Eur. J. Biochem. 227, 647-656 (1995) 0 FEBS 1995

Determination of kinetic constants for the interaction between the platelet glycoprotein IIb-IIIa and fibrinogen by means of surface plasmon resonance Walter HUBER, Jiirg HURST, Daniel SCHLATTER Richard BARNER Josef HUBSCHER William C. KOUNS and Beat STEINER

Pharma Division, Preclinical Research, F. Hoffmann-La Roche Ltd, Basel, Switzerland

(Received 16 November 1994) - EJB 94 1758/1

The binding reaction between purified human platelet glycoprotein IIb-IIIa and fibrinogen was investi- gated by real-time measurements using the surface-plasmon-resonance sensor technology. In these experi- ments, either glycoprotein IIb-IIIa or fibrinogen was immobilized on a sensor surface. The time-dependent change in surface coverage that occurred immediately upon contact with a solution of the complementary protein was then detected. The ability to record this dynamic event from its initiation allowed the collec- tion of kinetic and thermodynamic data over an extended time period. These data indicated that initially, in fast reaction, a reversible low-affinity complex with an equilibrium dissociation constant, Kd, of 155- 180 nM was formed. In a subsequent slower reaction this complex was transformed into a more stable high-affinity complex with a Kd of 20-70 nM. Efficient dissociation of the high-affinity complex could only be induced in the presence of a competitive inhibitor such as RGDV. These data demonstrate that the binding between glycoprotein IIb-IIIa and fibrinogen is not a single monophasic reaction, but is composed of at least two consecutive processes both with their own kinetics.

Keywords. Platelet aggregation ; adhesion receptor; protein-protein interaction ; ligand-induced conforma- tional change; affinity biosensor.

Biochemical affinity sensors represent a novel analytical tool for the characterization of biomolecular recognition processes such as the interaction between antibodies and antigens, recep- tors and ligands or DNA and proteins. The method uses capture molecules, covalently fixed on the surface of a sensor that bind the corresponding target molecule in solution. The target mole- cule to be analyzed, associating to and dissociating from the surface, changes the molecular coverage of the surface continu- ously. By measuring these changes in surface coverage as a function of time, the characteristic binding behavior between the respective reaction partners can be monitored. By observing the time course of the binding, it is possible to calculate not only the thermodynamic association and dissociation constants, but also the individual kinetic rate constants of the forward and the backward reactions. Based on the planar optical waveguide tech- nology three different affinity sensor systems have been de- scribed, namely surface plasmon resonance (Liedberg et al., 1983; LofAs et al., 1991) grating coupling (Nellen and Lukosz, 1990; Lukosz et al., 1988) and difference interferometry (Fat- tinger et al., 1993; Schlatter et al., 1993).

Human platelet glycoprotein Ilb-IIIa is a member of the inte- grin family of cell adhesion receptors. Glycoprotein IIb-IIIa is a Ca2+-dependent heterodimer complex (Jennings and Phillips, 1982). The heterodimeric receptor binds several adhesive pro-

Correspondence to w. Huber, F. Hoffmann-La Roche, Grenzacher-

Fax: +41 61 688 7617. Abbreviations. LIBS, Ligand-induced binding sites ; RU, response

unit; AR, sensor-response increase ; AR,,, sensor-response increase to equilibrium; AR,,,, sensor response after saturation of all binding sites, maximal response; Kd, equilibrium dissociation constant; k,,, associa- tion-rate constant ; k,,,, ku f f , dissociation rate constants.

strasse 124, CH-4002 Basel, Switzerland

teins such as fibrinogen (Marguerie et al., 1980; Bennett et al., 1982), von Willebrand factor (Fujimoto et al., 1982) and fibro- nectin (Plow and Ginsberg, 1981). Similar to some other mem- bers of the integrin family, glycoprotein IIb-IIIa recognizes li- gands via the amino acid sequence Arg-Gly-Asp (RGD; Pyetela et al., 1986). In addition, glycoprotein IIb-IIIa binds to the HHLGGAKQAGDV sequence (dodecapeptide) located at the carboxy-terminus of the y-chain of fibrinogen (Kloczewiak et al., 1984). Recent studies suggest that this latter sequence may be the primary mediator of platelet aggregation (Farrell et al., 1992; Farrell and Thiagarajan, 1994).

Following platelet activation, conformational changes occur in glycoprotein IIb-IIIa, which enable the complex to bind solu- ble fibrinogen (Sims et al., 1991). The binding of fibrinogen induces additional conformational changes in the complex that result in the exposure of neo-epitopes called ligand-induced binding sites (LIBS ; Frelinger et al., 1988). Neo-epitopes are also exposed on fibrinogen (Zamarron et al., 1991 ; Ugarova et al., 1993), thus demonstrating that the binding of ligands to gly- coprotein IIb-IIIa is a dynamic process that results in post-occu- pancy changes in both the receptor and the ligand. These post- occupancy events in the receptor-ligand complex may play an important role in ensuring that the platelet-bound fibrinogen is in the proper conformation to fully support platelet aggregation (Ugarova et al., 1993). Furthermore, over time, the bound fibrin- ogen becomes irreversibly bound to the platelet surface (Peerschke, 1992) and this may be an important process in sup- porting the contractile forces generated during the contraction of clots.

Platelet aggregation at the site of a vascular injury is critical for the formation of a normal hemostatic plug, particularly under arterial flow conditions. However, unregulated platelet deposi-

648 Huber et al. ( E m J . Biochem. 227)

tion can lead to pathological conditions such as myocardial in- farction, unstable angina and stroke. Clinical trials are currently underway to determine, whether potent and selective non-pep- tide glycoprotein IIb-IIIa antagonists are more effective than the currently available treatment of arterial thrombosis (Coller, 1992; Kouns et al., 1993). A detailed characterization of the kinetics of fibrinogen binding to glycoprotein IIb-IIIa could lead to a deeper understanding of the function of the receptor and aid in the development of new classes of antithrombotic compounds.

