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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2016 April 8; 291(15): 7858–7867.
Published online 2016 February 10. doi:  10.1074/jbc.M115.706861
PMCID: PMC4824994

The Platelet Integrin αIIbβ3 Differentially Interacts with Fibrin Versus Fibrinogen*

Abstract

Fibrinogen binding to the integrin αIIbβ3 mediates platelet aggregation and spreading on fibrinogen-coated surfaces. However, in vivo αIIbβ3 activation and fibrinogen conversion to fibrin occur simultaneously, although the relative contributions of fibrinogen versus fibrin to αIIbβ3-mediated platelet functions are unknown. Here, we compared the interaction of αIIbβ3 with fibrin and fibrinogen to explore their differential effects. A microscopic bead coated with fibrinogen or monomeric fibrin produced by treating the immobilized fibrinogen with thrombin was captured by a laser beam and repeatedly brought into contact with surface-attached purified αIIbβ3. When αIIbβ3-ligand complexes were detected, the rupture forces were measured and displayed as force histograms. Monomeric fibrin displayed a higher probability of interacting with αIIbβ3 and a greater binding strength. αIIbβ3-fibrin interactions were also less sensitive to inhibition by abciximab and eptifibatide. Both fibrinogen- and fibrin-αIIbβ3 interactions were partially inhibited by RGD peptides, suggesting the existence of common RGD-containing binding motifs. This assumption was supported using the fibrin variants αD97E or αD574E with mutated RGD motifs. Fibrin made from a fibrinogen γ′/γ′ variant lacking the γC αIIbβ3-binding motif was more reactive with αIIbβ3 than the parent fibrinogen. These results demonstrate that fibrin is more reactive with αIIbβ3 than fibrinogen. Fibrin is also less sensitive to αIIbβ3 inhibitors, suggesting that fibrin and fibrinogen have distinct binding requirements. In particular, the maintenance of αIIbβ3 binding activity in the absence of the γC-dodecapeptide and the α-chain RGD sequences suggests that the αIIbβ3-binding sites in fibrin are not confined to its known γ-chain and RGD motifs.

Keywords: adhesion, fibrin, fibrinogen, integrin, platelet

Introduction

The interactions of the platelet integrin αIIbβ3 with fibrinogen and fibrin are essential for hemostasis and thrombosis. Thus, fibrinogen binding to activated αIIbβ3 mediates platelet aggregation and platelet spreading on a fibrinogen-containing matrix (1). However, individual activated αIIbβ3 molecules also attach to fibrin fibers (2) such that aggregated platelets are major components of hemostatic blood clots and thrombi (3). Further, the interaction of αIIbβ3 with fibrin is involved in platelet-driven clot contraction (4, 5). In vivo, blood clotting is catalyzed by thrombin, which simultaneously induces fibrinogen binding to αIIbβ3 and the conversion of fibrinogen to fibrin. However, the relative importance of fibrinogen versus monomeric and polymerized fibrin as αIIbβ3 ligands remains unclear (6).

Fibrinogen binds to αIIbβ3 on agonist-stimulated platelets with a Kd of ~100 nm, nearly 100-fold less than the concentration of fibrinogen in plasma, implying that the fibrinogen binding site on αIIbβ3 is immediately occupied when platelets are activated in a plasma environment (7, 8). Fibrinogen contains several sequence motifs that can potentially mediate its interaction with αIIbβ3. Residues located at the C terminus of the fibrinogen γ chain (residues 400–411) are necessary for fibrinogen binding to platelets (8, 9). Human fibrinogen also contains two common Arg-Gly-Asp (RGD) integrin recognition motifs in its Aα chain (residues Aα95–97 and Aα572–574). However, deletion of these motifs does not impair the ability of fibrinogen to support platelet aggregation that is mediated by the KQAGDV sequence located at the C terminus of the fibrinogen γ chain (10, 11). Nonetheless, RGD-containing peptides inhibit αIIbβ3 function in vitro and are clinically effective antagonists of αIIbβ3 in vivo (1), presumably because they can compete with the γ chain motif for fibrinogen binding to αIIbβ3 (12).

