We have studied adhesive molecular interactions mediated by the platelet integrin αIIbβ3 using novel chemical and bio-physical approaches. First, we have developed methodology to functionalize microscopic spherical surfaces which can be used in various applications related to receptor–ligand interaction studies, such as biomembrane force probe, magnetic or optical tweezers, atomic force microscopy, and particle–cell binding reactions. Next, we applied a highly sensitive biophysical technique, optical tweezers, which is capable of measuring adhesive forces between two spherical microparticles that are repeatedly touched and separated.
To synthesize peptide-coated latex beads, we utilized a two-step double oxidation procedure based on the ability of oxidized dextran to form a Schiff base with primary amine groups to couple biologically active peptides to latex beads ( and ). The successful covalent coupling of dextran to latex beads is supported by three independent studies. First, fluorescent dextran covalently-bound to the beads did not detach after repeated washing cycles. By contrast, the chemically-untreated physisorbed TRITC-dextran was desorbed during washing, resulting in beads that were not visible by fluorescence microscopy (). Second, beads coupled with oxidized dextran did not change their size upon multiple washings, whereas beads bearing physisorbed dextran, although initially larger in diameter, became smaller with each washing cycle, behavior manifestly different from beads to which oxidized dextran was covalently attached (). It is noteworthy that the average diameter of the dextran-coated beads was consistently greater than that of the untreated control beads without dextran, further evidence for the presence of a stable, hydrated grafted polymer. Third, FTIR spectra provided direct evidence for chemically-induced structural changes in dextran, corresponding to successive steps of oxidation and reductive amination that resulted in the formation of covalent bonds between the polymer and the beads ().
investigated the effect of molecular weight on dextran grafting to surfaces. For dextran prepared from Leuconostoc
ssp., molecular weights ranging from 1 kDa to 100 kDa (close to that used in this study) were covalently grafted to surfaces and characterized by a range of methods including atomic force microscopy to determine roughness and coverage. The ~100 kDa dextran provided the lowest contact angle and thickest film. Moreover, the highest molecular weight dextran best mimicked the endothelial surface in measurements of bubble adhesion measurement. Furthermore, a detailed study of the oxidation kinetics of 110 kDa dextran was presented.6
To test the adhesive properties of dextran-coated latex beads, they were brought into intermittent contact with a surface coated with the plasma protein fibrinogen. In vivo
, fibrinogen is enzymatically converted to a fibrin polymer, a scaffold for blood clots and thrombi, and therefore is involved in multiple adhesive interactions in the vasculature and other tissues.28
The non-specific adhesive properties of covalently attached dextran were compared with physisorbed dextran and BSA, a relatively non-reactive blood plasma protein that is widely used as an inert blocker at bio-interfaces. The force histograms presented in and their quantitative parameters in clearly show that beads with physisorbed dextran were “stickier” to a fibrinogen-coated surface. On the other hand, beads with covalently attached dextran displayed remarkably low non-specific adhesiveness towards a fibrinogen-coated surface that was even less than that of BSA. The physisorbed dextran shows stronger adhesion to fibrinogen compared with covalently attached dextran because the bare polystyrene particles are attractive for proteins and the physisorbed (thinner) layer does not shield the core particle as well as the grafted dextran brush. Thus, the protocol for covalent coupling of oxidized dextran to NH2
-modified latex beads enabled us to synthesize micron-size particles with extremely low protein adhesion, a prerequisite for functionalizing them for use in bioassays.
Dextran-coated beads were further modified to introduce bioactive peptides with high binding selectivity. One peptide, RGD, is a binding motif for at least 8 of 24 integrins of different structure and cellular origin30
and X-ray crystallography has begun to reveal the structural basis for these interactions.31–33
Dextran coating containing immobilized RGD strongly promotes attachment and spreading of cells compared to dextran surfaces without RGD.34,35
Here, we asked whether cRGD, covalently-bound to dextran-coated latex beads, is able to interact specifically with the purified αIIbβ3, which is the most abundant platelet receptor for the RGD-containing protein ligands fibrinogen, von Willebrand factor, fibronectin, and vitronectin, and is required for platelet aggregation at the sites of vascular injury.18,20
In addition to RGD, αIIbβ3 interacts with residues located at the extreme C-terminus of the fibrinogen γ chain and represented by the H12 dodecapeptide used in this study. It is noteworthy that 10–15% of the fibrinogen γ chain occurs as an alternatively spliced variant (γ′), in which the C-terminal 400–411 motif is eliminated by adding new amino acids from 408 to 427.36
It is possible that in the absence of the γ chain C-terminal dodecapeptide, the RGD sequences in the Aα chain may play a role as the major integrin-binding site. Understanding the molecular basis for interaction of αIIbβ3 with RGD and the H12 sequence remains an unresolved issue whose kinetics and thermodynamics can be studied at the single-molecule level.