In this study, we have used surface plasmon resonance tech- nology to evaluate the kinetics of soluble glycoprotein IIb-IIIa binding to immobilized fibrinogen and of soluble fibrinogen binding to immobilized glycoprotein IIb-IIIa. Special attention was paid to the initial phase of the binding event, a possibility realized by the sensor technology allowing kinetic measure- ments in real time. The determined sets of kinetic data revealed new and interesting insights into mechanistic aspects of the in- teraction between glycoprotein IIb-IIIa and fibrinogen.

MATERIALS AND METHODS

Reagents and proteins. The synthesis of 11 mercaptounde- canol and 11-mercaptoundecanoic acid was performed as de- scribed (Bain et al,,, 1989). (Tridecafluoro-l,1,2,2,-tetrahydrooc- ty1)-1-dimethylchlorosilan was purchased from Petrarch. Fibrin- ogen (fibronectin free) was purchased from IMCO. The glyco- protein IIb-IIIa was isolated from human platelets that were too old for medicinal use according to the procedures described pre- viously (Steiner et al., 1989). Two different preparations of gly- coprotein IIb-IIIa were used. One glycoprotein IIb-IIIa prepara- tion contained the tetrapeptide RGDV (> 1 mM), which was used to remove g1;ycoprotein IIb-IIIa from the affinity column. The second preparation was obtained by dialyzing the glycopro- tein IIb-IIIa in order to remove RGDV.

Instrumentation. The measurements were carried out on an in-house-built surface-plasmon-resonance apparatus (Huber et al., 1992). The sensor device consists of the planar sensor chip and the micro cell. The sensor chip consists of a glass plate, which supports the waveguiding gold film which has a thickness of 46 nm. The micro cell defines a flow-through channel for the sample solution with a thickness of 20-30 pm and a volume of less than 1 pl. The flow of the sample solution was kept constant at 1 pl/s during the measurements, which were performed for the determination of kinetic constants. The plasmon-resonance instrument measures the resonance angle a, at which the surface- plasmon wave is excited at the interface between the gold film and the sample solution. This resonance angle depends critically on the effective refractive index of the guided plasmon and hence on the change of the refractive index within a thin layer (0-100 nm) close to the gold surface. The latter is changed upon adsorption on and desorption of molecules from the sur- face. The correlation between the shift in this resonance angle and the change in surface mass upon adsorption and desorption of proteins was determined to be 3.95 ng . mm-’ . degree-’ (Huber et al., 1992). One response unit (RU) given in the sen- sorgrams depicted in this publication corresponds to a surface mass change of 1 pg proteidmm’.

Chemical modifications of the gold surfaces of the sensor chips. Depending on the protein to be immobilized, glycoprotein IIb-IIIa or fibrinogen, the sensor surfaces were modified with different organic adlayers.

The surface for the immobilization of glycoprotein IIb-IIIa was prepared as follows. The sensor chip was incubated over- night in a solution of 11 -mercaptoundecanol in methanol (2 mM). The gold surface was thereby coated with a self-assem-

bled monolayer (Bain and Whitesides, 1989). The alkyl deriva- tives forming this self-assembled monolayer are covalently linked to the gold surface via the sulfur atoms and expose the hydroxy groups to the solvent. A second self-assembled adlayer was then coupled to this mercaptoundecanol layer by silanizing (Ulman and Tillman, 1989) the surface in a solution of (tride- cafluoro-l,1,2,2,-tetrahydroocty1)-1 -dimethylchlorosilan (0.5 % by vol.) in CC1,.

The surface for the immobilization of fibrinogen was modi- fied as follows. The gold surface of the sensor chip was treated overnight with a solution of 11-mercaptoundecanoic acid in methanol (2 mM). The self-assembled monolayer thereby formed is composed of undecanoic acid derivatives, which are covalently linked to the gold surface via sulfur atoms (Bain and Whitesides, 1989) and which expose the carboxylic acid groups to the solvent.

Immobilization of glycoprotein IIb-IIIa on the sensor surface. An aliquot of 5 p1 glycoprotein IIb-IIIa solution con- taining 150 pg/ml protein in buffer A [20 mh4 Tris/HCl, pH 7.4, 150 mM NaCI, 1 mM CaCl,, 1 mM MgCl,, 0.1 % (by mass) Tri- ton X-100, 0.05% (by mass) NaN,, 1 mM tetrapeptide RGDV] or buffer B (buffer A without RGDV) was diluted with 995 p1 buffer C [20 mM Tris/HCl, pH 7.4, 150 mM NaC1, 1 mM CaCl,, 1 mM MgC12, 0.05% (by mass) NaN,]. The corresponding chemically modified, hydrophobic gold surface of the sensor chip was brought into contact with this glycoprotein IIb-I1Ia so- lution immediately after dilution. The coating procedure was usually carried out within the plasmon-resonance instrument in order to follow the adsorption of glycoprotein IIb-IIIa and fi- nally to determine the amount of immobilized glycoprotein IIb- IIIa. After a contact of approximately 30min the surface was washed with buffer C. Non-specific binding sites on the surface were blocked with buffer C containing 3.5% (by mass) BSA.

Immobilization of fibrinogen. To follow the immobiliza- tion and to determine the amount of immobilized fibrinogen the immobilization of fibrinogen was carried out on the plasmon- resonance apparatus. The carboxylic acid groups of the mono- layer were converted into N-succinimidyl derivatives by treating the surface with an aqueous solution of N-(3-dimethylaminopro- py1)-N’-ethylcarbodiimide hydrochloride (100 mM) and N-hy- droxy-succinimide (25 mM) for 5 min. After washing the sur- face with water and acetate buffer (10 mM sodium acetate, pH 5.5) the surface was contacted with a solution containing fibrinogen (10 pg/ml) dissolved in acetate buffer. The amount of fibrinogen immobilized in a given experiment was adjusted by varying the incubation time with the fibrinogen solution. Typi- cally, the sensor surface was coated with 1 .O- 1.5 ng/mm2 fibrin- ogen. Non-specific binding sites on the surface were blocked with buffer B containing 3.5% (by mass) BSA.