Because of the experimental challenges involved in measuring the interaction of platelets with fibrin, most research has focused on the role of fibrinogen in platelet adhesion and aggregation. Thus, much less is known about the former interaction. However, differences in the ability of αIIbβ3 antagonists to inhibit clot contraction versus platelet aggregation suggest that the interactions of αIIbβ3 with fibrinogen and fibrin are different (13,18). The sites at which αIIbβ3 interacts with fibrin and fibrinogen also appear to be substantially different. Thus, whereas fibrinogen lacking the C-terminal γ chain residues is unable to support platelet aggregation, its ability to support clot contraction is unaffected (19, 20). Moreover, substitution of each RGD motif in the fibrinogen α chain with RGE has no effect on clot contraction (21), and although clot contraction is somewhat delayed when the RGD and C-terminal γ chain motifs are mutated, it is eventually indistinguishable from that mediated by intact fibrin. These observations suggest that a site or sites in addition to the RGD and C-terminal γ chain motifs participate in the interaction of platelets with fibrin during thrombus growth and clot contraction. Candidate sites proposed previously include residues 15–42 in the β chain (22), as well as residues 316–322 (23, 24) and 370–381 (25, 26) in the γ chain (Fig. 1).

FIGURE 1.
Diagram of the fibrinogen molecule with the Aα (blue), Bβ (red), and γ (green) chains indicated. Putative αIIbβ3 binding sites are denoted with the color of the corresponding fibrinogen chain. The location of the ...

Because the interactions of platelets with fibrin appear to be important for the generation of a hemostatic plug or thrombus that is able to withstand the hemodynamic stresses present in circulating blood, mechanistic insights into αIIbβ3-fibrin interactions may provide new avenues for the development of novel treatment modalities for the thrombotic complications of cardiovascular disease and ischemic stroke. Here, we have tested the hypotheses that αIIbβ3-mediated platelet interactions with fibrin may be stronger than those with fibrinogen because of broader specificity, faster kinetics, and higher affinity and result from the exposure of cryptic αIIbβ3 binding sites when fibrinogen is converted to fibrin.

Experimental Procedures

Production of Recombinant Fibrinogens

Recombinant humanfibrinogens were expressed in transfected BHK cells as described previously (27). Briefly, recombinant fibrinogens were isolated from the conditioned medium of BHK cells that were transfected with expression vectors encoding the Aα, Bβ, and γ chain cDNAs. Purified recombinant fibrinogens were isolated in a two-column procedure using protamine-agarose and GPRPC-agarose affinity chromatography.

Model System to Study the Interaction of αIIbβ3 with Fibrinogen and Fibrin

αIIbβ3 interactions with human fibrinogen and fibrin were studied at the single-molecule level using a biophysical methodology that we have developed called optical trap-based force spectroscopy (28,31). In this method, each of two contacting surfaces is coated with one type of interacting molecule. Here, purified human αIIbβ3 was immobilized on micron-size stationary silica beads, whereas fibrinogen or monomeric fibrin was bound to freely moving latex beads. Under visual microscopic control, a bead coated with either fibrinogen or monomeric fibrin was trapped in a fluid chamber by a focused laser beam and moved in an oscillatory manner so that it tapped a stationary αIIbβ3-coated pedestal anchored to the bottom surface of the flow chamber (Fig. 2A). When the immobilized fibrinogen or fibrin monomer on the bead interacted with αIIbβ3 on the pedestal, tension was produced when the bead was displaced from the laser focus until the αIIbβ3-fibrinogen or αIIbβ3-fibrin bond ruptured. The applied force was then displayed as a signal proportional to the strength of αIIbβ3-fibrinogen or αIIbβ3-fibrin binding (29, 31). Such signals were on the order of piconewtons and quantitatively characterize the interactions of αIIbβ3 with fibrin and fibrinogen at the nanomechanical molecular level.

FIGURE 2.
A, schematic representation of the model system used to study bimolecular interactions between αIIbβ3 and fibrinogen or fibrin. A latex bead coated with fibrinogen or fibrin is trapped by a focused laser beam and oscillated toward or away ...

