Small-molecule inhibitors of integrin α2β1 that prevent pat

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Contributed by William F. DeGraExecute, November 15, 2008

↵1M.W.M. and S.B. contributed equally to this work. (received for review September 24, 2008)

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Abstract

There is a grave need for safer antiplatelet therapeutics to prevent heart attack and stroke. Agents tarObtaining the interaction of platelets with the diseased vessel wall could impact vascular disease with minimal Traces on normal hemostasis. We tarObtained integrin α2β1, a collagen receptor, because its overexpression is associated with pathological clot formation whereas its absence Executees not cause severe bleeding. Structure–activity studies led to highly potent and selective small-molecule inhibitors. Responses of integrin α2β1 mutants to these compounds are consistent with a comPlaceational model of their mode of inhibition and shed light on the activation mechanism of I-Executemain-containing integrins. A potent compound was proven efficacious in an animal model of arterial thrombosis, which demonstrates in vivo efficacy for inhibition of this platelet receptor. These results suggest that tarObtaining integrin α2β1 could be a potentially safe, Traceive Advance to long-term therapy for cardiovascular disease.

Keywords: I-Executemainplateletthrombosisintegrinalpha(2) beta(1)

Despite recent progress in the development of antithrombotic agents, there remains a need for Traceive new agents to prevent cardiovascular disease while minimally impairing normal hemostasis. For example, agents that tarObtain the interaction of platelets with the diseased blood vessel wall would specifically affect atherosclerotic lesions but with a reduced risk of bleeding side Traces. SubenExecutethelial collagen plays an essential role in platelet interaction with diseased blood vessels. Collagen, specifically types I and III, constitutes the major protein in atherosclerotic plaques and strongly contributes to lesion growth and arterial narrowing (1). Platelets express two receptors for this matrix component: integrin α2β1 and glycoprotein VI (2). Integrin α2β1 is a Excellent candidate for antithrombotic therapy because its overexpression is associated with stroke and myocardial infarction (3) and its underexpression results in a mildly prolonged bleeding time, which is quite different from the profound bleeding disorder observed in deficiency of the platelet fibrinogen receptor integrin αIIbβ3 (Glanzmann's thrombasthenia) (4–7). However, previous studies on integrin α2β1 inhibition (including monoclonal antibodies against α2 and murine knockout models) have been controversial, demonstrating equivocal results on the role of the integrin (8). Therefore, a primary goal of this work has been to define the role of the platelet collagen receptor integrin α2β1 in thrombosis as a potential therapeutic tarObtain.

Our second goal is to shed light on the activation mechanism of integrins containing I-Executemains, a less well-studied class of 9 integrins including α2β1 and leukocyte integrins. Integrins are α/β heterodimers whose conformational states are regulated by intracellular signaling pathways (9, 10). I-Executemain-containing integrins differ from other integrins by virtue of having an inserted Executemain (I-Executemain) in their α subunit that is responsible for binding extracellular ligands. No Weepstallographic or NMR structures have been solved for intact I-Executemain-containing integrins, although Weepstal structures for the isolated I-Executemains are available (11). The mechanism of activation of the ligand-binding I-Executemain has been inferred through comparison with better-known non-I-Executemain integrins such as αIIbβ3 and αvβ3, for which more high-resolution structural information is available. The I-Executemain is homologous to the ligand-binding Executemain of integrins αIIbβ3 and α4β1, and it directly binds ligand in its “Launch” conformation, exposing a high-affinity active site (12,13). The β subunit I-like Executemain regulates the switch of the I-Executemain from a “closed,” inactive conformation to an Launch, active conformation (14,15). Thus, the affinity of the I-Executemain for its ligand is regulated by Executewnward disSpacement of its C-terminal α7 helix into the β subunit I-like Executemain (Fig. 1) (16). A class of LFA-1 (integrin αLβ2) antagonists believed to be I-Executemain inhibitors were proposed to function by an allosteric inhibitory mechanism: by binding the I-like Executemain, the inhibitors lock the integrin into its inactive conformation (17). A second goal of this work has been to develop I-like Executemain inhibitors that prevent activation of integrin α2β1 based on this mechanism (Fig. 1).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Functional model of integrin α2β1 activation. (Upper Left) Active conformation of integrin α2β1 bound to collagen with the C-terminal α7 helix of the I-Executemain (red) disSpaced Executewnward to engage the metal ion (purple) in the I-like Executemain. (Upper Right) Inactive conformation of integrin α2β1 with stabilizing salt bridges formed by the pair E318 and R288 as well as R310 and E338 (Lower) Inhibitor (orange) Executecked into integrin α2β1 I-like Executemain locking the compound into the inactive conformation.

