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Changes in Adsorbed Fibrinogen upon Conversion to Fibrin Kenyon M. Evans-Nguyen,† Ryan R. Fuierer,† Brian D. Fitchett,‡ Lauren R. Tolles,† John C. Conboy,‡ and Mark H. Schoenfisch*,† Department of Chemistry, UniVersity of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, and Department of Chemistry, UniVersity of Utah, Salt Lake City, Utah 84112 ReceiVed NoVember 14, 2005. In Final Form: February 23, 2006 The conversion of adsorbed fibrinogen to fibrin in the presence of the enzyme thrombin was studied using surface plasmon resonance (SPR), a quartz crystal microbalance (QCM), sum frequency generation (SFG), atomic force microscopy (AFM), and an elutability assay. Exposure of adsorbed fibrinogen to thrombin resulted in a mass loss at the surface consistent with fibrinopeptide release and conversion to fibrin. Changes in hydration upon conversion of adsorbed fibrinogen to fibrin were determined from comparisons of acoustic (QCM) and optical (SPR) mass adsorption data. Conversion to fibrin also resulted in the adsorbed layer becoming more strongly bound to the surface and more compact. The elutability of adsorbed fibrinogen by Triton X-100, studied with SPR, decreased from 90 ( 5 to 6 ( 2% after conversion to fibrin. The height of the adsorbed monolayer, as determined by AFM, decreased from 5.5 ( 2.2 to 1.7 ( 0.8 nm. We conclude that thrombin-catalyzed fibrinopeptide release triggers significant changes in fibrinogen conformation beyond peptide cleavage.
Introduction Although the use of biomaterials has become widespread, significant problems with thrombosis accompany the utility of blood-contacting devices such as catheters, intravascular sensors, and extracorporeal cardiopulmonary bypass tubings.1,2 When a material foreign to the body contacts blood, plasma proteins rapidly adsorb at the blood-material interface.3-5 Subsequent platelet adhesion and activation of coagulation are mediated by this adsorbed protein layer, often resulting in surface-induced thrombosis and associated embolic complications.2,6 Efforts to improve the clinical viability of blood-contacting materials are dependent upon research dedicated to enhancing the understanding of protein adsorption and subsequent function at surfaces. Fibrinogen is of particular significance in the coagulation response to implants due to its roles in platelet adhesion and formation of fibrin polymer, the major structural component of the provisional wound-healing matrix.3,7,8 Fibrinogen, a 340 kD plasma rod-shaped protein roughly 45 nm long with an approximate diameter of 5 nm,9,10 is divided into four distinct regions: the E domain at the center of the molecule; two flanking D domains located at the terminal ends of the molecule; the coiled-coil arms (i.e., intertwined R helices) connecting the D and E domains; and the RC domains that loosely extend outward from the D domains. Fibrin polymer is formed through a series * To whom correspondence should be
[email protected]. † University of North Carolina at Chapel Hill. ‡ University of Utah.
addressed.
E-mail:
(1) Ratner, B. D. J. Biomed. Mater. Res. 1993, 27, 283-7. (2) Horbett, T. A. BMES Bull. 1999, 23, 5-9. (3) Anderson, J. M. Annu. ReV. Mater. Res. 2001, 31, 81-110. (4) Andrade, J. D. In Surface and Interfacial Aspects of Biomedical Polymers; Andrade, J. D., Ed.; Plenum Press: New York, 1985; Vol. 2. (5) Andrade, J. D.; Hlady, V. In Blood in Contact With Natural and Artificial Surfaces; Leonard, E. F., Turitto, V. T., Vroman, L., Eds.; New York Academy of Sciences: New York, 1987; Vol. 516, pp 158-172. (6) Horbett, T. A. CardioVasc. Pathol. 1993, 2, 137S-148S. (7) Feng, L.; Andrade, J. D. In Proteins at Interfaces II: Fundamentals and Applications; Horbett, T. A., Brash, J. L., Eds.; American Chemical Society: Washington DC, 1995. (8) Blomback, B. Thromb. Res. 1996, 83, 1-75. (9) Weisel, J. W. AdV. Protein Chem. 2005, 70, 247-299. (10) Mosesson, M. W. Semin. Thromb. Hemostasis 1998, 24, 169-174.
