Binding of fibrinogen to platelet integrin αIIbβ3 in solution as monitored by tracer sedimentation equilibrium

JOURNAL OF MOLECULAR RECOGNITION, VOL. 9.31-38 (1996)

Binding of Fibrinogen to Platelet Integrin aIIbp3 in Solution as Monitored by Tracer Sedimentation Equilibrium German Rivas*, Kirsten Tangemann, Allen P. Minton+, and Jurgen Engel Biozentrum, University of Basel, Basel, Switzerland and 'the Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, MD, USA

Fibrinogen showed essentially no binding (KD> 1 mM) to platelet aIIb@ integrin in solution in the presence of Triton or octylglucoside above critical micellar concentrations. Under these conditions the integrin was an a$ monomer. After removal of the detergent from the Triton containing buffer (25 mM W i C I ; , 150 mM NaCI, 1 mM CaCI, 1 m~ MgClz, pH 7.4) the integrin formed aggregates with hemmers as the most prominent species, as demonstrated by analytical ultracentrifugation and electron microscopy. Tracer sedimentation equilibrium experiments indicate that fibrinogen binds to the integrin aggregates, but with a surprisingly large KD (at least 3 p ~ ) . This value is 10- to 100-fold higher than values determined by solid phase assays or with integrins reconstituted onto lipid bilayers.

Keywords: fibrinogen; integrin; binding; tracer sedimentation equilibrium

Introduction

Integrins are an important class of transmembrane receptors which are essential for the specific recognition of extracellular matrix (ECM) proteins (Hynes, 1992). They consist of a heterodimer of LY and p subunits, both with membrane spanning regions. A large extracellular domain contains the recognition surfaces and binding sites for Ca2+ and other cations. A small cytosolic domain interacts with cytoskele- tal elements (Carrel1 et al., 1985; Nermut et al., 1988; Hynes, 1992). The platelet integrin aIIbp3 (228 kDa) is the best biochemically characterized member of this family (Calvete et al., 1992a,b; Rivas and Gonzalez-Rodriguez, 1991; Rivas et al., 1991a). It serves as the receptor for fibrinogen in activated platelets, thus playing an important role in platelet aggregation and haemostasis (Kieffer and Phillips, 1990; Marguerie et al., 1980; Phillips et al., 1991; Plow and Ginsberg, 1989). Besides fibrinogen, aIIbp3 also recognizes other ECM proteins, such as fibronectin, von Willebrand factor, vitronectin, and thrombospondin (Hynes, 1992; Phillips et al., 1991).

Binding of fibrinogen to its receptor has been monitored by different methods and very different concentrations for 50% inhibition (IC5,,) and dissociation equilibrium constants K, have been reported (Table 1). In part, differences may be due to methodological factors. For example, intrinsic problems of the widely used solid phase assays are the undefined state of the coated substrate, possibility of

*Author to whom correspondence should be addressed at: Centro de Investigaciones biol6gicas, CSIC, Velbquez 144, 28006-Madrid, Spain. Fax: 34-1-562-75 18. Email: [email protected]. Abbreviations used: ECM, extracellular matrix; TRITC, tetramethylrho- damine-5-isothiocyanate; EITC, eosin-5-isothiocyanate; TIRFM, total internal reflection microscopy.

changes in the binding properties of the ligand due to structural changes associated with the adsorption, lack of proportionality between signal and the amount of bound material (Tangemann and Engel, 1995), possible irreversible binding phenomena and removal of bound material

Table 1. Binding of fibrinogen to aIIbp3 at different organiza- tional levels

Organization t o r IC, level (*M)

Platelets 2-5a.i 0.5b*c

Platelet 0.012d membrane Liposomes 0.015' Lipid bilayers 0.030'.' Solid phase 0.17@,"

0.03' 0.024h 0.01 2' 0.007i*m

(no detergent) 5 look (detergent)

a Marguerie eta/., (1980). Kirschbaum eta/., (1992). Legrand etal., (1985).

Solution > 3k

Method Centrifugation'* Filtrationb Centrifugation

['2511fibrinogen TIRFM ELSA with fibrinogen or allbp3 coated on plastic

Sedimentation equilibrium

Phillips and Baughan (1983). Parise and Phillips (1985b).

Nachman etal., (1982, 1984).

' Mueller ef a/., (1993). 9 Aumailley eta/., (1991).

' Charo eta/., (1991). i Pfaff et a/., (1994). ' Present work. ' Two step mechanism with the second one irreversible. " IC, values.

CCC 0952-3499/96/01003 1-08 1996 by John Wiley & Sons, Ltd.

Received 29 June 1995 Accepted (revised) 20 October 1995

32 G . RIVAS ET AL.

during the washing steps (Ingham et al., 1985). Other reasons for the observed differences are different degrees of activation of the receptor (Hynes, 1992). differences in multivalency of fibrinogen and integrins due to aggregation or binding to other components (Smyth et al., 1992), the effect of detergents in solubilized receptor preparations, and the effects of divalent ions (Smith et al., 1994).