Determination of the kinetic and thermodynamic param- eters of the binding of glycoprotein IIb-IIIa to immobilized fibrinogen. The kinetic constants were determined as follows. A sensor chip with immobilized fibrinogen was brought into contact with buffer D [buffer B containing 1 % (by mass) BSA]. After reaching a stable baseline the buffer D was exchanged by the sample solution, which contained glycoprotein IIb-IIIa in buffer D. The concentration was chosen in such a way that the resulting equilibrium surface concentration corresponded to an occupancy of approximately 30-70 % of the maximal glycopro- tein-IIb-IIIa-binding sites exposed by the immobilized fibrino- gen. The flow of the buffer solution was kept at a high level (1 plls) to prevent depletion of glycoprotein IIb-IIIa at the sen- sorlsolution interface. The change in the resonance angle was monitored consecutively with time intervals of 1-20 s. The measurements were continued for 5 - 10 min. Afterwards, the glycoprotein IIb-IIIa solution was replaced by buffer D. The de-

Huber et al. ( E m J. Biochern. 227) 649

sorption of glycoprotein IIb-IIIa occurring under these condi- tions was followed by measuring the change in plasmon-reso- nance angle. After a contact time of a few minutes buffer D was exchanged by sample solution containing a high concentration (> 50XKd) of glycoprotein IIb-IIIa. To save protein, when work- ing with these highly concentrated protein solutions, the flow was usually decreased to 0.1 pl/s as soon as the sample cell vol- ume was completely exchanged. The surface was kept in contact with this highly concentrated glycoprotein IIb-IIIa solution until the sensor had approached near saturation (full saturation was usually calculated by extrapolating the sensor-response curve to infinite time). Finally, the sensor was again brought into contact with buffer D.

From such experiments the kinetic data can be extracted (Ed- dowes, 1987; Schlatter et al., 1993). Within the simple kinetic model, one can assume that the net adsorption monitored with the surface-plasmon-resonance sensor is due to reversible asso- ciation and dissociation reactions occurring between the immo- bilized species A ( e g fibrinogen) and the species in solution B (e.g. glycoprotein IIb-IIIa). Such an interaction is expressed by Eqn (1).

k m

A + B C A B . (1)

Based on this model the expected time-dependent change in the concentration of the surface-bound complex exponentially ap- proaches an equilibrium surface concentration and hence the time-dependent sensor response exponentially approaches an equilibrium sensor response (Eddowes, 1987). The rate of a change in the surface concentration of the analyte, assuming that the reaction is not limited by mass transport to the surface, can be expressed as the difference between the rate of association and the rate of dissociation (Eqn 2).

d [ABIldt = k,, X [AB],,,, X [B] - (ken X [B] + koR) X [AB]. (2)

Since the concentration of the complex ([AB]) bound to the sur- face is directly proportional to the sensor response (AR), all ab- breviations in Eqn (2), which denote the concentration of the complex can be replaced by the change in sensor response AR (Eqn 3).

From the slope and the intercept of the straight line, which re- sults from plotting the slope of the sensor-response curve at given time t (dARldt) versus the sensor responses at the respec- tive times (AR), one can calculate the rate constants for the asso- ciation and the dissociation reaction. The slopes of the response curve at times t (dARldt) were evaluated by fitting an exponen- tial equation to the experimental data points. The derivative of the fitting equation was used to calculate the slopes of the re- sponse curve at different times t . AR,,,, represents the sensor response after saturation of all binding sites on the surface. The concentration of the dissolved species ([B]) is held constant by maintaining a high sample flow within the sample cell.

The determination of the AR,, value is sometimes difficult because high protein concentrations in solution and long contact times on the surface are needed to reach saturation. The determi- nation of AR,,,, can, however, be circumvented by carrying out binding studies with one and the same sensor surface (the latter is regenerated after each experiment) but with different concen- trations of dissolved species B. From the sensor response at a given concentration one can extract the term,

k,,, X [B] + k,, = t (4) which represents the time constant, z, of the exponential change

of the sensor response. These time constants, t, were again eval- uated by fitting an exponential equation to the response curves. By plotting the z determined for different concentrations [B] versus the respective concentrations, a straight line should be obtained, the intercept and the slope of which correspond to the rate constant of the dissociation and association reaction, respec- tively.

An independent way to determine the rate constant of the dissociation reaction is based on the investigation of the dissoci- ation reaction of a formed complex. In the case of reversible processes, the complex at the interface will completely dissoci- ate, if the sensor surface is brought into contact with ligand-free buffer solution, which is free from species B. One expects an exponential decay of the surface-bound complex and hence an exponential change of the sensor response. The time constant of this exponential change, which represents the rate constant of the dissociation reaction (koC), was evaluated by fitting an expo- nential equation to the experimental data points.

Another type of experiment was performed to determine equilibrium dissociation constants. A fibrinogen-coated surface was subsequently brought into contact with glycoprotein IIb-IIIa solutions. An equilibrium sensor response could be evaluated by fitting a mathematical equation to the initial experimental curve and extrapolating it to infinite time. After each binding study the surface was regenerated and contacted with a solution con- taining a different concentration of glycoprotein IIb-IIIa. The sensor responses evaluated for the different concentrations were then used in a Scatchard-type analysis (Scatchard, 1949). Thereby, the sensor response was taken as a measure for the concentration of bound ligand. Since a flowing sample solution is used, the concentrations of free ligand are known. From this type of Scatchard analysis one can extract the equilibrium disso- ciation constants.