Coating Surfaces with αIIbβ3

Purified human αIIbβ3 (Abcam) at 1 mg/ml in 0.055 m borate buffer, pH 8.5, containing 60 mm n-octyl-β-d-glucoside and 3 mm CaCl2 was activated with 1 mm MnCl2 for 30 min at 37 °C. The activated αIIbβ3 was bound covalently to 5-μm spherical silica pedestals anchored to the bottom of a chamber as previously described (29, 31). Briefly, pedestals coated with a thin layer of polyacrylamide were activated with 10% glutaraldehyde (30 min, 37 °C), after which the activated αIIbβ3 (1 mg/ml in the activation mixture) was immobilized for 2 h at 4 °C. The chamber was then washed with 20 volumes of the same buffer to remove noncovalently adsorbed protein; blocked with 2 mg/ml BSA in 0.055 m borate buffer, pH 8.5, containing 150 mm NaCl, 3 mm CaCl2 for 30 min at 4 °C; and equilibrated with 20 volumes of cold (4 °C) 0.1 m HEPES buffer, pH 7.4, containing 150 mm NaCl, 3 mm CaCl2, 1 mm MnCl2, 2 mg/ml BSA, and 0.1% (v/v) Triton X-100 for 30 min at 4 °C before rupture force measurements were performed. The αIIbβ3 coating concentration (1 mg/ml) at which the cumulative probability of fibrinogen binding reached saturation was determined experimentally (Fig. 2B).

Coating Beads with Fibrinogen and Monomeric Fibrin

Purified human fibrinogen (Hyphen Biomed, Neuville-sur-Oise,France) and recombinant human fibrinogen variants were prepared as previously described (27) and were covalently attached to 1.75-μm carboxylate-modified latex beads (Bangs Laboratories, Fishers, IN) using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride as the cross-linking agent. As the first step in the procedure, carboxyl groups on the beads were activated by mixing 1 ml of 0.1 m MES buffer, pH 5.2, 20 μl of a 10% bead suspension, and 3 mg of dry carbodiimide, followed by constant rotation for 15 min at 4 °C. Next, the beads were centrifuged, washed once with binding buffer (0.055 m borate buffer, pH 8.5), and resuspended in 1 ml of 20 μg/ml fibrinogen in the binding buffer containing 150 mm NaCl. After 30 min of incubation with stirring at room temperature, the beads were sedimented and resuspended in a blocking solution (2 mg/ml BSA in the binding buffer). Before each experiment, fibrinogen-coated beads were freshly prepared and mildly sonicated to disrupt aggregates. To generate beads coated with monomeric fibrin, the fibrinogen-coated beads were treated with human α-thrombin (1 unit/ml, 37 °C, 1 h) followed by centrifugation and resuspension in 0.1 m HEPES buffer, pH 7.4, containing 150 mm NaCl, 3 mm CaCl2, 1 mm MnCl2, 2 mg/ml BSA, and 0.1% (v/v) Triton X-100. This treatment has been previously shown to generate biologically active fibrin molecules on a surface (33, 34).

Measurements of αIIbβ3-Fibrin(ogen) Rupture Forces,Data Processing, and Analysis

To measure the rupture force between αIIbβ3 and fibrinogen or fibrin, a suspension of fibrinogen- or fibrin monomer-coated beads was injected into a microscopic flow chamber with αIIbβ3-coated pedestals attached to the bottom. A fibrinogen- or fibrin-coated bead, trapped by the laser light, was brought to a distance of 2–3 μm from the integrin-coated pedestal. After oscillation of the bead was initiated, the bead was brought into contact with the pedestal by micromanipulation using a keyboard-controlled piezoelectric stage. Data collection was initiated at the first contact between the bead and the pedestal. Rupture forces following repeated contacts between the pedestal and the bead were collected for several minutes and were displayed as normalized force histograms for each experimental condition. Because only a small percentage of contact/detachment cycles result in effective αIIbβ3-fibrinogen or αIIbβ3-fibrin monomer binding/unbinding, data from more than 10 different bead-pedestal pairs, representing from 103 to 1.5 × 104 individual measurements, were combined for each experimental condition. Individual forces measured during each contact-detachment cycle were collected into 10-pN-wide bins. The number of events in each bin was plotted against the average force for that bin after normalizing for the total number of interaction cycles. The percentage of events in a particular force range (bin) represents the probability of rupture events at that tension. Optical artifacts observed with or without trapped latex beads produce signals that appear as forces below 10 pN (31). Accordingly, rupture forces in this range were not considered when these data were analyzed. Measurements were performed in 0.1 m HEPES buffer, pH 7.4, containing 150 mm NaCl, 3 mm CaCl2, 2 mg/ml BSA, and 0.1% (v/v) Triton X-100. Fibrinogen and fibrin monomer-coated bead were oscillated in a triangular waveform at 10 Hz with the pulling velocity of 24 μm/s, corresponding to a loading rate of ~2,600 pN/s with a trap stiffness of 0.11 ± 0.03 pN/nm as computed from the bandwidth of Brownian motion. Contact duration between interacting surfaces was 25 ± 1 ms. Force signals were collected at 2000 scans per second (0.5-ms time resolution).