As a highly precise tool to assess the molecular mechanisms of the αIIbβ3–peptide interactions, we used an original optical tweezers-based model system developed in our laboratory to study individual receptor–ligand interactions.21,23,37
This system permits the measurement of discrete rupture forces up to about 100 pN, which are in the range of forces known to break non-covalent bimolecular interactions observed in biology.38
An important feature of the laser tweezers system is that it was specifically designed to ensure that the majority of observed rupture events are due to single bimolecular attachments. Evidence that this is indeed the case has been previously reported in detail.23
Briefly, this evidence includes the observation that rupture events always occur in one step, whereas the rupture of multiple attachments should occur in sequential multiple steps. Here, we found that >80% of the ruptures occurred in one step and the remainder occurred in two or more steps manifested as jagged signals (not shown). Only single-step interactions were included in our analysis. Further, the distribution of rupture forces of multiple identical attachments should appear as a series of peaks that are multiples of a single value of force and have probabilities inversely proportional to the number of bonds. As seen in and , for interactions with rupture forces greater than 40 pN, we observed only a single well-defined peak in the force histograms. Lastly, to ensure that the majority of observed rupture events are due to single αIIbβ3–peptide bonds, surface-coating conditions are tuned to keep protein density low. Thus, the frequency of binding events comprised <20% of total interface contacts, the exception being H12–αIIbβ3 interactions, which had a cumulative binding probability of ~40% due to increased surface density ().
Two conclusions are immediately apparent from an analysis of the rupture force histograms for cRGD–αIIbβ3 and H12–αIIbβ3 ( and ). First, the peptide-functionalized beads are much more reactive than bare dextran-coated beads. Coupling cRGD or H12 resulted in strong interactions with αIIbβ3, producing a broad range of forces up to 90 pN, appearing in two relatively distinctive ranges, one with exponentially decreasing probability and the other as a Gaussian-like peak. Importantly, the bimodal force distributions we observed are similar to those we previously detected for the interaction of αIIbβ3 with fibrinogen, but with lower binding strengths, as might be expected since the coupled peptides only partially correspond to the binding interface of the whole fibrinogen molecule.23
It is noteworthy that the broad force range is an intrinsic property of single-molecule force spectroscopy, originating from the stochastic nature of the interactions, molecular heterogeneity, and extraordinary sensitivity of the optical trap. Second, the interactions we observed are specific, inasmuch as they were sensitive to the presence of RGDS, a competitive inhibitor of fibrinogen binding to αIIbβ3.39
RGDS at 1 mM reduced the probability of interaction between αIIbβ3 and beads coated with cRGD or H12 by 45% and 66%, respectively, comparable to the 45% reduction in the interaction of αIIbβ3 with beads coated with fibrinogen by the same concentration of RGDS.23
The RGDS competitor more effectively suppressed higher forces, indicating that they reflected the most specific portion of the integrin–peptide interactions. Similarly, we found that beads coated with non-reactive peptide cRAD were only slightly stickier than beads coated with dextran but uncoupled to peptide. The latter result also confirms that re-oxidizing and further processing dextran neither physically degrades the latex bead surface nor changes the dextran chemically to make it less inert.
The bimodal rupture force distribution observed for cRGD–αIIbβ3 and H12–αIIbβ3, as we previously suggested,23,37
indicates that the complex of αIIbβ3 bound to ligands likely exists in two states with different mechanical stabilities. Based on the effect of RGDS on αIIbβ3 binding to beads coupled with cRGD, the interaction can be segregated into relatively non-specific interactions with rupture forces <25–30 pN and more specific interactions whose rupture forces exceed this value. We used the latter part of the rupture force distribution, which appeared as a Gaussian-like peak, to determine the binding strength of αIIbβ3 to cRGD, defined as the most probable force or a peak position on the X
-axis. Under our experimental conditions, the binding strength was ~60 pN. This value is smaller than the unbinding force of 93 pN determined by AFM for the single molecular complex of an RGD-containing peptide and αIIbβ3 on the platelet membrane.40
The difference is likely attributable to the substantially higher loading rate used in the AFM studies (12 000 pN s−1
) compared to ours (2000 pN s−1
), given the logarithmic dependence of measured rupture forces on the rate at which load is applied to the molecular complex.41
Comparison of the cRGD–αIIbβ3 vs.
the H12–αIIbβ3 interactions reveals two major differences, one in the binding probability and the other in the binding strength. The 2-fold difference in the binding probability (18% for cRGD and 38% for H12) may be attributable to the different surface density of the immobilized peptides, despite the fact that the binding procedures for the two peptides were similar. H12 has two potentially reactive amine groups, the α-amine of the N-terminal glycine and the ε-amine of lysine; thus, it is twice as reactive with oxidized dextran as cRGD which has only one reactive amine group. Further, there is a moderate, but statistically significant, difference between the values of binding strength for cRGD and H12, indicating that the cRGD–αIIbβ3 complex is more mechanically stable. Although the results are not fully comparable due to a number of major experimental differences, this conclusion is consistent with the results of AFM experiments using live platelets showing that the zero-force kinetic off-rate (Koff
) was much greater for H12 than for an RGD-containing peptide, indicating that the H12–αIIbβ3 complex dissociates faster upon mechanical separation.42