbg), the aggregates did not adhere to the surface. Fbg Adsorption on PE and Platelet Adhesion in the Presence of β-Endorphin. The spectra of Fbg adsorbed on PE in the presence of β-endorphin at the basal and stress levels did not show any significant variation with respect to that of the adsorbed Fbg by itself. On the other hand, the SEM analysis of platelets adhesion revealed a few isolated platelets on the material surface in all of the performed experiments, i.e., with Pw-KR with basal and stress levels of β-endorphin (Figure 10, parts a and b). Moreover, β-endorphin at both basal and stress levels, in absence of Fbg, did not induce platelet adhesion on PE: very few platelets were in fact observed and they did not adhere to the surface. Conclusions The role of epinephrine in inducing Fbg conformational changes, and, in turn, platelet adhesion and aggregation, was assessed when its concentration reached the stress level. In particular, the following main features can be pointed out by this study: The response of blood used for haemocompatibility tests was affected by the stress condition of donors or, in other words, by some hormones secreted thereof (i.e., epinephrine).

(1) Horbett, T. A.; Brash, J.-L. In Physicochemical and biochemial studies; Brash, J.-L., Horbett, T. A., Eds.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987; Chapter 1. (2) Dumitriu, S. In Polymeric Materials; Marcel Dekker Publisher: New York, 1994; Chapter 7. (3) Jahangir, A. R.; McClung, W. G.; Cornelius, R. M.; McCloskey, C. B.; Brash, J. L.; Santerre, J. P. J Biomed. Mater. Res. 2002, 60, 135. (4) Brash, J. L. J. Biomater. Sci. Polym. Ed. 2000, 11, 1135. (5) Sheppard, J. I.; McClung, W. G.; Feuerstein, I. A. J. Biomed. Mater. Res. 1994, 28, 1175. (6) Grunkemeier, J. M.; Tsai, W. B.; McFarland, C. D.; Horbett, T. A. Biomaterials 2000, 21, 2243. (7) Tsai, W. B.; Grunkemeier, J. M.; Horbett, T. A. J. Biomed. Mater. Res. 1999, 44, 130. (8) Kieffer, N. In The role of platelets in blood-biomaterial interactions; Missirlis, Y.-S., Wautier, J.-L,, Eds.; Kluwer Academic Publishers: The Netherlands, 1993; Chapter 7. (9) Barbucci, R.; Lamponi, S.; Aloisi, A. M. J. Biomed. Mater. Res. 1999, 46, 186. (10) Lamponi, S.; Aloisi, A. M.; Barbucci, R. Biomaterials 1999, 19, 1791. (11) Lamponi, S.; Barbucci, R.; Aloisi, A. M. J. Biomed. Mater. Res. Appl. Biomater. 1999, 48, 9. (12) Breznitz, S.; Ben-Zur, H.; Berzon, Y.; Weiss, D. W.; Levitan, G.; Tarcic, N.; Lischinsky, S.; Greeberg, A.; Levi, N.; Zinder, O. Brain BehaV. Immun. 1998, 12, 34. (13) Ryu, H.; Lee, H. S.; Shin, Y. S.; Chung, S. M.; Lee, M. S.; Kim, H. M.; Chung, H. T. Am. J. Chin. Med. 1996, 24, 193. (14) Anfossi, G.; Trovati, M. Eur. J. Clin. InVest. 1996, 26, 353. (15) Lanza, F.; Beretz, A.; Stierle`, A.; Hanau, D.; Kubina, M.; Cazenave, J. P. Am. J. Pysiol. (Heart Circ. Physiol.) 1998, 255(24), H1276. (16) Rao, G. H. R.; Escolar, G.; White J. G. Thrombosis Res. 1986, 44, 65. (17) Rao, G. H. R.; Escolar, G.; White J. G. Platelets 1990, 1, 145 (18) Muldoon, M. F.; Herbert, T. B.; Patterson, S. M.; Kameneva, M.; Raible, R.; Manuck, S. B. Arch. Intern. Med. 1995, 155, 615. (19) Castillo, V.; Navas, E.; Naranjo, R.; Jimenez-Jimenez, L. Rev Esp. Anestesiol. Reanim. 1997, 44, 52. (20) Gendreau, R. M. In Spectroscopy in the Biomedical Sciences; Gendreau, R. M.. Ed.; CRC Press: Boca Raton, FL, 1986; Chapter 2. (21) Kieffer, N. In The role of platelets in blood-biomaterial interactions; Missirlis, Y. F., Wautier, J.-L. Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1993; Chapter 2. (22) Lindon, J. N.; McManama, G.; Kushner, L.; Merril, E. W.; Salzman, E. W. Blood 1986, 68, 355. (23) Chinn, J. A.; Posso, S. E.; Horbett, T. A.; Ratner, B. D. J. Biomed. Mater. Res. 1991, 25, 535.

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Effect of Stress Hormones - American Chemical Society

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