Results

Interactions of αIIbβ3 with Immobilized Fibrinogen versus Fibrin

The interactions of surface-attached αIIbβ3 with immobilized fibrinogen and monomeric fibrin were studied in parallel. Control αIIbβ3-fibrinogen interactions manifested characteristic bimodal rupture force histograms with rupture forces of up to 140 pN (Fig. 3A), similar to those we previously observed (31). This rupture force distribution consists of the sum of exponentially decreasing weak to moderate (20–60 pN) forces, and a Gaussian-like distribution of strong (>60 pN) rupture forces peaking at 70–80 pN. The two clusters of rupture forces represent at least two types of single-molecule αIIbβ3-fibrinogen interactions that differ in kinetics, loading rate dependence, and susceptibility to inhibition or activation (29). Thus, fibrinogen-reactive αIIbβ3 exists in at least two interconvertible conformational states that form αIIbβ3-fibrinogen complexes having distinct affinities and discrete binding and unbinding kinetics (28, 30).

FIGURE 3.
Rupture force spectra for αIIbβ3 binding to either wild-type fibrinogen or wild-type fibrin in the absence and presence of inhibitors. A, αIIbβ3 binding to immobilized wild-type fibrinogen. B, αIIbβ3 binding ...

The rupture force spectrum for αIIbβ3 bound to monomeric fibrin was remarkably different from αIIbβ3 bound to fibrinogen, although it also had two observable binding regimes. The major difference was a significant 3-fold higher cumulative binding probability throughout the entire force range (Fig. 3B and Table 1). This difference was not due to a difference in the surface density of fibrinogen and fibrin monomer because the fibrin monomer was produced in situ by treating fibrinogen-coated beads with thrombin so that the surface densities of fibrinogen and monomeric fibrin were essentially identical. Moreover, because the monomeric fibrin was covalently attached to the bead surface, substantial fibrin oligomerization was not possible. Further, because the pedestals were saturated with αIIbβ3 (Fig. 2B), the difference in the probability of specific binding under standard experimental conditions was determined primarily by the intrinsic reactivity (on rates) of fibrinogen and fibrin toward αIIbβ3. The possibility that physically absorbed thrombin was responsible for the higher nonspecific surface reactivity was excluded in control experiments where fibrinogen-coated beads were treated with thermally or d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone-inactivated thrombin without a change in reactivity toward αIIbβ3-coated pedestals (not shown). The average cumulative binding probability of >30% and the overall rupture force profile with barely discernible force peaks suggest that despite the absence of polymerization on the interacting surface, a substantial portion of the αIIbβ3-fibrin interactions were multimolecular. This was likely due to the high αIIbβ3 binding activity of fibrin monomers and perhaps to the incidental formation of short oligomers between primarily clustered/aggregated fibrin molecules as well. The absence of distinct force peaks precludes determining the binding strength for bimolecular αIIbβ3-fibrin interactions. Moreover, it is likely that strong multiple fibrin-αIIbβ3 interactions were near or beyond the upper limit of forces measurable by the optical trap (~130–140 pN) and therefore were missing or distorted in the rupture force histograms. Indirect confirmation of this assumption comes from the observation that beads coated with fibrin, unlike those that were coated with fibrinogen, were often irreversibly attached to αIIbβ3-coated pedestals shortly after they were brought in contact, implying that their binding strength exceeded the power of the optical trap.