The aim of the present work was to study equilibrium binding of aIIbp3 with fibrinogen in solution in the presence and absence of detergents and divalent ions, under conditions such that the states of ligand and receptor are well defined. The influence of integrin aggregation was also studied. We used the technique of sedimentation equilibrium, a well established method for the quantitative characterization of macromolecular interactions in solution (Harding et al., 1993; Minton, 1990; Rivas and Minton, 1993; Schuster and Laue, 1994).

Experimental

Triton X-100 and its reduced form were obtained from Fluka. Octylglucopyranoside (octylglucoside) was pur- chased from Calbiochem. Biobeads SM-2 were from Bio Rad. NaIz5I was from Amersham Corp., and iodobeads from Pierce Chemical Corp. The chromophores tetramethylrho- damine-5-isothiocyanate (TRITC) and eosin-5-isothiocyanate (EITC) were obtained from Molecular Probes Inc. All other chemicals were of analytical grade.

Proteins. Human fibrinogen, grade L, was obtained either from Kabi or from IMCO. In both cases clottability was higher than 90%. Further purification was done according to Laki (195 1). Human platelet integrin aIIbp3 was purified from platelets as previously described (Rivas et al., 1991b). All the proteins were equilibrated in 25 mM Tris/HCI, 150 m~ NaCI, 1 mM CaCI,, 1 mM MgCl,, pH7.4 buffer (TBS-Ca2+/Mg2+ buffer), in some cases with a defined detergent concentration.

Labelling procedures. Chromophoric labelling of fibrinogen and aIIbp3 was done as previously described (Usobiaga et al., 1987; Rivas, 1989; Muller et al., 1993). The degree of labelling was 3-4 mol of chromophore/mol of protein in all cases. Iodination of fibrinogen and aIIbp3 was performed according to Parise and Phillips (1985b). The iodinated fibrinogen was more than 90% clottable. Specific activity was in the range 1-5 x lo9 cpm/mg protein. Labelled and unlabelled proteins were identical as judged by chromatography and sedimentation velocity and equilibrium experiments.

Electron microscopy. Samples were diluted with TBS- Caz+’Mg2+ buffer with 1 mM Mn2+ and 0.035% Triton to a final concentration of 30 pg/mL. To visualize oligomers of integrin, Triton was removed with Bio beads.

Binding to fibrinogen was achieved by incubating 0.4 p~ of integrin oligomers with 1 p~ of fibrinogen for 2 h at room temperature. For rotary shadowing, aliquots were

diluted with TBS-Ca2+/Mgz+ with Mn”, mixed 1:l (v/v) with glycerol and sprayed onto freshly cleaved mica. Rotary shadowing with platinudcarbon was at an angle of 9”.

Sedimentation equilibrium of aIIbPJ preparations. Short column (ca 0.8-1 mm) equilibrium experiments were performed at various rotor speeds (ranging between 3000 rpm and 10000 rpm) and 10°C using an analytical ultracentrifuge Optima XL-A (Beckman Instruments Inc.) with six-channel 12 mm charcoal-filled epon centrepieces (Laue, 1992). Radial scanning at 280-290 nm of aIIbPj samples equilibrated in TBS-Ca2+/Mg2+ buffer were done after 12 h at intervals of 3 h until negligible difference between two successive scans indicate attainment of equilibrium. To obtain the weight-average molecular mass (M,) of aIIbp3 integrin equilibrium data were analysed by using the programs XLAEQ and EQASSOC (supplied by Beckman; see Minton, 1994). The partial specific volume of aIIbp3 was 0.7 16 cm3/g, calculated from the amino acid and sugar composition of the integrin (Eirin et al., 1986), according to Durchschlag (1986) and Laue et al., (1993).

With detergent-solubilized cuIIbp3, the sedimentation equilibrium experiments were performed as described above but with 0.1% reduced Triton or 50 m~ octylglucoside in the equilibrium buffer. In this case the experimental weight-average molecular mass and experimental partial specific volume are related to the values for the protein by (Tanford et al., 1974):

Mw=Mp (1 +dd) (1)

(2)

where ijd is the partial specific volume of detergent [0.913 cm3/g for Triton (Laue et al., 1993) and 0.835 cm3/g for octylglucoside (Reynolds and McCaslin, 1985)] and d,, is the detergent bound to aIIbp, in g of detergentlg aIIbp3 [0.3 g/g for Triton (Rivas et al., 1991a) and 0.11 g/g for octylglucoside, the latter calculated assuming that one micelle of detergent is bound per molecule of integrin (Rivas et al., 1991a)l.

v = ( vp + ddv*)/( 1 + dd)