of steps that begins when fibrinogen is exposed to the enzyme thrombin, which is activated during coagulation. Thrombin is a serine protease that catalyzes the cleavage of two sets of specific arginine-glycine bonds at the center of fibrinogen, releasing two sets of two types of peptides, fibrinopeptides A and B (FpA and FpB, respectively). Fibrinopeptide cleavage yields fibrin, which has newly formed regions of positive charge, known as “knobs”, in the region from which FpA and FpB are cleaved. Fibrin molecules associate in a staggered orientation as the knobs in the E domain interact with regions of negative charge in the D domain, known as “holes”. Additionally, intermolecular interactions between D domains promote fibrin interactions. Fibrin molecules associating in this half-staggered fashion yield extended strands of fibrin, which branch out and polymerize further, to produce fibrin polymer. The weblike structure of this polymer acts as the structural scaffolding in the blood clotting process. In view of fibrinogen’s role in mediating such vital processes, fibrinogen adsorption has been widely studied at a variety of surfaces using numerous techniques.7 In particular, the conformation of fibrinogen at surfaces and its correlation to adsorbed fibrinogen function is of critical interest. Horbett and co-workers studied how effectively adsorbed fibrinogen was eluted by surfactants and attributed differences in “elutability” to changes in protein conformation.11-13 They concluded that less elutable fibrinogen (i.e., more tightly bound to the surface) correlated with decreased platelet binding activity. Sit and Marchant were able to directly observe the conformation of fibrinogen at surfaces using atomic force microscopy (AFM).14 They found that fibrinogen spreads more extensively at hydrophobic and positively charged surfaces, relative to negatively charged surfaces. Likewise, Agnihotri and Siedlecki used AFM to directly observe the spreading of fibrinogen at hydrophobic and negatively charged surfaces as a function of time.15 As evidenced by changes in the (11) Chinn, J. A.; Phillips, R. E., Jr.; Lew, K. R.; Horbett, T. A. J. Colloid Interface Sci. 1996, 184, 11-19. (12) Chinn, J. A.; Posso, S. E.; Horbett, T. A.; Ratner, B. D. J. Biomed. Mater. Res. 1991, 25, 535-555. (13) Horbett, T. A. In Biopolymers at Interfaces; 2nd ed.; Malmsten, M., Ed.; Marcel Dekker: New York, 2003; Vol. 110, pp 393-413. (14) Sit, P. S.; Marchant, R. E. Thromb. Haemostasis 1999, 82, 1053-1060. (15) Agnihotri, A.; Siedlecki, C. A. Langmuir 2004, 20, 8846-8852.
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protein’s dimensions (i.e., height), they found that adsorbed fibrinogen spread in a two-phase process: rapidly upon initial adsorption, but then slowing exponentially as more fibrinogen adsorbed. Using sum frequency generation (SFG) spectroscopy, a method used to assess the symmetry of organic functional groups at an interface,16 Jung et al. monitored changes in fibrinogen conformation at a negatively charged surface as a function of pH.17 They found that the interactions of the RC domain with the surface vary significantly with pH and influence the overall adsorbed state of fibrinogen. The quartz crystal microbalance (QCM) has also been used to provide valuable information about protein adsorption including the amount (i.e., mass) adsorbed and viscoelasticity properties.18,19 In combination with the frequency signal that provides information about the mass of material adsorbed, the energy dissipation of the adsorbed material has been used to monitor changes in viscoelasticity when adsorbed proteins cross-link,20 and conformational changes in adsorbed proteins.21 More recent studies have focused on the initial stages