TABLE 1
Cumulative binding probability of fibrinogen (Fg) and fibrin (Fn) with the integrin αIIbβ3 in the absence and presence of H12 and the cyclic RGD peptide (cRGD)

Role of the γA/γ′ Chain Polymorphism in the Interaction of αIIbβ3 with Fibrinogen and Fibrin

It is generally accepted that αIIbβ3-fibrinogen interactions occur via the C-terminal γ chains and that neither of the two Aα chain RGD motifs is required (1). To directly address the role of the γ chain sequences in the interaction of αIIbβ3 with fibrin, we performed force spectroscopy measurements with recombinant homodimeric γA/γA and γ′/γ′ fibrinogen and fibrin. The latter proteins are alternatively spliced variants in which the C terminus of the fibrinogen γ chain, the physiologic αIIbβ3 binding site on fibrinogen, is altered by adding new amino acids from 408 to 427 (35), thereby impairing the binding of soluble fibrinogen to αIIbβ3 (36). Although γ′/γ′ fibrinogen remained reactive with αIIbβ3, its overall reactivity was ≈2-fold less than that of γA/γA fibrinogen (Fig. 4, A and C, and Table 2). Replacing γA chains with γ′ chains also attenuated rupture forces >60 pN and partially reduced weaker interactions as well. That fibrinogen γ′/γ′ maintained its ability to interact with αIIbβ3 implies that the C terminus of the γ chain is not the only motif involved in the interaction of immobilized fibrinogen with αIIbβ3. The differences in the rupture force spectra between Figs. 3A and and44C reflect differences in the reactivity of plasma-purified versus recombinant fibrinogen with αIIbβ3, the latter displaying lower reactivity.

FIGURE 4.
A–D, rupture force spectra for αIIbβ3 binding to recombinant fibrinogen or fibrin containing either two γ′ chains (A and B) or two γA chains (C and D). Fibrin monomers were generated as described in Fig. ...
TABLE 2
Cumulative binding probability of the corresponding recombinant fibrinogen (rFg) and fibrin (rFn) variants with the integrin αIIbβ3 in the absence and presence of H12

γA/γA fibrin and γ′/γ′ fibrin, each derived from the respective fibrinogen, exhibited greater binding probabilities and stronger rupture forces compared with the parental fibrinogen molecules. However, the average cumulative binding probability of γ′/γ′ fibrin was ≈40% less than γA/γA fibrin (Fig. 4, B and D, and Table 2), indicating that the C-terminal γA sequence 408–411 is involved in the interaction of αIIbβ3 with monomeric fibrin as well as fibrinogen. Further, a dodecapeptide corresponding to the C terminus of the γ-chain inhibited the interaction of αIIbβ3 with γA/γA fibrin by ≈60%, whereas under the same conditions it suppressed fibrinogen-αIIbβ3 interactions by 90% (Fig. 4, E and F, and Table 2). These results indicate that the C-terminal γA sequence 400–411 mediates the interaction of αIIbβ3 with both immobilized fibrinogen and fibrin; however, the relative contribution of this sequence is less substantial for fibrin-αIIbβ3 rather than for fibrinogen-αIIbβ3 interactions.

Role of the (A)α Chain RGD Motifs in the Interaction of αIIbβ3 with Fibrinogen and Fibrin

The interaction of soluble fibrinogen with αIIbβ3 is mediated by the C terminus of the fibrinogen γ chain (10, 11). Nonetheless, soluble fibrinogen binding to αIIbβ3 is inhibited by RGD-containing peptides (1) whose binding site on αIIbβ3 overlaps with that of peptides corresponding to C-terminal γ chain residues (12). It is also noteworthy that the affinity of RGD-containing peptides for αIIbβ3 is greater than that of γ chain peptides (8, 9, 37). Because the fibrin(ogen) (A)α chain contains two RGD motifs at residues 95–98 and 572–575, it is possible that the residual interaction of immobilized γ′/γ′ fibrinogen and γ′/γ′ fibrin with αIIbβ3 may be mediated by one or both of these otherwise cryptic (A)α chain RGD motifs exposed when fibrinogen is immobilized on a surface or when fibrinogen is converted to fibrin (38). To address this possibility, we tested the consequences of mutating the RGDF motif at Aα95–98 and the RGDS motif at Aα572–575 to RGEF and RGES, respectively.

The effects of mutating either RGD motif in intact immobilized fibrinogen were qualitatively and quantitatively similar, implying that neither motif preferentially interacts with αIIbβ3 (Figs. 5 and and66 and Table 2). In comparison with control recombinant γA/γA fibrinogen (Fig. 4A), AαD97E and AαD574E fibrinogens were 69 and 62% less reactive toward αIIbβ3, respectively (Figs. 5A and and66A and Table 2). Thus, both motifs can support the interaction of immobilized fibrinogen with αIIbβ3. Likewise, mutating the RGD motifs in monomeric fibrin reduced its interaction with αIIbβ3 by 55% (Figs. 4B, ,55B, and and66B and Table 2). Nonetheless, like wild-type fibrin, the cumulative probability of αIIbβ3 binding to mutated monomeric γA/γA fibrin remained ≈2-fold greater than the cumulative probability of αIIbβ3 binding to mutated fibrinogen (Figs. 5, A and B, and and6,6, A and B, and Table 2).