Tracer sedimentation equilibrium. Experiments to characterize heteroassociation between fibrinogen and aIIbp3 were performed according to Rivas et al., (1994), following the changes in M , of a fixed amount of labelled fibrinogen, acting as a chromophoric or radioactive tracer, upon incubation with increasing amounts of aIIbp3. In the case of the chromophorically labelled material, short column (ca 0.8-1 mm) experiments were performed at 3000-10 OOO rpm and 10°C in a XL-A Optima (Beckman Instruments Inc.) analytical ultracentrifuge (Laue, 1992) as described above, except that absorbance gradients were measured at wavelengths of 495-550 nm, where integrin does not absorb. When iodinated fibrinogen was used in the mixture, 70 pL samples were centrifuged at sedimentation equilibrium using preparative ultracentrifuges (Darawshe et al., 1993; Rivas et al., 1992, 1994) at various speeds between 3000 rpm and 6000 rpm and 10°C. Equilibrium gradients of labelled fibrinogen and its complexes with cvIIbB in the mixtures were analysed to yield the apparent weight-

BINDING OF FIBRINOGEN TO PLATELET INTEGRIN aIIbp3 33

average molecular mass of fibrinogen (M,,)* in the mixture, as a function of composition, according to (Hsu and Minton, 1991; Rivas et al., 1994):

where Fto, denotes the fibrinogen concentration (in absorbance or cpm units), rf is a reference radial distance (cm), cV> is the weight-average partial specific volume of the mixture, p is the solvent density, o is the angular velocity, R is the molar gas constant, and T the absolute temperature. The weight-average partial specific volume of the mixture was 0.720 cm3/g, calculated as described in the appendix of Rivas et al., (1994), from the partial specific volumes of fibrinogen (0.722 cm3/g; Schultze and Heremans, 1966) and integrin (0.7 16 cm3/g).

Flot(r)=Ftot(rf)exp fMw,F(l - <V>p)&? - 6)/2RT] (3)

Characterization of aIIbp3 preparations

1500 1 T I

Mw.ap 1200 C I I

600 1

I I I I 0.4 4.4 44

Coallbp3 (pM)

Figure 1. Weight-average molecular mass of allbp3 integrin plotted as a function of weight integrin concentration C,,,,. (0) allbp3 in the absence of detergent in TBS-Ca2+/Mg2+; (0) allbp3 equilibrated in the same buffer with 0.1% reduced Triton (see text).

*The weight-average molecular mass of fibrinogen in the mixture is defined (Chatelier and Minton, 1987; Hsu and Minton, 1991; appendix of Rivas el 01.. 1994) by:

W#F + ~WC&. c, Mc, WF+~Wc&.ct)

M = -~__-__ w. F

where W, denotes the weight concentration of the different species x where the fibrinogen is present, MF and Mc, are the molecular masses of fibrinogen alone and the fibrinogen-integrin complexes C,, respectively (where i denotes the number of fibrinogen molecules bound per integrin hexamer in the complex), and AEC,, is the fractional contribution of fibrinogen to the weight of the complexes:

A~ca=iM&Mc,.

Triton. The best fit value of Mw=310+20 kDa for the detergent-solubilized aIIbp3, corresponding to Mp=238 kDa for the glycoprotein, which is in good agreement with the expected molecular mass of monomeric aIIbp3 and did not change in a concentration interval of 0.9-8.8 FM (molar units of ap integrin protomer). The finding of monomeric receptor agrees with the electron microscopic pictures of the same population [Fig. 2(A)], and also with previous physical studies (Parise and Phillips, 1985a; Rivas et al., 1991a; Weisel et al., 1992). In the absence of detergent aIIbp3 tends to form aggregates with a molecular mass of 1400+ 100 kDa, which corresponds approximately to a complex of six aIIbp3 molecules (Fig. l), in agreement with the results of electron microscopic analysis [Fig. 2(B)] and previous studies (Parise and Phillips, 1985a; Rivas et al., 1991a; Weisel et al., 1992). This value was found to be constant to protein concentrations of 4.4 FM, but at concentrations of 8 . 8 ~ ~ or higher (not shown), aIIbp3 tended to form larger aggregates in the absence of detergent.

Lack of fibrinogen binding to cuIIbp3 in the presence of non-ionic detergents

Fibrinogen was mixed with triton solubilized aIIbp3 and binding was monitored by tracer sedimentation equilibrium. Concentration gradients of fibrinogen at sedimentation equilibrium in TBS-Ca2+/Mg2', 0.1% Triton buffer, alone and in the presence of aIIbp3 are shown in Fig. 3. The data clearly indicate a lack of complex formation between fibrinogen and detergent-solubilized aIIbp3 at 4.4 p~ total concentration of the integrin protomer. The same lack of association was found in buffers containing 50 m~ octylglucoside instead of Triton (data not shown). The data were substantiated by experiments in the analytical ultracentrifuge in which fibrinogen was labelled with the chromophores EITC or TRITC (Fig. 4). In the presence of detergent no complex formation was observed at integrin concentrations of up to 11 p~

Binding of fibrinogen to aIIbp3 in detergent-free solution

Binding was demonstrated by two independent sedimentation equilibrium methods. In the first one, a fixed amount (0.6 FM) of chromophorically labelled fibrinogen was centrifuged at sedimentation equilibrium with different amounts of detergent-free aIIbp3. In Fig. 4, each data point represents the average molecular mass of labelled fibrinogen obtained by fitting an ideal single-species equation to the entire gradient of a single tube, as described earlier. An increase in molecular mass of the tracer fibrinogen with increasing amounts of aIIbp3 integrin would reflect an association between both components. In the absence of integrin or at insufficient concentrations of integrin (Fig. 4) the molecular mass of fibrinogen was almost equal to that expected for monomeric fibrinogen (3302 15 ma) . The value increased to 700 kDa in the presence of a seven-fold molar excess of aIIbp3 indicating complex formation (Fig.