of fibrin formation at surfaces using QCM,22 surface plasmon resonance (SPR),23-25 coagubility of beads coated with fibrinogen,26 and AFM.27 Our group has reported that initial fibrin formation at surfaces is strongly dependent on the surface properties of the underlying substrate.22 We have also characterized thrombincatalyzed fibrinopeptide cleavage from adsorbed fibrinogen, finding that the surface dependence of fibrin formation is likely due to differences in thrombin interaction with fibrinogen.23 Herein, we report on the structural changes in the adsorbed fibrinogen layer after exposure to thrombin. Both fibrinogen adsorption on a hydrophobic model substrate and the conversion of adsorbed fibrinogen to fibrin were characterized by SPR and QCM. Additionally, SPR was used to determine the adhesion strength of adsorbed fibrinogen and fibrin via surfactant elution studies. Differences in adsorbed fibrinogen and fibrin layers were further characterized using SFG and AFM. Alkanethiol and alkylsilane self-assembled monolayers28,29 (SAMs) were used as model surfaces. Experimental Methods Materials. Plasminogen-depleted human plasma fibrinogen (FIB1) and human R-thrombin (HT1002a) were purchased from Enzyme Research Laboratories (South Bend, IN). Dodecanethiol and hexadecanethiol were products of Sigma Scientific (St. Louis, MO) and (16) Koffas, T. S.; Amitay-Sadovsky, E.; Kim, J.; Somorjai, G. A. J. Biomater. Sci. Polym Ed. 2004, 15, 475-509. (17) Jung, S.-Y.; Lim, S.-M.; Albertorio, F.; Kim, G.; Gurau, M. C.; Yang, R. D.; Holden, M. A.; Cremer, P. S. J. Am. Chem. Soc. 2003, 125, 1278212786. (18) Cavic, B. A.; Chu, F. L.; Furtado, L. M.; Ghafouri, S.; Hayward, G. L.; Mack, D. P.; McGovern, M. E.; Su, H.; Thompson, M. Faraday Discuss. 1997, 107, 159-176. (19) Marx, K. A. Biomacromolecules 2003, 4, 1099-1120. (20) Hook, F.; Kasemo, B.; Nylander, T.; Fant, C.; Sott, K.; Elwing, H. Anal. Chem. 2001, 73, 5796-5804. (21) Hook, F.; Rodahl, M.; Kasemo, B.; Brzezinski, P. Proc. Natl. Acad. Sci. 1998, 95, 12271-12276. (22) Evans-Nguyen, K. M.; Schoenfisch, M. H. Langmuir 2005, 21, 16911694. (23) Evans-Nguyen, K. M.; Tolles, L.; Gorkun, O.; Lord, S. T.; Schoenfisch, M. H. Biochemistry 2005, 44, 15561-15568. (24) Dyr, J. E.; Rysava, J.; Suttnar, J.; Homola, J.; Tobiska, P. Sens. Actuators, B 2001, 74, 69-73. (25) Rysava, J.; Dyr, J. E.; Homola, J.; Dostalek, J.; Krizova, P.; Masova, L.; Suttnar, J.; Briestensky, J.; Santar, I.; Myska, K.; Pecka, M. Sens. Actuators, B 2003, B90, 243-249. (26) Whitlock, P. W.; Clarson, S. J.; Retzinger, G. S. J. Biomed. Mater. Res. 1999, 45, 55-61. (27) Sit, S. P.; Marchant, R. E. Surf. Sci. 2001, 491, 421-432. (28) Ostuni, E. Y., L.; Whitesides, G. M. Colloid Surf. B 1999, 15, 3-30. (29) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. ReV. 2005, 105, 1103-1169.
EVans-Nguyen et al. were used as received. All protein aliquots and fibrinogen fragments were stored at -80 °C. Fibrinogen aliquots were thawed at 37 °C, stored at 5 °C when not in use, and used within 1 week of thawing. Fibrinogen concentrations were determined using a Perkin-Elmer Lambda 40 Spectrophotometer, assuming an A280 of 1.5 for a 1 mg/mL fibrinogen solution.9 Thrombin aliquots were used on the same day that they were thawed. (1-Mercaptoundec-11-yl) tri(ethylene glycol) was purchased from Prochimia (Gdansk, Poland). Thiol solutions were prepared with absolute ethanol. HEPES-buffered saline with calcium (HBSC; 0.1 M HEPES, 0.15 M NaCl, 1 mM CaCl2, pH 7.4) was used to prepare protein solutions and as the buffer for all experiments unless noted otherwise. Water was purified using a Milli-Q UV Gradient A10 System (Millipore Corp.; Billerica, MA) to a final resistivity of 18.2 MΩ/cm and a total organic content of