Determination of the kinetic and the thermodynamic parameters for the binding of fibrinogen to immobilized gly- coprotein IIb-IIIa. The experiments were similar to those car- ried out with the reverse experimental system. For these experi- ments the buffer of the fibrinogen solution was changed to buffer E (buffer D without Triton X-100). For reasons given in the following sections, the procedure to determine the equilibrium dissociation constants was also changed. With immobilized gly- coprotein IIb-IIIa the surface was contacted subsequently with sample solutions containing different concentrations of fibrino- gen without regenerating the surface. For a given concentration the contact was maintained until a stable sensor response was reached. For each concentration a step-wise increase in the sen- sor response was observed. The plateau values of this step-wise increase corresponded to the equilibrium sensor responses, which were used for the Scatchard analysis as described above.

Regeneration of sensor surfaces. The surface of the sensor was regenerated by using buffer solutions containing RGDV (1 mM). To regenerate the fibrinogen-coated surface buffer F [buffer A containing 1 % (by mass) BSA] was used. To regener- ate the glycoprotein IIb-IIIa surface buffer G (buffer F without Triton X-100) was used to avoid desorption of glycoprotein IIb- IIIa from the surface. After the contacts with RGDV-containing solutions the surfaces were always subjected to the respective RGDV-free buffers before starting a new adsorption experiment.

RESULTS

Activity of immobilized glycoprotein IIb-IIIa. Fig. 1 depicts a sensorgram that was recorded during the immobilization of glycoprotein IIb-IIIa onto the chemically modified sensor sur- face of a plasmon-resonance sensor. During the time interval of

650 Huber et al. (Eul: J . Biochem. 227)

1 .o

0.8 - 3 [r ;;; 0.6

a 8 0.4 [r

v) c 0

“0 T- 0.2

0.0

0 500 1000 1500 2000

Time (s)

Fig. 1. Sensorgram monitored during the immobilization of glyco- protein IIb-IIIa on the sensor surface. A and B indicate time intervals during which the sensor was in contact with different solutions (A, buffer C; B, buffer C with glycoprotein 1%-IIIa, 0.75 pg/ml). Protein surface coverage of 1 pg/mm2 is equivalent to 1 RU.

Table 1. Comparison of the mass of glycoprotein IIb-IILa immobi- lized on a sensor surface with the maximal mass of fibrinogen bound to the surface. The percentage of binding sites that were occupied after full saturation was calculated from these data by considering the dif- ferent mass of the two proteins (230 kDa for glycoprotein IIb-IIIa and 340 kDa for fibrinogen).

Sensor no. Immobilized Bound fibrinogen Occupied glycoprotein after saturation binding sites IIt-IIIa WLJ

ng/mrnz YO

1 0.47 0.63 91 2 0.61 0.80 89 3 0.71 0.95 91 4 0.79 0.79 68 5 0.83 0.79 64 6 0.87 0.71 55 7 0.91 0.83 62 8 0.95. 0.79 56

contact of the sensor with the glycoprotein IIb-IIIa solution an increase in the sensor response, which indicates the adsorption of glycoprotein IIb-IIIa onto the surface, was observed. The shape of the response curve indicates that full saturation of the surface with glycoprotein IIb-IIIa is a slow process. The amount of immobilized protein can be changed by varying the concen- tration of glycoprotein IIb-IIIa in solution or by varying the time interval of contact between sensor surface and protein solution. Table 1 shows the fibrinogen-binding capacity of sensor surfaces containing immobilized glycoprotein IIb-IIIa in the range 0.47 - 0.95 ng/mmz. The maximal amount of fibrinogen that bound to the various glycoprotein IIb-IIIa surfaces was determined by using fibrinogen concentrations greater than 2.5 pM. By com- paring the mass of immobilized glycoprotein IIb-IIIa with the maximum amount ‘of bound fibrinogen one can directly calculate the percentage of the immobilized glycoprotein IIb-IIIa that bound fibrinogen (Table 1).

Activity of immobilized fibrinogen. The activity of immobi- lized fibrinogen was determined in an analogous way by record-

ing the mass of fibrinogen deposited during immobilization and by comparing this mass with the maximum mass of glycoprotein IIb-IIIa, which bound specifically to immobilized fibrinogen. Under the experimental conditions, 25 -30% of the immobilized fibrinogen was able to bind solubilized glycoprotein IIb-IIIa.

Determination of rate constants for the binding reaction be- tween immobilized fibrinogen and glycoprotein IIb-IIIa. Fig. 2A depicts a typical sensorgram that was recorded to deter- mine the kinetics of the interaction between immobilized fibrin- ogen and glycoprotein IIb-IIIa. The increase in sensor response observed during the contact with a continuous flow (1 pl/s) of buffer D containing solubilized glycoprotein IIb-IIIa at a con- centration of 29 nM indicates a net adsorption of glycoprotein IIb-IIIa to immobilized fibrinogen. During the subsequent con- tact with buffer D the decrease in sensor response indicates a net desorption of glycoprotein IIb-IIIa from the immobilized fi- brinogen. The binding sites for glycoprotein IIb-IIIa presented by the immobilized fibrinogen can be saturated by contacting the surface with a highly concentrated glycoprotein IIb-IIIa solution (> 3.2 pM). Since full saturation of such a sensor surface needed very long contact intervals between the surface and the solution, the sensor response at full saturation was, in general, not deter- mined experimentally but by a curve-fitting procedure, which allowed for the extrapolation of the experimental response to infinite time (Fig. 2B). Desorption of glycoprotein IIb-IIIa from a saturated surface was again observed in contact with buffer D. The desorption was accelerated and completed by treating the sensor surface with buffer F, containing the tetrapeptide RGDV (Fig. 2 A). Buffer F, containing 1 mM RGDV, fully displaced the glycoprotein IIb-IIIa from the surface. The treatment with RGDV produced a surface that exhibited identical response characteristics when recontacted with a new glycoprotein IIb- IIIa solution of the same concentration.