34

7 0 0 :

600

M,,

W a ) 5oo.

400

300

G. RIVAS ET A L

7 - 1 - f I

k j ki I i

f+-.- <===- #' ---ilp-g&-o , I - F b g

~ c Fbg + allbe3

Figure 2. Electron micrographs after rotary shadowing of crllbp3 integrin. (A) lntegrin in TBS-Caa+IMgz+ buffer containing 0.035% Triton. (B) Same buffer but detergent removed with Bio beads. (C) lntegrin (0.4 JLM) mixed with fibrinogen (1 JLM in TBS-Ca2+/Mgz+ buffer with 1 mM Mn2+ in the absence of detergent.

4). At this point the total fibrinogen concentration was 0.6 p~ and the total molar aIIbp3 protomer concentration was 4.4 p ~ . The data indicate that only a small fraction of the maximum amount of fibrinogen was bound at these concentrations and that binding continued to increase steeply at the highest integrin concentrations (Fig. 4). The formation of larger aggregates of integrin at higher protein concentrations (Fig. 1) precluded its use in the ultra- centrifugal experiment. The fibrinogen-aIIbp3 association was abolished when the experiments were done in the

16000

14000

12000

10000

8000

6000

4000

2000

0

=Pm

I I I 1 I I

0

b

8

I I I I I I

8.25 8.30 8.35 8.40 8.45 8.50

r (cm)

Figure 3. Lack of fibrinogen binding to Triton solubilized allbfl. Gradients of radioactivity in tracer equilibrium centrifugation (4500 rpm, 10°C) for fibrinogen alone (0.6 p ~ ) in TBS-Ca2+/Mgz+ buffer alone (O), and mixed with (ullb3 (0.2.2 JLM; A, 4.4 JLM).

BINDING OF FIBRINOGEN TO PLATELET INTEGRIN aIIb@ 35

fibrinogen. Near the meniscus the fibrinogen concentration was decreased but its molecular mass was that of a monomer [lower solid curve in Fig. 5(A)]. This shallow gradient corresponds to free monomeric fibrinogen in the region where no integrin is present, as indicated in Fig. 5(B). The steeper gradient near the bottom of the cell represents the sum of free, given by the theoretical curve, and bound fibrinogen. This conforms with an independent experiment monitoring the cuIIbp3 concentration under otherwise identical conditions [Fig. 5(B)]. The integrin was essentially absent near the meniscus and accumulated in the bottom region due to its high molecular mass. The profile in Fig. 5(A), corresponding to monomeric fibrinogen in the absence of integrin, was restored when the binding experiment was performed in the presence of 1 m~ EGTA. Also, a 100-fold excess of unlabelled fibrinogen was able to displace [i251] fibrinogen from the cuIIbpJ binding sites.

160000

140000

120000

100000

80000

60000

40000

20000

0 6 4 0 645 650 655 6 60 6 6 5 6 7 0

= (cm) I I I I I

B

6.40 6.45 6.50 6.55 6.60 6.65 6.70

(cm) Figure 5. Binding of iodinated fibrinogen to allbp3 in detergent- free solutions. (A) Equilibrium gradients of radioactivity (5000 rpm, 10°C) for fibrinogen alone (0.6 p , ~ 01, fibrinogen mixed with allbp3 (8.8 p . ~ ) in either 1 mM Ca2+lMg'+ (0) or 1 mM EGTA (A), allbp3 and a 200-fold molar excess of cold fibrinogen in 1 rnM Ca2+/Mg2+ (W. The solid curves correspond to the theoretical equilibrium gradient of the fibrinogen monomer. (B) lntegrin equilibrium gradient under the same experimental conditions.

Therefore we concluded that the binding was calcium dependent, reversible and specific.

Discussion Equilibrium binding parameters are most important for the assignment of biological functions of integrin receptors, including their specificity and regulation. Binding of the well characterized cuIIbp3 receptor to its most important physiological ligand fibrinogen has therefore been studied by many assays (Table 1). In these studies the receptor was incorporated in platelet membranes, synthetic lipid bilayers, liposomes or absorbed to plastic surfaces. The present work is the first study in which both reaction partners were in solution. The data therefore provide information on the binding of the detergent solubilized monomeric integrin and on its aggregated soluble form after removal of the detergent.

The molecular mass of cuIIbp3 integrin in the presence of 0.1% Triton or 50 m~ octylglucoside was 238 kDa indicating its monomeric state (the (YP dimer is designated monomer). Less expected was the inability of the detegent solubilized monomer to bind fibrinogen. Data were col- lected up to concentrations of 8.8 p~ of integrin protomer, at which no binding was observed. Therefore, the K, must be larger than 100 p ~ .

