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Nanoparticles That Display Short Collagen-Related Peptides. Potent Stimulation of Human Platelet Aggregation by Triple Helical Motifs Mabel A. Cejas, Cailin Chen, William A. Kinney,* and Bruce E. Maryanoff Vascular Research Team, Johnson & Johnson Pharmaceutical Research and Development, Spring House, Pennsylvania 19477-0776. Received March 23, 2007; Revised Manuscript Received May 23, 2007
Collagen plays a key role in the activation and adhesion of blood platelets via their cell-surface receptors. Normally, collagen-related peptides (CRPs), even one as long as a 30-mer (10 Gly-Pro-Hyp (GPO) repeats), are unable to effectively express collagen’s platelet-activating behavior. We attached two short CRPs, AcHN-(Gly-Pro-Hyp)nGlyOH with n ) 5 (1) and n ) 10 (2), via the C-terminus to amino-functionalized latex nanoparticles to create a multimeric display of triple helical motifs. These nanomaterials were characterized by dynamic light scattering and environmental scanning electron microscopy. The nanoparticles bearing the 31-mer CRP sequence, 2, but not the 16-mer sequence, 1, effectively induced the aggregation of human platelets, with a potency level approaching that of native type I collagen. Our results highlight the importance of presenting triple helical CRP motifs of sufficient length on a suitable scaffold in order to stimulate platelets.
Blood platelets are crucial for maintaining normal hemostasis. Through their activation and aggregation, platelets arrest bleeding caused by vascular injury and then encourage wound healing. However, excessive platelet-rich thrombus formation, especially in high-sheer arterial environments, can promote chronic cardiovascular diseases, such as unstable angina, acute myocardial infarction, and stroke (1). Collagen plays a pivotal role in initiating platelet activation and adhesion via two main collagen receptors: glycoprotein VI (GPVI) and the integrin R2β1 (2-5). There are several types of collagen present in the blood vessel wall, with fibrillar type I and type III predominating in the subendothelial matrix. When a vessel’s endothelial layer is damaged, such as in atherosclerosis, the underlying collagen fibers are exposed, thereby inducing platelet aggregation and triggering adverse cardiovascular events. Synthetic triple helical peptides containing the main collagen repeat Gly-Pro-Hyp (GPO) have been used as models to study the structural basis of platelet activation by collagen (6-13). Rao et al. (6) were the first to report the promotion of platelet adhesion and aggregation by synthetic collagen-related peptides (CRPs). By using a branched assembly of three peptide chains containing a (GPO)8 CRP and an R1(IV)1263-1278 cell adhesion sequence, they demonstrated that a triple helix is required for platelet activation and suggested a role for the extra sequence. However, the potency observed in their study was much less than that of native collagen (by ∼1000-fold). A different approach involving the polymerization of short CRPs by cross-linking to obtain quaternary structures resulted in materials that were highly platelet-aggregatory (7), through direct action on GPVI (8). Interestingly, monomeric CRPs absorbed onto a glass surface can be as potent as cross-linked CRPs in aggregating platelets (9). Relative to structure-function requirements, Asselin et al. (10) found that short GPO containing monomeric CRPs are just partial agonists of GPVI, and in a review Nieswandt and Watson (11) present evidence that lengthy CRPs in a multimeric format are important for the clustering of GPVI and for robust signaling. The structural hypothesis for * To whom correspondence should be addressed. Phone: (215) 6285908. Fax: (215) 628-4985. E-mail:
[email protected].
the collagen-GPVI interaction is supported by an X-ray investigation on the GPVI dimer (12). Further understanding of the structural requirements for platelet signal transduction through GPVI could emerge from a system in which the CRPs are covalently attached to an inert support in a well-defined (nonrandom) manner. Such a construct would supply information about the importance of multivalency, spatial orientation, and chain length on collagen function. Thus, we sought to immobilize short CRPs of varying lengths onto a stable surface to present multiple peptide helices to platelets in a defined orientation. We now report a study in which we have covalently attached two different short CRPs by the C-terminus to the surface of latex nanoparticles. Some of these nanoparticle constructs, with a multimeric display of triple helical motifs, were able to induce the aggregation of human platelets with a potency level approaching that of native type I collagen. Latex nanoparticles were chosen as a support because they are available in diameters similar to the diameters of the exposed collagen fibers in a damaged blood vessel (∼50 nm, Figure 1A) (14). In addition, they contain various chemical functionalities for attachment of the probe peptide ligands of interest. Nanoparticle scaffolds have been used effectively in drug delivery and diagnostics (15), as chemical sensors (16), and in presenting biomacromolecules for molecular recognition (17, 18). Recently, the preparation of gold CRP-coated nanoparticles and their interaction with collagen fibers has been described (19). Peptides AcHN-(Gly-Pro-Hyp)nGly-OH, with n ) 5 (1) and n ) 10 (2), were synthesized on solid phase using standard FastMoc chemistry with an ABI 433A synthesizer (20). The N-terminus of the peptides was acetylated to avoid polymerization during the reaction with the nanoparticles. These CRPs were purified by HPLC and characterized by MALDI-TOF MS and CD spectroscopy. Their CD spectra showed a positive band at 225 nm, which is characteristic of a collagen triple helix (21, 22). The 225 nm band was more intense for 31-mer 2 vs 16mer 1, indicating that 2 has a more robust triple helical structure. Each peptide was conjugated through its carboxyl terminus (via carbodiimide coupling) to 200 nm latex nanoparticles bearing primary amine-terminated, six-carbon spacer units. The degree of functionalization was determined by measuring the
10.1021/bc070105s CCC: $37.00 © 2007 American Chemical Society Published on Web 06/21/2007
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Figure 1. ESEM images of (A) collagen fibrils from a murine vessel, (B) reference nanoparticles, (C) 16-mer (1) modified nanoparticles, and (D) 31-mer (2) modified nanoparticles.