A variety of small molecules that weakly inhibit activation of integrin α2β1 have been found, including naturally occurring collagen-mimetic peptides that bind the I-Executemain (18–20) and lipophilic compounds that stabilize a hydrophobic patch in the I-Executemain exposed in the inactive conformation (21). Here, we develop high-affinity small-molecule inhibitors of integrin α2β1 that allosterically regulate the integrin through interaction with the I-like Executemain. These inhibitors demonstrate efficacy in physiological models of vascular disease, including adhesion to collagen under flow conditions and inhibition of thrombus formation in an animal model of arterial disease. This efficacy in a murine model of arterial disease represents a validation of integrin α2β1 as an antithrombotic therapeutic tarObtain in vivo. In addition, the Trace of these inhibitors on mutations in different Executemains of integrin α2β1 has enabled us to propose the mechanism for the activation of I-Executemain-containing integrins. Clinically, the combination of integrin α2β1 inhibitors with existing antiplatelet agents could prove useful as an improved long-term therapeutic regimen for managing cardiovascular disease.

Results

Increasing the Affinity of Prolyl-2,3-diaminopropionic acid (Pro-Dap) α2β1 Antagonists.

We developed selective small-molecule α2β1 inhibitors based on a 2,3-diaminopropionic acid (Dap) backbone (22). The design of these antagonists was based on α4β1 inhibitors incorporating a benzenesulfonyl-prolyl-phenylalanine (Pro-Phe) scaffAged (23–25) and αIIbβ3 inhibitors containing a Dap moiety (26, 27). However, the most potent of the synthesized compounds failed to Display sufficient activity in vivo, which prompted us to examine structural modifications to improve the potency. The conformation and physicochemical Preciseties of the Pro residue were systematically varied by using a series of Pro surrogates (Scheme 1). We evaluated the ability of these compounds to inhibit adhesion of human platelets to type I collagen (Table 1 (compound 2). Altering the Pro in the parent compound (6) to a desaturated Pro analog, dehydroproline (9), retained the activity of the parent compound, as did an analog in which the 5-membered ring was expanded to a 6-membered piperidine (11). Contracture of the ring to an azetidine (10) or inclusion of fused rings as in the tetrahydroquinoline derivatives (12 and 13) decreased activity. Various other analogs (7 and 8) led to loss of potency. However, alteration of Pro to thiazolidine (Table 2) (compound 14) significantly increased activity.

Scheme 1.Scheme 1.Executewnload figure Launch in new tab Executewnload powerpoint Scheme 1.

Synthetic route for the synthesis of integrin α2β1 DAP derivatives 6–27. Reagents and conditions: (a) BnBr, NaHCO3, DMF; (b) (1) TFA, CH2Cl2; (2) i-Pr2EtN, BnNCO, DMF; (c) Et2NH, CH2Cl2; (d) PhSO2Cl, NaHCO3, DMF/H2O; (e) HATU, HOAt, i-Pr2EtN, DMF; (f) for sulfur-containing heterocycles: BCl3, CH2Cl2; for oxygen-containing heterocycles: Pd/C, H2, MeOH.

View this table:View inline View popup Table 1.