After removal of the detergents by Bio beads the integrin formed aggregates of M,= 1400 kDa, which corresponds to integrin hexamers. Sedimentation equilibrium experiments indicated that the molecular mass remained constant in a concentration range of 0.4-4.4 p ~ . A slight increase was only observed above this value. Electron microscopy supported the view that the most prominent form of aggregates is a hexamer although aggregates of other sizes were also found. These structures had already been seen in electron micrographs by other authors (Nermut et al., 1988; Rivas et al., 1991a; Weisel et al., 1992) and were named rosettes because of their shape. Integrin molecules assem- bled into rosettes are apparently connected by association of their hydrophobic membrane spanning domains (Nermut et al., 1988). Their extracellular domains with the ligand binding sites are pointing outward from the surface of the aggregates.

The hexameric rosettes in detergent-free solution showed weak fibrinogen binding. Because of the low affinity, it was not possible to reach a plateau value at the highest integrin concentration exployed (4.4 p ~ ) . Instead the average molecular mass continued to increase steeply at this point. Additionally, the value of 700 kDa at 4.4 p~ integrin is certainly much smaller than the plateau value. Because the binding of fibrinogen to the rosette does not approach saturation at the highest fibrinogen concentrations we can achieve in these experiments, we cannot measure the equilibrium constant. However, an upper bound for the equilibrium association constant may be calculated using the method described by Lakatos and Minton (1991). Given the molar radioactivity of fibrinogen and the molar absorbance of integrin, the data plotted in Figs 5(A) and (B) enable one to calculate the molar concentrations of free fibrinogen, bound fibrinogen, and total integrin at each radial position. The ratio of bound fibrinogen to total

36 G. RIVAS ETAL.

0.055 7 0.050

0.045 1 0.040 1 T A 0.035 1 I. @ [integrin]

I l l / I

90 100 110 120 130 140 150 160 170 180

IF~LII,,~ (nM)

figure 6. Binding of iodinated fibrinogen to allbp3 in detergent- free solutions. Plot of bound fibrinogen/total integrin vs free fibrinogen, calculated as described in the text. The solid line shows the best straight line weighted fit to the data. The error bars represent 220; where d=a2,+l(dy/dx)uXl2.

integrin at a given position is plotted as a function of free fibrinogen at that position in Fig. 6.

For a simple independent binding site model, the fractional saturation of sites on integrin for the binding of fibrinogen, y, is given by:

(4)

where K, is the equilibrium association constant (= l/Kd). It is clear from the linear dependence of y on [Fbg], that Ka[FbglfmG 1, and Eqn (4) reduces to:

Y=nKa [FbglfEe (5 ) The slope of the best fit straight line plotted in Fig. 6 is 2.8f 1.2 x lo5 M - ' , where the indicated uncertainty corresponds to f 2SE of estimate. The lower limit of n= 1 corresponds to an upper limit of 2 . 8 ~ lo5 M - ' for K,, or a lower limit of -3 FM for Kd.

The low binding affinity of the solubilized aIIbp3 cannot be explained by an irreversible denaturation by the detergent because of a full recovery of activity after incorporation of the Triton solubilized integrin into lipid bilayers for total internal relefection microscopy (TIRFM, Miiller et al., 1993). A local and reversible inactivation of the integrin by Triton may not be fully excluded but it was shown by several electron microscopic studies (Nermut et al., 1988; Weisel et al., 1992; Miiller et al., 1993) that a gross alteration of the structure of integrins by Triton solubilization may be excluded. Detergent solubilized integrin preparations were also used for several of the other assays in Table 1 which gave similar K,, values as the TIRF'M method. The low binding affinity of integrin monomers was also confirmed by an electron microscopic analysis by Weisel et al., (1992). The absence of binding activity of the aIIbp3 monomers in the presence of detergent and the low activity of the hexameric rosettes produced after removal of the detergent must therefore be regarded as intrinsic properties of the integrin in solution. Moreover, the affinity for fibrinogen measured in this work is not so different than that reported for intact platelets (Table 1). also substantially lower than that for in vitro solid phase assays and for reconstituted systems. Although the

differences in level organization and the complexities of integrin transmembrane function (Miyamoto et al., 1995) precludes a direct comparison between those studies, it is worth noting that the fibrinogen binding capacity measured by Phillips and Baugham (1983) with intact platelet membranes is 60-fold less than that in whole platelets (Marguerie et al., 1980); also in the case of liposomes Parise and Phillips (1985b) found a relatively low binding capacity: 0.12 mol of fibrinogen/mol of integrin. Receptor inactivation it seems to be the most likely explanation for that reduction; however, the fact that those binding studies were performed with submicromolar amounts of interacting partners means that the existence of a much lower affinity class of binding sites cannot be excluded. The importance of weak protein interactions in cellular adhesion has recently been pointed out (van der Merwe and Barclay, 1994).