FIGURE 5.
Rupture force spectra for αIIbβ3 binding to recombinant fibrinogen and fibrin in which the RGDS motif at (A)α572–575 was mutated to RGES [(A)αD574E]. A, αIIbβ3 binding to AαD574E fibrinogen. ...
FIGURE 6.
Rupture force spectra for αIIbβ3 binding to recombinant fibrinogen and fibrin in which the RGDF motif at (A)α95–98 was mutated to RGEF [(A)αD97E]. A, αIIbβ3 binding to AαD97E fibrinogen. ...

The binding sites for RGD-containing peptides and the γ chain dodecapeptide on αIIbβ3 overlap (12). Consequently, RGD and dodecapeptide binding to αIIbβ3 is mutually exclusive (39). Accordingly, we posited that free dodecapeptide should further reduce the reactivity of fibrinogen and fibrin with mutated RGD motifs toward αIIbβ3. As shown in Figs. 5C and and66C and Table 2, binding of AαD574E- and AαD97E-fibrinogen to αIIbβ3 was reduced by 73 and 84%, respectively, by the presence of 1 mm dodecapeptide. Moreover, as would be predicted from data shown in Table 1, the inhibitory effect of the free dodecapeptide on mutant fibrin-αIIbβ3 interactions was significantly less pronounced than in the corresponding mutant fibrinogens (64% for αD574E and 66% for αD97E, p < 0.01) (Figs. 5D and and66D and Table 2). Thus, these results indicate that both (A)α chain RGD motifs play a role in the interaction of αIIbβ3 with immobilized fibrinogen and fibrin. However, because 1 mm γC-dodecapeptide2 did not completely abrogate the interaction of αIIbβ3 with αD97E and αD574E fibrinogen or fibrin, it is likely that additional αIIbβ3-binding sites are exposed when fibrinogen and monomeric fibrin are immobilized.

Differential Susceptibility of αIIbβ3-Fibrinogen and αIIbβ3-Fibrin Interactions to Inhibitors

Besides being quantitatively different, fibrinogen-αIIbβ3 and fibrin-αIIbβ3 interactions respond differently to well characterized αIIbβ3-fibrinogen antagonists. αIIbβ3 binding to fibrinogen-coated beads was highly sensitive to competitive inhibition by the γC-dodecapeptide (>60% inhibition), whereas αIIbβ3 binding to fibrin monomer-coated were inhibited by only 30% (Fig. 3C and Table 1). Similarly, a cyclic RGD peptide more effectively inhibited the interaction of αIIbβ3 with fibrinogen (~80%) than with fibrin monomer (~60%, p < 0.01) (Fig. 3E and Table 1). Further, it is apparent from inspection of the rupture force histograms that both inhibitors completely abrogated αIIbβ3-fibrinogen interactions with rupture forces >60 pN, whereas the weaker force signals were only partially reduced. By contrast, αIIbβ3-fibrin interactions were essentially unaffected by the γC-dodecapeptide at rupture forces >60 pN (Fig. 3D) and substantially less affected by cyclic RGD (Fig. 3F), consistent with a readily apparent difference in αIIbβ3 binding to fibrinogen and fibrin.

To determine whether these observed differences were exclusive to peptides derived from the fibrinogen molecule, we repeated experiments using the αIIbβ3 antagonists abciximab and eptifibatide. Both inhibitors suppressed αIIbβ3 binding to fibrinogen and fibrin in a dose-dependent manner, but αIIbβ3-fibrin interactions were less sensitive compared with αIIbβ3-fibrinogen interactions. The concentration dependences of the inhibitory effects of abciximab and eptifibatide on the αIIbβ3-fibrin and αIIbβ3-fibrinogen interactions were fitted with exponentials, revealing that αIIbβ3-fibrin interactions were 4.5-fold less susceptible to inhibition as judged from the slopes (Fig. 7). Taken together, these results reiterate that the interaction of αIIbβ3 with fibrin is quantitatively and qualitatively different from its interaction with fibrinogen.