Figure 2. Percentage of platelet response to collagen, peptides, and nanoparticles before and after functionalization with CRPs.
amount of peptide that was cleaved off the nanoparticles following acid hydrolysis.1 The amino acid analysis indicated that ∼10% of the amino groups (molar basis) on the surface of the particles were conjugated to the peptides. The particle size of the resulting material was estimated by dynamic light scattering (DLS).2 The average diameter of the reference particles suspended in water was determined to be approximately 200 nm, as expected. The values for the functionalized nanoparticles in solution were about 1000 nm, due to conglomeration. The morphology of the various nanoparticles was visualized by environmental scanning electron microscopy (ESEM, Figure 1). The images illustrate that both functionalized nanoparticles tended to adhere to one another (Figure 1C,D) probably because of peptide-peptide interaction, while the reference nanoparticles remained loose (Figure 1B). The sizes of conglomerates here are larger than those determined by DLS, perhaps because of different experimental conditions. For DLS the nanoparticle clusters were moving freely in solution, whereas for ESEM the 1 In preliminary experiments, smaller nanoparticles (20 nm) were found to be impractical because they did not centrifuge well and tended to associate. 2 See Supporting Information.
material was deposited on a solid support by drying a suspension. The display presented by multiple triple helices attached to the clustered nanoparticles is strikingly similar to the surface created by a severed murine blood vessel (compare part A of Figure 1 with parts C and D of Figure 1). The effect of collagen nanoparticles on platelet function was evaluated in an in vitro platelet aggregation assay. Washed platelets were prepared from human platelet-rich plasma (from healthy volunteers), and platelet aggregation measurements were performed (21).2 As depicted in Figure 2, free peptides 1 and 2, unmodified nanoparticles, and nanoparticles functionalized with 16-mer 1 failed to aggregate platelets at concentrations below 1 µg/mL.3 In sharp contrast, nanoparticles functionalized with 31-mer 2 markedly induced platelet aggregation in a dosedependent manner (EC50 ) 0.60 µg/mL). The potency of this material was comparable to that of equine type I collagen (EC50 ) 0.40 µg/mL). Experiments with three 31-mer nanoparticle preparations proved to be highly reproducible (experiments A-C). In the 3 This concentration is based on peptide content in peptide-containing materials. Unmodified nanoparticles were processed in the same way, and used in the same volume, as the modified nanoparticles.
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case of experiment B, the peptide was dissolved in MES buffer and kept at 4 °C for 1 h. The triple helix conformation was verified by CD before conjugation to the particles. For experiments A and C, the peptide was coupled without previous equilibration. The fact that all preparations yielded similar results in the platelet assays indicates the formation of a triple helix structure on the particle surface. Interestingly, while both conjugates form conglomerates of similar size, only the conjugate bearing the 31-mer peptide (2) is effective in the platelet aggregation assay, suggesting that functional activity depends on the nature of the peptide triple helix and not just on the multiple display. Our study demonstrates that a CRP with a 31-mer sequence can mimic the function of collagen when this peptide is displayed in a multivalent fashion by linking its C-terminus to a nanoparticle scaffold. These results support the importance of multiple triple helical motifs for robust stimulation of platelets by CRPs via the GPVI receptor (7-9). Looking ahead, the covalent attachment of short CRPs to nanoparticles should provide a useful means to acquire new information about the structural requirements of such peptides for collagen-like function in cell biology.
ACKNOWLEDGMENT We thank Pratik P. Joshi (Center for Advance Microscopy, University of Miami) for ESEM measurements. We also acknowledge the valuable guidance provided by Marian Kruszynski, Tami Raguse, and Albert Schmidt. Supporting Information Available: Details on materials and methods, peptide synthesis, CD spectroscopy, nanoparticle functionalization, DLS, and platelet aggregation. This material is available free of charge via the Internet at http://pubs.acs.org.
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