Several reasons may be suggested for the difference between the affinity of integrin in a membrane or in an absorbed state and the same integrin in solution. A possibility, as already mentioned, is the lack of integrin in solution. A possibility, as already mentioned, is the lack of activation of the integrin in its solubilized form. Based on results with solid phase assays it was reported (Du et al., 1991; Kouns et al., 1992) that activation can be achieved by exposure of aIIbp3 to RGD by affinity chromatography over a RGD affinity column. In several cases integrins were reported to be activated by addition of activating antibodies (Ginsberg et al., 1990). A second possibility is that the restricted geometry of the rosettes may not allow fibrinogen to bind multivalently, as it may in the case of integrins adsorbed on surfaces or incorporated into membranes. It is well established that multivalent binding greatly enhances binding affinity [see. e.g., Crothers and Metzger (1972) and Reynolds (1979)l. Multivalency in the membrane bound state most likely also leads to the biphasic binding behaviour of fibrinogen to the integrin (Marguerie et al., 1980; Miiller et al., 1993; Huber et al., 1995). The biphasic binding kinetics complicates the interpretation of the binding data in this system. Another possible reason for the low affinity of aIIbp3 integrin for fibrinogen in our experiments may be the lack of manganese ions, which have very recently been reported to activate several integrins (Smith et al., 1994); however, preliminary studies in our laboratory suggest that the addition of manganese ion does not increase affinity in this system. Finally, the present work demonstrates the utility of the methodology of tracer sedimentation equilibrium to characterize complex hetero- associating systems; moreover, this experimental method should be of particular interest in the analysis of relatively low affinity reversible associations, where quasi-equilibrium methods, such as filter binding, that depend upon kinetic trapping of an equilibrium state after removal from equilibrium conditions, are of questionable validity.

Acknowledgements

We thank the Swiss National Science Foundation for financial support and Therese Schultzness (Basel) for the EM pictures. G. R. thanks EMBO for a postdoctoral fellowship and Dr JosC Gonzalez (Instituto de Quimica Fisica, CSIC, Madrid) for the excellent laboratory facilities to purify the integrin.

BINDING OF FIBRINOGEN TO PLATELET INTEGRIN aIIbD

References

31

Aumailley, M., Gurrath, M., Muller, G., Calvete, J. J., Timpl, R. and Kessler, H. (1991). Identification of the Arg-Gly-Asp sequence in laminin A chain as a latent cell-binding site being exposed in fragment P1. FEES Lett. 290,50-54.

Calvete, J. J., Mann, K., Alvarez, M. V., Lopez, M. M. and Gonzalez-Rodriguez, J. (1992a). Proteolytic dissection of the isolated platelet fibrinogen receptor, integrin GPllb/llla. Localization of GPllb and GPllla sequences putatively involved in the subunit interface and in intra-subunit and intrachain contacts. Biochem. J. 282,523-532.

Calvete, J. J., Schafer, W., Mann, K., Henschen, A. and Gonzalez- Rodriguez, J. (1992b). Localization of the cross-linking sites for RGD and KQAGDV peptides to the isolated fibrinogen receptor, the human platelet integrin glycoprotein llb/llla. Influence of peptide length. fur. J. Biochem. 206,759-765.

Carrell, N. A., Fitzgerald, L. A., Steiner, 6.. Erickson, H. P. and Phillips, D. R. (1985). Structure of human platelet membrane glycoproteins Ilb and llla as determined by electron microscopy. J. Biol. Chem. 260,17743-17749.

Charo, I. F., Nannizzi, L., Phillips, D. R., Hsu, M. A. and Scarborough, R. M. (1991). Inhibition of fibrinogen binding to GPllb-llla by a GPllla peptide. J. Biol. Chem. 266, 141 5-1421.

Chatelier, R. C. and Minton, A. P. (1987). Sedimentation equilibrium in macromolecular solutions of arbitrary concentration. II. Two protein components. Biopolymers 26,

Crothers, D. M. and Metzger, H. (1972). The influence of polyvalency on the binding properties of antibodies. lmmu- nochemistry 9,341-357.

Darawshe, S., Rivas, G. and Minton, A. P. (1993). Rapid and accurate microfractionation of the contents of small centrifuge tubes: application in the measurement of molecular weights of proteins via sedimentation equilibrium. Anal. Biochem. 209,130-1 35.

Du, X., Plow, E. F., Frelinger 111, A. L., OToole, T. E., Loftus, J. C. and Ginsberg, M. H. (1991). Ligands 'activate' integrin allbp3 (platelet GPllb-Ma). Cell65.409-416.

Durchschlag, H. (1986). Specific volumes of biological macro- molecules and some other molecules of biological interest. In Thermodynamic Data for Biochemistry and Biotechnol- ogy, ed. by H. J. Hinz, pp.45-128. Springer-Verlag, Berlin.

Eirin, M. T., Calvete, J. J. and Gonzalez-Rodriguez, J. (1986). New isolation procedure and further biochemical characterization of glycoproteins Ilb and llla from human platelet plasma membrane. Biochem. J. 240,147-153.