FIGURE 7.
Cumulative probability of αIIbβ3-fibrinogen and αIIbβ3-fibrin binding plotted against the concentrations of abciximab (A) and eptifibatide (B) added to the reaction chamber. The plots were generated by fitting the experimental ...

Discussion

The existing paradigm is that fibrinogen is necessary and sufficient for platelet aggregation, although it is known that other ligands can also support platelet aggregation. However, clotting in vivo is more complex and various in vitro and in vivo models are beginning to clarify the differences. Our presumption is that in vivo, platelet aggregation may initially begin with fibrinogen binding to αIIbβ3 but quickly evolves to monomeric or oligomeric fibrin binding as fibrinogen is converted to fibrin by thrombin generated on or near the surface of activated platelets (Fig. 8). Moreover, platelet aggregation/adhesion and fibrin polymerization may be initiated simultaneously, in contrast to the traditional sequential view that fibrinogen-mediated platelet aggregation is followed by fibrin formation (40). In other words, even platelet-rich areas in a thrombus contain fibrin and much of the platelet aggregation and adhesion processes are mediated by fibrin, not fibrinogen. Thus, fibrin likely plays a major unappreciated role in platelet aggregation and adhesion in vivo, and it is important to understand how the interactions of platelets with fibrin differ from those with fibrinogen.

FIGURE 8.
Cartoon illustrating the potential role of fibrin in platelet aggregation. Platelet aggregation initially begins with fibrinogen (Fg) binding to αIIbβ3 but quickly evolves into αIIbβ3 interaction with monomeric, oligomeric, ...

A number of reports in the literature indicate that the αIIbβ3 binding sites on fibrin may be substantially different from fibrinogen, suggesting that fibrinogen conversion to fibrin is accompanied by exposure of otherwise cryptic binding sites for αIIbβ3. An increase in the number of the αIIbβ3 binding sites and enlargement of the platelet interacting surface in a fibrin clot may lead to strong platelet adhesion on fibrin followed by conformational perturbations of the integrin (41, 42) and enhanced and/or selective outside-in signaling (43). In addition to the C-terminal portion of the fibrinogen γ chain (γ400–411 including the binding spot γ408–411 AGDV), a well established αIIbβ3 binding site, major candidates for sites that mediate fibrin binding αIIbβ3 are residues α95–98 (RGDF) and α572–575 (RGDS) located in the α chain because RGD sequences are the most general integrin binding motif. Nonetheless, additional αIIbβ3 binding sequences may be exposed when fibrinogen is converted to fibrin as well. Thus, platelet spreading on surfaces coated with fibrin lacking β chain residues 15–42 is substantially impaired (22). Further, antibodies against the γ316–322 region inhibit clot retraction (23), and a site designated P3 at γ370–383 has been identified that also mediates fibrin clot retraction and binds αIIbβ3 (25, 26). Mutational analyses have confirmed that both γ408–411 AGDV and P3 are required for the full adhesive activity of fibrin for platelets. It is noteworthy that P3 residues γ373–383 are fibrin-specific and are exposed upon the transformation of fibrinogen to fibrin (32), whereas the AGDV sequence is exposed constitutively in both fibrin and fibrinogen.

Because it is methodologically difficult to do quantitative measurements of platelet binding to insoluble fibrin, conventional experimental approaches have been ineffective. Accordingly, our studies make use of a biophysical technology (optical trap-based force spectroscopy) to measure receptor-ligand binding and unbinding at the single-molecule level. Earlier, this technology enabled us to determine quantitative parameters of integrin-fibrinogen interactions at interfaces and to study their molecular mechanisms (28, 29, 31, 37). Because we can convert fibrinogen immobilized on a surface to fibrin monomer (33, 34), we applied this technology to obtain quantitative characteristics and to identify fibrin structures involved in αIIbβ3-fibrin interactions. By directly measuring the binding strength between individual fibrin and αIIbβ3 molecules, we found clear differences from the interaction of αIIbβ3 with fibrinogen. To identify sites by which fibrin interacts with αIIbβ3, we used two independent approaches. First, rupture force spectroscopy was performed in the absence and presence of specific αIIbβ3 antagonists (cyclic RGD and dodecapeptide γ400–411, as well as the αIIbβ3 antagonists abciximab and eptifibatide). Second, we made use of recombinant fibrinogen and fibrin mutants lacking either the γ408–411 AGDV motif [fibrin(ogen) γ′/γ′] or the RGD motifs AαD97E and AαD574E.