Fig. 2B depicts the two fits to the experimentally observed sensor-response curves during net adsorption. A mono-exponen- tial function could be fitted to the response curve observed at the lower concentration (29 nM). However, it has to be empha- sized that such pure mono-exponentiality of the sensor response was only obtained during the first 5-10 min of the sensor re- sponses. The mono-exponential rise indicates that reversible as- sociation and dissociation are the dominant processes that take place at the interface of the sensor during this time interval. The net adsorption that occurred during this phase of the experiment, approached an equilibrium surface concentration that gave rise to the equilibrium sensor response (AR,,). The latter was deter- mined by extrapolating the exponential fit to infinite time. The response curve observed with a solution containing glycoprotein IIb-IIIa at a concentration that was higher than the saturation concentration was not purely mono-exponential. This change in shape is rationalized by the depletion in fibrinogen occumng at the interface due to the reduced flow. The recorded response curve was used to determine the saturation sensor response (ARmaX). Fig. 2C shows the evaluation of the kinetic data accord- ing to Eqn (3) by using the time-dependent sensor response at the lower concentration and the AR,, value obtained by the extrapolation of the saturation response (Fig. 2A and 2B). The plot of the dARldt values against AR values produced a straight line. From the slope and the intercept of this line and the AR,, value, the k,,, and k,, values were calculated according to Eqn (3). Mean values for k,, and k,, determined from five inde- pendent measurements with two different sensors are given in Table 2.

It is obvious from the saturation curve shown in Fig. 2A and 2B that the determination of real AR,, values is sometimes difficult; it needs long time intervals of contact between the so-

Huber et al. ( E m J. Biochem. 227) 651

A

- 2 1.2

s 0.9

v

a, ln

Q 0

0.6 D 0 7

0.3

0.0

0 600 1200 1800 2400 3000

Time (s)

B

1.5 - 3 -r g 1.0

A

- 3 g 1.0 - X

S a 0 U a

0 200 400 600 800 1000

Time (s)

0.0 0.0 0.2 0.3 0.4 0.5

lo3 * Response (RU)

‘ig. 2. Determination of the rate constants for the binding reaction Netween immobilized fibrinogen and glycoprotein IIb-IIIa using :qn (3). (A) Adsorption of glycoprotein Ib-IIIa to immobilized fibrino- en. The letters A, B, C and D indicate time intervals during which the ensor was in contact with different solutions (A, buffer D; B, buffer D Jith glycoprotein IIb-IIIa, 29 nM; C, buffer D with glycoprotein IIb- [Ia, 3.2 pM; D, buffer F). (B) Fitting of the sensor response. A mono- xponential and a bi-exponential equation were fitted to the sensor re- ponse at the lower and at the higher concentration and extrapolated to ifinite time intervals. (C) Slopes at different times of the sensor re- ponse at the lower concentration versus the sensor response at the re- pective times.

Table 2. Rate constants (ken, k,, kor) and dissociation equilibrium constants (Kd) for the interaction between immobilized fibrinogen and glycoprotein IIb-IIIa.

Calculation k,, koe k,,. fast k,,. slow Kd based on component component

nM M-1, s-t s-l

Eqn (3) 3.3 2 1 5.1 t 1 2.6? 0.6 1.9 t 0.6 155 2 25

Eqn (4) 5.OX1O4 3.6X10-3 72 Scatchard 180

x 104 x10-3 xio-2 x10-3

i 0 ’ I

0 100 200 300 400 500 600

[Glycoprotein Ilb-llla] (nM)

Fig. 3. Evaluation of the rate constants (/con, kOm) of the binding reac- tion between solubilized glycoprotein IIb-IIIa and immobilized fi- brinogen using Eqn (4). The time constants, t, of the mono-exponential response curves observed when the sensor was brought into contact with different glycoprotein IIb-IIIa concentrations are plotted versus the re- spective concentrations.

lution and the sensor surface and solutions with very high pro- tein concentrations. However, one can show that small (5 %) de- viations in the AR,,, do not significantly influence the calculated rate constants. As inidcated in Materials and Methods, such a determination can be circumvented by correlating the time con- stants, z, of the exponential rise for a given sensor in contact with solutions containing different concentrations of glycopro- tein IIb-IIIa with the respective concentrations according to Eqn 4 (Fig. 3). The correlation between the two parameters is perfectly linear. The rate constants extracted from the slope and the intercept (Table 2) are well comparable with the rate con- stants determined by using Eqn 3.

It is also straightforward to determine rate constants of the dissociation (kow) of glycoprotein IIb-IIla from immobilized fi- brinogen by using the time-dependent sensor response observed during the time intervals of pure desorption. Since such a de- sorption should be a first-order reaction, one would expect that the sensor response decays mono-exponentially with a time con- stant corresponding to the rate constant of the desorption reac- tion. It was found, however, that the time-dependent sensor sig- nal was composed of two exponential components, each of them with its own time constant. An interpretation of this finding is given in Discussion. Fig. 4 shows two such sensor responses and the respective mathematical fit of a bi-exponential equation to the experimental curves. Values of the respective time constants are given as k,, values in Table 2. The variation within these values is very small, although the different dissociation experi-

Huber et al. (EUK J. Biochern. 227)

1.4

1.2 1 = 1.0 -5. a,

0 Q v)

2 0.8

2 0.6

mz 0.4

0.2

0.0 I I I I I I I 0 100 200 300 400 500 600

Time (s)

A 6

5 h

3

a, fn c

c r 4

P

?