Ginsberg, M. H., Frelinger 111, A. L., Lam, S. C.-T., Forsyth, J., McMillan, R., Plow, E. F. and Shattil, S. J. (1990). Analysis of platelet aggregation disorders based on flow cytometric analysis of membrane glycoprotein Ilb-llla with conformation-specific monoclonal antibodies. Blood 76,2017-2023.

Harding, S. E., Horton, A. and Rowe, A. (Eds) (1993). Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, London.

Hsu, C. S. and Minton, A. P. (1991). A strategy for efficient characterization of macromolecular hetero-associations via measurement of sedimentation equilibrium. J. Mol. Recogn 4, 93-104.

Huber, W., Hurst, J., Schlatter, D., Barner, R., Hubscher, J., ' Kouns, W. C. and Steiner, 6. (1995). Determination of kinetic

constants for the interaction between the platelet glycoprotein Ilb-Ma and fibrinogen by means of surface plasmon resonance. Eur. J. Biochem. 227,647-656.

Hynes, R. 0. (1992). Integrins. Versatility, modulation, and signaling in cell adhesion. Cell69, 11-25.

Ingham,. K. C., Landwehr, R. and Engel, J. (1985). Interaction of fibronectin with C lq and collagen. Effect of ionic strength and denaturation of the collagenous component. fur. J. Biochem. 148,219-224.

Kieffer, N. and Phillips, D. R. (1990). Platelet membrane glycoproteins: functions in cellular interactions. Annu. Rev. Cell Biol.6,329-357.

1097-1 113.

Kirschbaum, N. E., Mosesson, M. W. and Amrani, D. L. (1992). Characterization of the gamma chain platelet binding site on fibrinogen fragment D. Blood79,2643-2648.

Kouns, W. C., Hadvary, P., Haering, P. and Steiner, 6. (1992). Conformational modulation of purified glycoprotein (GP) Ilb-llla allows proteolytic generation of active fragments from either active or inactive GPllb-llla. J. Biol. Chem. 267, 18844-18851.

Lakatos, S. and Minton, A. P. (1991). Interaction between globular proteins and F-actin in isotonic saline solution. J. Biol. Chem. 266,1870743713.

Laki, K. (1951). The polymerization of proteins: the action of thrombin on fibrinogen. Arch. Biochem. Biophys. 32,

Laue, T. M. (1992). Short-column sedimentation equilibrium. Technical Information DS-835, Beckman Instruments Inc., Palo Alto, CA.

Laue, T. M., Shah, 6. D., Ridgeway, T. M. and Pelletier, S. L. (1993). Computer-aided interpretation of analytical sedimentation data for proteins. In Analytical Ultracentrifugation in Biochemistry and Polymer Science, ed. by S. E. Harding, A. Horton and A. Rowe, pp. 90-125. Royal Society of Chemistry, London.

Legrand, C., Dubernard, V. and Nurden, A. T. (1985). Character- ization of collagen-induced fibrinogen binding to human platelets. Biochim. Biophys. Acta 812,802-812.

Marguerie, G. A., Edgington, T. S. and Plow, E. F. (1980). Interaction of fibrinogen with its platelet receptor as a part of a multistep reaction in ADP-induced platelet aggregation. J. Biol. Chem. 255, 154-161.

Minton, A. P. (1990). Quantitative characterization of reversible molecular associations via analytical centrifugation. Anal. Biochem. 190,1-6.

Minton, A. P. (1994). Conservation of signal: a new algorithm for the elimination of the reference concentration as an inde- pendently variable parameter in the analysis of sedimentation equilibrium. In Modern Analytical Ultra- centrifugation, ed. by T. M. Schuster and T. M. Laue, pp. 81-93. Birkhauser, Boston.

Miyamoto, S., Akiyama, S. K. and Yamada, K. M. (1995). Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science 267,883-885.

Muller, B., Zerwes, H.-G., Tangemann, K., Peter, J. and Engel, J. (1993). Two-step binding mechanism of fibrinogen allbp3 integrin reconstituted into planar lipid bilayers. J. Biol. Chem. 268,6800-6808.

Nachman, R. L. and Leung, L. L. K. (1982). Complex formation of platelet membrane glycoproteins Ilb and llla with fibrinogen. J. Clin. Invest. 69, 263-269.

Nachman, R. L., Leung, L. L. K., Kloczewiak, M. and Hawiger, J. (1974). Complex formation of platelet membrane glycoproteins Ilb and llla with the fibrinogen D domain. J. Biol. Chem. 259,8584-8588.

Nermut, M. V., Green, N. M., Eason, P., Yamada, S. S. and Yamada, K. M. (1988). Electron microscopy and structural model of human fibronectin receptor. EMBO J. 7,

Parise, L. V. and Phillips, D. R. (1985a). Platelet membrane glycoprotein Ilb-llla complex incorporated into phospholipid vesicles. J. Biol. Chem. 260, 1750-1756.