Our results can be summarized as follows. The effect of replacing the γA with the γ′ chains in fibrin was similar to competitive inhibition by free γC-dodecapeptide, confirming involvement of the γC integrin-binding site in fibrin binding to αIIbβ3. Interactions of fibrin γ′/γ′ with αIIbβ3 were strongly inhibited by cyclic RGD, implying that the fibrin RGD motifs play a role as well. γ′/γ′ fibrin was more reactive with αIIbβ3 than γ′/γ′ fibrinogen, suggesting that cryptic integrin-binding sites, perhaps one or both α chain RGD motifs are exposed in fibrin and synergize with the γC binding site. This assumption was supported using the fibrin mutants αD97E or αD574E, which were slightly less reactive than fibrin γA/γA. Maintenance of the αIIbβ3 binding activity of fibrin γ′/γ′ in cyclic RGD or mutation of the αRGD motifs confirms that the αIIbβ3 binding sites in fibrin are not confined to the γC-terminal 400–411.

Our results also suggest the following general conclusions. First, the binding specificity of αIIbβ3 with fibrinogen depends on whether fibrinogen is soluble or surface-attached. At the surface, fibrinogen lacking the γ408–411 AGDV site is still reactive with αIIbβ3, indicating exposure of additional αIIbβ3-binding sites upon immobilization. Second, binding interactions of surface-attached fibrin and fibrinogen with αIIbβ3 are clearly different, both qualitatively and quantitatively. Fibrin binds to αIIbβ3 with a higher frequency at the same surface density, implying a higher association constant. This quantitative difference between fibrin and fibrinogen also reflects differences in specificity. The relative αIIbβ3 binding activity of the variant fibrin γ′/γ′, lacking the γC-terminal AGDV motif, suggests that this structure may not be a major integrin-binding site in fibrin. Third, the reduced interaction of fibrinogen and fibrin mutants lacking α chain RGD motifs implies that these motifs are involved in the interaction of αIIbβ3 with fibrin and immobilized fibrinogen as opposed to soluble fibrinogen. Binding to these motifs may even be preferred as we (37) and others (8, 9) have shown that the affinity of RGD for αIIbβ3 is greater than of AGDV. Fourth, the absence of RGD in the presence of free γC-dodecapeptide did not completely abrogate the interactions of fibrin with αIIbβ3, suggesting the existence of additional sites in fibrin other than γAGDV, αRGDF, and αRGDS. This point could be formally addressed by producing a compound fibrinogen mutant lacking the γ-chain C-terminal dodecapeptide and both α-chain RGD motifs. At this time, however, there are no laboratories producing and purifying such mutant fibrinogens. Thus, procuring this construct is not currently feasible. Fifth, αIIbβ3-fibrin interactions are less susceptible to αIIbβ3 antagonists than αIIbβ3-fibrinogen, suggesting that drugs that inhibit platelet interactions with fibrin, as well as fibrinogen, could be more effective antithrombotic agents.

The principal goal of this study was to improve our understanding of how platelets and αIIbβ3 participate in thrombosis and to aid in advancing our therapeutic capabilities for cardiovascular diseases. Our results may help to explain, in part, why the currently available αIIbβ3 antagonists are less effective than expected. Importantly, they provide a new target for development of more effective αIIbβ3 antagonists.

Author Contributions

R. I. L., J. W. W., and J. S. B. conceived and designed the research; D. H. F. and R. I. L. performed the experiments; and R. I. L., D. H. F., J. W. W., and J. S. B. analyzed the data and wrote the paper.

Acknowledgment

We thank Dr. Artyom Zhmurov for the image in Fig. 1.

*This work was supported by National Institutes of Health Grants PO1 HL40387, RO1 HL090774, and UO1 HL116330 and a grant from the American Society of Hematology. The authors, except for DHF, declare they have no conflicts of interest with the contents of this article. D. H. F. and Oregon Health and Science University have a significant interest in Gamma Therapeutics, a company that may have a commercial interest in the results of this research and technology. This potential individual and institutional conflict of interest has been reviewed and managed by Oregon Health and Science University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

2The abbreviations used are:

γC
C-terminal portion of the fibrinogen and fibrin γ chain
H12
γ chain dodecapeptide.

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