0 3

: 2 N

1

0

1 -10 L l I 1 I I

0 200 400 600 800 1000

Time (s)

Fig. 5. Binding of fibrinogen to glycoprotein IIb-IIIa immobilized on the sensor surface. The filled symbols indicate the data points measured during net adsorption and net desorption on a freshly prepared surface. Mono-exponential equations are fitted to the data points. The open sym- bols indicate the sensor response when the surface was brought into contact for the second time with a fibrinogen solution. A bi-exponential equation is fitted to this response curve. (A) and (B) indicate time in- tervals during which the sensor surface was in contact with buffer E containing fibrinogen or with buffer E, respectively.

ments start from different surface coverages. The relative weight of the two components within a given decay, however, depends on the time interval of contact between immobilized fibrinogen and glycoprotein IIb-IIIa and on the occupancy of the binding sites. The component representing the slower dissociation is more dominant after longer time intervals at higher occupancy.

Determination of rate constants for the binding reaction be- tween immobilized glycoprotein IIb-IIIa and fibrinogen. Ki- netic measurements were also carried out using the inverse ex- perimental system, i.e. with the glycoprotein IIb-IIIa immobi- lized on the surface. Two glycoprotein IIb-1IIa preparations were used, one which did not contain RGDV (in buffer B) and one, which contained RGDV (in buffer A). The surface of the sensor showing the sensorgram depicted in Fig. 5 was coated with RGDV-free glycoprotein IIb-IIIa. If the first contact of this sur-

I

A I

Fig. 4. Desorption of glycoprotein IIb-IIIa from fibrinogen immobi- lized on the sensor surface. (A) High surface concentration of glyco- protein Ilb-IIIa; (B) low surface concentration of glycoprotein 1%-llla. Bi-exponential equations are fitted to the data points.

0 50 100 150 200 250

Time (s)

B 20

15

fn v

P 10

N 0 7

5

0 100 200 300 400 500

[Fibrinogen] (nM)

Fig. 6. Adsorption of fibrinogen to immobilized glycoprotein IIb- IIIa. (A) Response curves recorded with solutions of different fibrinogen concentrations (A, 236 nM; B, 131 nM; C , 43.6 nM; D, 14.5 nM) after treating the surface extensively with fibrinogen and/or RGDV solutions. Bi-exponential equations were fitted to the data points. (B) The pairs of time constants, t, evaluated from the sensor responses by the fitting procedure are plotted versus the respective fibrinogen concentrations.

face with a fibrinogen solution was kept very short ( t4400 s), the fibrinogen could be removed quantitatively from the surface by washing with buffer E. The increase in sensor response due to net adsorption and the decrease in sensor response due to net desorption can be fitted satisfactorily with a mono-exponential equation (Fig. 5). However, subsequent contacts of such a sur- face with fibrinogen solutions of the same concentration resulted in a sensor-response curve that deviated substantially from the one observed during the first contact (Fig. 5). Only a bi-expo- nential equation could be fitted to this second sensor-response curve. The two components of the bi-exponential rise of the sen- sor response have time constants that differ by a factor of ap- proximately 10. Moreover, the fibrinogen molecules that bound during the second and all the subsequent contacts with the im- mobililized glycoprotein IIb-IIIa to such a sensor surface could only be removed with difficulty by washing the surface with buffer E. Faster regeneration of the surface was possible by using a buffer containing RGDV at high concentrations (buffer G). Due to the continual change of the sensor surface, which resulted in a continual change of the shape of the sensorgram, it was impossible to extract meaningful1 data from the initial

Huber et al. (Eul: J. Biochem. 227) 653

Table 3. Rate constants (,ton, ,tom ,ton) and thermodynamic data (Kd) evaluated for the binding of fibrinogen to immobilized glycoprotein IIb-IIIa. Data for k,, and k,, were evaluated by a deconvolution of the bi-exponential sensor response during adsorption and by plotting the time constants, T, of the single exponential functions against the concen- tration of fibrinogen (Eqn 4). k,, values were evaluated from the bi- exponential sensor responses during net desorption. Equilibrium dissoci- ation constants, Kdr were calculated from the ratio kor/k0. or were deter- mined by measuring equilibrium sensor responses and subjecting them to a Scatchard analysis.

Component k,. ko, k 0 r K d

nM M-1. s-l s-l

Fast 3.1X105 5.1X10-* 4.0?1X10-2 165 Slow 2.OX lo4 2.4X 4.2? 2X 21 Scatchard 65

measurements. However, the bi-exponential sensor responses became reproducible after a series of adsorptiodregeneration steps, if the time intervals of the fibrinogen adsorption and of the surface regeneration (contact with RGDV-containing buffer G followed by RGDV-free buffer E) were kept constant. Typical responses, which were then monitored with the sensor in contact with different fibrinogen concentrations, are depicted in Fig. 6A. From the fit of a bi-exponential equation to the data points a pair of time constants (7) was extracted for each concentration and plotted versus the respective concentrations (Fig. 6 B). Two linear regressions were obtained (Table 3).

Hence, dissociation rate constants were also determined from the sensor responses during time intervals of net desorp- tion, i.e. during contact with fibrinogen-free buffer solutions. These sensor responses were again bi-exponential decay curves. The time constants of the two mono-exponential decays, which contribute to the bi-exponential decay are given as k,, values in Table 3.

The conditioning of the sensor surface in order to reach re- producibility did not necessarily need a contact with fibrinogen as described above. It was also possible to obtain equally stable conditions when the sensor surface was brought into contact with an RGDV-containing buffer over an extended time interval or when the glycoprotein IIb-IIIa was immobilized in the pres- ence of RGDV.

Determination of equilibrium dissociation constants using equilibrium sensor responses. Equilibrium binding constants are commonly determined by the Scatchard-type analysis. The Kd values are thereby determined by measuring the amount of ligand that binds to an activated solid phase and the amount of ligand that remains free using different concentrations of ligand. These parameters can in principle also be extracted from the sensor response detected with different ligand concentrations. Due to the fast continuous flow over the sensor surface the con- centration of ligand in solution remains always constant. Fig. 7 depicts Scatchard plots for both experimental systems, i.e. with either fibrinogen or glycoprotein IIb-IIIa immobilized on the sensor surface. The different ways to extract the equilibrium sen- sor responses are discussed in Materials and Methods. The two graphs exhibit a linear relationship between the amount of bound ligand (equilibrium sensor response) and the ratio of bound and free ligand. The equilibrium dissociation constants were calcu- lated from the slope of these straight lines and are indicated in Tables 2 and 3.