Parise, L. V. and Phillips, D. R. (1985b). Reconstitution of the purified platelet fibrinogen receptor. Fibrinogen binding properties of the glycoprotein Ilb-llla complex. J. Biol. Chem. 260,1069&10707.

Pfaff, M., Tangemann, K., Muller, B., Gurrath, M., Muller, G., Kessler, H., Timpl, R. and Engel, J. (1994). Selective recognition of cyclic RGD peptides of NMR defined conformation by alpha Ilb beta 3, alpha v beta 3, and alpha 5 beta 1 integrins. J. Biol. Chem. 269,20233-20238.

Phillips, D. R. and Baugman, A. K. (1983). Fibrinogen binding to human platelet plasma membranes. Identification of two steps requiring divalent cations. J. Biol. Chem. 258,

317-324.

4093-4099.

38 G. RIVAS ET AL

10240-1 0246. Phillips, D. R., Charo, 1. F. and Scarborough, R. M. (1991).

GPllbllla: the responsive integrin. Cell 65,359-362. Plow, E. F. and Ginsberg, M. H. (1989). Cellular adhesion: GPllb-

llla as a prototypic adhesion receptor. Prog. Hemostasis Thromb. 9,117-156.

Reynolds, J. A. (1979). Interaction of divalent antibody with cell surface antigens. Biochemistry 18,264-269.

Reynolds, J. A. and McCaslin, D. R. (1985). Determination of protein molecular weight in complexes with detergents without knowledge of binding. Methods Enzymol. 61, 58-62.

Rivas, G. (1989). Isolation and molecular characterization of the integrin GPllb/llla (the platelet fibrinogen receptor) and its constituent subunits GPllb and GPllla. Doctoral Thesis, Universidad Autonoma, Madrid, Spain.

Rivas, G. A. and Gonzalez-Rodriguez, J. (1991). Calcium binding to human platelet integrin GPllb/llla and to its constituent glycoproteins. Effect of lipids and temperature. Biochem. J. 276,3540.

Rivas, G. and Minton, A. P. (1993). New developments in the study of biomolecular associations via sedimentation equilibrium. Trends Biochem. Sci. 18, 284-287.

Rivas, G. A., Aznarez, J. A., Usobiaga, P., Saiz, J. L. and Gonzalez-Rodriguez, J. (1991a). Molecular characterization of the human platelet integrin GPllb/llla and its constituent glycoproteins. Eur. Biophys. J. 19,335-345.

Rivas, G. A., Calvete, J. J., and Gonzalez-Rodriguez, J. (1991b). A large-scale procedure for the isolation of integrin GPllb/llla, the human platelet fibrinogen receptor. Prot. Express. Purif. 2,248-255.

Rivas, G., Ingham, K. C. and Minton, A. P. (1992). Calcium-linked association of human complement Cls. Biochemistry 31, 11707-11710.

Rivas, G., Ingham, K. C. and Minton, A. P. (1994). Calcium-linked hetero-association between human complement Cls and Clr. Biochemistry33,2341-2348.

Schultze, H. E. and Heremans, J. L. (1966). In Molecular Biology of Human Proteins, Vol. 1, pp. 176-181. Elsevier, Amster- dam.

Schuster, T. M. and Laue, T. M. (Eds) (1994). Modern Analytical Ultracentrifugation. Birkhauser, Boston.

Smyth, S. S., Hillery, C. A. and Parise, L. V. (1992). Fibrinogen binding to purified platelet glycoprotein Ilb-llla (integrin allbp3) is modulated by lipids. J. Biol. Chem. 267, 15568-15577.

Smith, J. W., Piotrowicz, R. S. and Mathis, D. (1894). A mechanism for divalent cation regulation of /B-integrins. J. Biol. Chem. 269,960-967.

Tanford, C., Nozaki, Y., Reynolds, J. A. and Makino, S. (1974). Molecular characterization of proteins in detergent solutions. Biochemistry 13, 2369-2376.

Tangemann, K. and Engel, J. (1995). Demonstration of non- linear detection in ELISA resulting in up to 1000-fold too high affinities of fibrinogen binding to integrin allbp3. FEBS Lett. 358,179-181.

Usobiaga, I?, Calvete, J. J., Saiz. J. L., Eirin, M. T. and Gonzalez- Rodriguez, J. (1987). Molecular characterization of human platelet glycoproteins llla and Ilb and the subunits of the latter. Eur. Biophys. J. 14; 21 1-218.

van der Merwe, P. A. and Barclay, A. N. (1994). Transient intercellular adhesion: the importance of weak protein- protein interactions. Trends Biochem. Sci. 19,354-358.

Weisel, J. W., Nagaswami, C., Vilaire, G., and Bennett, J. S. (1992). Examination of the platelet membrane glycoprotein Ilb-llla complex and its interaction with fibrinogen and other ligands by electron microscopy. J. Biol. Chern. 267, 16637-16643.

Documents

Binding of fibrinogen to platelet integrin αIIbβ3 in solution as monitored by tracer sedimentation equilibrium