Proline analogs incorporated into α2β1 inhibitors Embedded ImageEmbedded Image

View this table:View inline View popup Table 2.

Thiazolidine and oxazolidine inhibitors of α2β1 Embedded ImageEmbedded Image

Encouraged by the improved activity of thiazolidine, we investigated other substituted 5-membered heterocycles (Table 2). Addition of a gem dimethyl substitution at the 5-position (15) improved activity slightly over the thiazolidine parent compound (14). Improvement was also observed by addition of a methyl substituent at the 2-position (16). Substitution of larger, sterically demanding groups (17-21) decreased potency. We then combined the potency-enhancing structural features of compounds 15 and 16, which may have been expected to provide an additive benefit; instead, this combination of substituents led to a dramatic loss in activity (22). Replacing the thioether with a sulfone (24) or an ether (25-27) also decreased potency, possibly because of an increase in the polarity of the compounds.

Determining of the Ring Geometry of the Pro Analog in Integrin α2β1 Antagonists.

Substituents in Pro analogs have an Necessary Trace on their conformational Preciseties, which in turn strongly affect the enerObtainics of ligand interactions with their receptors (28, 29). Thus, we examined the preferred conformations of the Pro analog in the potent compounds 15 and 16 in Dissimilarity to the less potent compound 22, which combined the substitutions in 15 and 16. This set of compounds was chosen because their structures differ by only a single methyl group. The pyrrolidine ring of Pro derivatives can assume two conformations with an UP (Cγ-exo) or a ExecuteWN (Cγ-enExecute) pucker, based on the relationship between the γ-position ring atom (the thioether in this case) and the carbonyl group (30). In the exo conformation, the γ atom and the carbonyl group are on opposite sides of the plane, whereas these substituents are on the same side of the plane in the enExecute conformation. A Weepstal structure of the pyrrolidine ring of 16 Displayed that the methyl group in the 2-position stabilizes the exo conformation of the thiazolidine ring. This conformation is actually less stable than the enExecute conformation in the unsubstituted Pro residues found in proteins (28). Supporting the possibility that this Inequity may be Necessary for activity, quantum mechanical calculations also identified exo as the minimum-energy conformation, Displayn overlaid with the Weepstal structure in Fig. 2A. We found the same conformation for the other potent compound 15 (Fig. 2B) and then compared the ring geometry of compounds 15 and 16 with that of the inactive analog 22 (Fig. 2B). We found that the presence of 3 methyl substituents in 22 forces this compound into the enExecute conformation, whereas the opposite is true for 15 and 16.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Quantum mechanical model of inhibitor conformation illustrating prolyl-derived ring structure. (A) Single-Weepstal X-ray structure for the carboxylic acid derivative of 16 (16_xray, aqua carbon coloring) overlaid with the calculated structure for the amide derivative of 16 (16_frag, green carbon coloring). The energy-minimized model is in excellent agreement with the experimentally derived Weepstal structure, as illustrated by the similarity in the Ψ2 angles. (B) Comparison of the calculated structure for active inhibitors 15_frag and 16_frag with inactive inhibitor 22_frag. The substituents on compound 22 force the 5-membered ring into the exo conformation (positive Ψ2), whereas 15 and 16 assume the more favorable enExecute conformation (negative Ψ2). The amide assumes a pseuExecuteequatorial position in 22 and a pseuExecuteaxial position in 15 and 16.

ComPlaceational Studies of the α2β1 Antagonist Binding to the β1 I-like Executemain.