A 9.0

h

E 7.5 - C a, cn

6.0

P LL . 4.5

e a 3.0

5 1.5

._ L

- 3

v) c 0

2 0.0

0 1 2 3 4 5 6 7

10' * Response (RU)

B

3.0 - - m - = 2.5

2 2.0 2

K 1.5 (3 3 1.0 E

4 - K .-

a 0 - . W

0 Q (I)

g 0.5

2 0.0 LL

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

10' * Response (RU)

Fig. 7. Scatchard plots for the determination of equilibrium dissoci- ation constants Kd using equilibrium sensor responses. (A) Glycopro- tein IIb-IIIa immobilized on the sensor surface. (B) Fibrinogen immobi- lized on the sensor surface.

DISCUSSION

In this study we have used the plasmon-resonance technol- ogy to measure the kinetic parameters of fibrinogen binding to purified platelet glycoprotein IIb-IIIa. To investigate this recep- tor-ligand interaction, one set of experiments was performed using immobilized fibrinogen and another using immobilized glycoprotein IIb-IIIa. The main advantage of this sensor technol- ogy over other techniques is that all the reactions occurring at the solid/liquid interface can be recorded in real time without the need for labeling of the participating molecules.

Monitoring the immobilization process was of particular im- portance to establish the optimal immobilization procedure for glycoprotein IIb-IIIa. The adsorption of glycoprotein IIb-IIIa on a lipid-like adlayer formed by fluoroalkane chains was found to be the only way to generate stable surfaces of high fibrinogen binding capacity. We hypothesize that the tight adherence of gly- coprotein IIb-IIIa to the lipid-like adlayer is due to the interac- tion of the hydrophobic transmembrane domains of the receptor with the fluoroalkane chains of the self-assembled monolayer. Due to this selective interaction via specific molecular domains, which are well separated from the fibrinogen-binding site of the glycoprotein (Phillips et al., 1988), the glycoprotein IIb-IIIa

654 Huber et al. ( E m J. Biochem. 227)

molecules may be immobilized in a uni-directionally oriented way. The finding that more than 90% of the fibrinogen-binding activity (Table 1) could be retained using this immobilization technique strongly supports this hypothesis.

The density of immobilized glycoprotein IIb-IIIa is also a critical parameter that had to be controlled to reach high relative binding activity. A relative activity of more than 90% was ob- tained, when the density of immobilized glycoprotein IIb-IIIa was lower than 0.'7 ng/mm2. At higher densities the apparent rel- ative activity decreased significantly. A rationale for this decrease can be given by simply considering the shapes and dimensions of glycoprotein IIb-IIIa (Wippler et al., 1994) and fibrinogen (Davie et al., 1991). Assuming homogeneous distri- bution of the glycoprotein, a model calculation shows that the apparent decrease of fibrinogen-binding activity at high receptor densities is most likely due to steric hindrance. In contrast to glycoprotein IIb-I[Ia, the immobilization of fibrinogen did not occur via a specific domain but via free amino groups, which are expected to be randomly distributed over the surface of the protein. Therefore, it is not surprising that this immobilization procedure resulted in a substantial loss of binding activity. Only 25 -30 % of the immobilized fibrinogen molecules were capable of binding glycoprotein IIb-IIIa.

The type of system considerably influenced the kinetics of complex formation. On glycoprotein IIb-IIIa, probably immobi- lized in a directionally controlled manner via the hydrophobic transmembrane domains, the fibrinogen binding occurs to easily accessible binding sites, whereas the binding of glycoprotein IIb-IIIa to randomly immobilized fibrinogen might be partially hindered by the surface or by adjacent molecules. Thus, it is not surprising that the rate of association (k,,") for the formation of the initial complex is much faster on immobilized glycoprotein IIb-IIIa than on immobilized fibrinogen. As a consequence, the measured k,,, values for the two systems differ by an order of magnitude (Tables 2 and 3). The formation of the complex is not a fully reversible process. This was most apparent in experi- ments in which the dissociation of a prefomied complex was investigated. In both experimental systems a full dissociation of the complex was observed only when the two proteins had been in contact for a short time interval. After prolonged contacts an efficient dissociation had to be forced by using high concentra- tions of an inhibitor (e.g. RGDV) that was able to displace fi- brinogen from glycoprotein IIb-IIIa. The finding that the dissoci- ation process was slowed down by a prolonged contact between glycoprotein IIb-IIIa and fibrinogen indicated that two coupled reactions were involved : the formation of an initial complex and its subsequent stabilization. This postulation is consistent with the finding that the sensor response during net dissociation has a bi-exponential shape. The observed bi-exponentiality can be explained by the presence of two types of complexes, each with its own dissociation kinetics (Tables 2 and 3). The rate constants kow differ from each other by a factor of 20, when fibrinogen is immobilized and by a factor of 100, when glycoprotein IIb-IIIa is immobilized on the surface. The k,, values determined for the weak complex with the fast dissociation kinetics were only slightly different for the two systems (2.6X10-2s-1 and 4.0X 10-2s-'). However, the k,, values of the stabilized com- plex, the complex .with the slow dissociation kinetics, were sig- nificantly different (1.9X10-3 s-' and 4.2X10-4s-') indicating that the receptor-ligand complex is stabilized more efficiently when glycoprotein IIb-IIIa is immobilized on the sensor surface. All the bound fibrinogen, or in the case of immobilized fibrino- gen all the bound glycoprotein IIb-IIIa can be removed by add- ing a specific glycoprotein IIb-IIIa antagonist (RGDV) to the buffer solution. Thus, only specific molecular interactions are

detected and the observed stabilization process is not due to non- specific interactions with the surface.

Another significant and informative difference between the two types of arrangements became evident following the perfor- mance of a series of repetitive experiments on these surfaces. If fibrinogen was immobilized on the sensor surface, the same glycoprotein-IIb-IIIa-binding properties were observed in the first and all the subsequent experiments, i.e. following regenera- tion of the surface with RGDV, identical response curves were obtained. In contrast, we found that the sensor surface contain- ing immobilized glycoprotein IIb-IIIa was changed after the first contact with fibrinogen. Although all the bound fibrinogen could be removed (either by a spontaneous desorption using fibrino- gen-free solutions or by an inhibitor-forced displacement), the regenerated glycoprotein IIb-IIIa surface exhibited different binding properties during subsequent experiments. The bi-expo- nential shape of the sensor-response curve recorded during fur- ther adsorption experiments indicated the evolution of two gly- coprotein IIb-IIIa populations on the surface, each with its own binding kinetics. It can be excluded that the two populations were formed during the immobilization, since the response curve monitored during the first fibrinogen-binding experiment always showed pure mono-exponentiality. Since after the first contact with fibrinogen both glycoprotein IIb-IIIa populations were pre- sent on the sensor surface, it can be assumed that after dissoci- ation of the complex the reversion of the high-affinity to the low-affinity conformer is a relatively slow process.

One of the two glycoprotein IIb-IIIa populations showed very fast association and dissociation rate constants in the order of 3.1X105M-' s-l (k0J and 5.1X10-2s-' (koE) or 4.0X10-2s-' (kOc) and a Kd of 165 nM. We assume that this population is identical to the glycoprotein IIb-IIIa originally immobilized on the sensor surface. The second population of immobilized glyco- protein IIb-IIIa is formed following contact with fibrinogen or RGDV. It has been demonstrated earlier that the binding of RGDS or fibrinogen to glycoprotein IIb-IIIa induces conforma- tional changes within the receptor (Du et al., 1991 ; Kouns et al., 1992) that result in the exposure of neo-epitopes called LIBS. It is, therefore, likely that these ligand induced conformational changes cause a slowing down of the association as well as of the dissociation process. The respective rate constants were more than one order of magnitude lower (ken = 2.0X104M-' s-l,

k,, = 2.4X10 - 3 s-', k,, = 4.2X10-4s-1) than those of the initial complex. Thus, the equilibrium dissociation constant of the high-affinity complex was approximately 21 nM. From these re- sults one can conclude that purified active glycoprotein IIb-IIIa first forms a low-affinity complex with fast reaction kinetics. In a subsequent step, this initial complex is transformed into a more stable complex. The association of fibrinogen to the conforma- tionally changed glycoprotein IIb-IIIa molecules is a slow pro- cess but leads directly to a stable complex, where fibrinogen is bound with a nearly eightfold higher affinity.

Due to the bivalency of fibrinogen, a critical discussion of the results should consider the possible formation of cross-linked ligand-receptor complexes. The parallel occurrence of bivalent and monovalent binding would lead to similar results (Hogg et al., 1987). Arguments against the possibility of cross-linking can be gained from a comparison of the results obtained with the two systems. Cross-linked ligand-receptor complexes can only be formed with the bivalent ligand (fibrinogen) in solution. However, bi-exponential response curves were observed during net desorption for both systems and their analysis led to similar k,, values. Bi-exponential response curves were not observed on freshly prepared glycoprotein surfaces. Only contact with li- gand-induced irreversible changes in the binding behaviour. The bivalency of the fibrinogen cannot be responsible for these

Huber et al. (Eur. J. Biochem. 227) 655

changes, since they were also induced by the monovalent ligand RGDV.

The existence of a low-affinity and high-affinity complex between glycoprotein IIb-IIIa and fibrinogen is not only indi- cated by the kinetic measurements. The dissociation equilibrium constants extracted via Scatchard plots from equilibrium sensor responses can be interpreted in the same way. In an experimental procedure, which allowed for a long contact between fibrinogen and immobilized glycoprotein IIb-IIIa, a Kd value of 65 nM was determined. However, we have also determined a Kd value of 180 nM for the complex, which is formed during the very first contact between glycoprotein IIb-IIIa and immobilized fibrino- gen by extrapolating the sensor response observed during this initial phase to infinite time intervals. It has to be emphasized that all the Kd values determined in this study are within the range of Kd values determined using different methods. Reported Kd values using whole platelets range from 30 nM (DiMinno et al., 1983) to 5.6 pM (Komecki et al., 1981), those for purified or partially purified glycoprotein IIb-IIIa from 4.8 nM (Baldas- sare et al., 1985) to 60.2 nM (Bajt et al., 1992). The kinetics of the binding reaction between glycoprotein IIb-IIIa and fibrino- gen was recently studied using glycoprotein IIb-IIIa reconsti- tuted in immobilized planar lipid bilayers and fluorochrome-la- beled fibrinogen (Muller et al., 1993). The analysis of the kinet- ics revealed the reversible formation of a precomplex, which is further stabilized in an irreversible process. The calculated Kd value (50 nM) and the respective rate constants (ken = 4.4X104M-’ 6, kor= 2.2X10-3 s-’) for the precomplex for- mation are comparable to the values for an already stabilized complex described in the present study. Since the time resolution of our experiments allowed us to focus on the very first phase of the binding reaction between glycoprotein IIb-IIIa and fibrin- ogen, an even less stable complex could be identified, which forms immediately following the initial contact of the two pro- teins.

In conclusion, our results demonstrate that optical sensors are a suitable technique for the investigation of biomolecular interactions. The fast kinetic measurements (fast with respect to conventional assay technologies) give insights into the dynamics of binding events. In this study, we demonstrated that the bind- ing of fibrinogen to glycoprotein IIb-IIIa is not a single mono- phasic reaction but is composed of at least two consecutive steps. It will be interesting to investigate, which domains of the two proteins are involved in the stabilization process.

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