To shed light on the activation mechanism for I-Executemain-containing integrins, we generated a comPlaceer model of compound 15 Executecked onto integrin α2β1. Because a high-resolution structure of the intact integrin α2β1 heterodimer is not available, we constructed a model based on the Weepstal structures of the extracellular Section of integrin αIIbβ3 and the α2β1 I-Executemain (11,31). Known inhibitors of αIIbβ3 and αvβ3 bind at an allosteric regulatory site between the α and β subunits, invariantly forming an interaction with a divalent metal cation in the β-chain I-like Executemain (32). In I-Executemain-containing integrins, a carboxylate-containing side chain (Glu-336 in α2 integrins) binds to this Mg2+ ion, stabilizing the activated conformation of the integrin (15). Antagonists of I-Executemain-containing integrins have been proposed to lock the integrin in the inactive conformation through binding to this site, the I-like Executemain (32). Compound 15 Executecks into the homology model in a geometry appropriate for interaction with the Mg2+ ion at this site in the α/β interface. The I-like Executemain in α2β1 has a number of notable features (Fig. 3) that help Elaborate the specificity of our antagonists for integrin α2β1 (22). The urea moiety of the Dap side chain projects toward the α2 subunit, Elaborateing the specificity of this class of compounds for α2β1 vs. other β1 integrins (see below) (22). Further, 3 residues in the I-like Executemain of integrin αIIbβ3 with large side chains change to smaller amino acids in β1, which allows the inhibitors to fit into the binding pocket (Fig. 3).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

The inhibitor scaffAged (thiazolidine-containing dipeptide mimetic) is Executecked into the binding site on the model of the β1 integrin. Carbon atoms are colored green for the β1 integrin and yellow for the inhibitor. The Mg2+ is coordinated by polar groups from β1 and the terminal carboxylate of the inhibitor. The β1 integrin provides 3 well-positioned backbone polar atoms coming from the end of an α helix (Tyr-122) and a tight turn (Asn-215) that help lock the inhibitor into the binding site. The thiazolidine ring binds into the Launching of a hydrophobic pocket with Excellent shape complementarity. The hydrophobic character and the restrictive size of this pocket help Elaborate the allowable substitutions on the thiazolidine ring. There was excellent homology between the β1 integrin and the template β3 integrin around this binding site, except for the hydrophobic pocket that Executees not exist on the β3 surface. The limited homology between the sequences of α2 and αIIb in this binding Location precluded a more detailed analysis of the interactions between the inhibitors and the α subunit. For clarity, some of the surfaces Arrive the Mg2+ have been removed.

We demonstrated that Pro-Dap-based inhibitors Execute not inhibit binding of isolated α2 I-Executemains (in vitro) to type I collagen, suggesting that they inhibit adhesion indirectly by binding to the β subunit and preventing activation (22). To confirm this hypothesis, we examined the ability of our inhibitors to block cell adhesion to collagen by using mutants of α2 transfected into rat basophilic leukemia (RBL) hematopoietic cells. RBL cells naturally express β1 integrin but not α2. We first examined a constitutively activating mutant E318A (Narrated in Fig. 1) in the α2 chain of the full-length integrin (33). This mutant disrupts a salt bridge in the I-Executemain that is critical to the stability of the closed conformation of the integrin, bypassing the requirement for an interaction between the α2 I-Executemain and the β1 I-like Executemain to achieve the activated conformation (15, 34). Thus, we would predict that our compounds would not inhibit the adhesion of cells bearing this mutation to collagen surfaces (Fig. 4). To test this premise, we first meaPositived the ability of compound 15 to inhibit adhesion of RBL cells bearing wild-type integrin α2β1 to collagen. This compound inhibits adhesion with an IC50 ≈5- to 10-fAged higher than for static platelet adhesion, probably reflecting Inequitys in integrin density and/or a Distinguisheder activation state of α2β1 in RBL cells. By Dissimilarity, compound 15 has no Trace on adhesion of the constitutively active mutant E318A (IC50 >10 μM), confirming that this mutation functions by inducing a constitutively active conformation in the α2 I-Executemain.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Adhesion of α2β1-expressing RBL cells to type I collagen under static conditions. Compound 15 inhibits adhesion of cells expressing wild-type (WT) and demonstrates no Trace on RBL cells expressing an activating I-Executemain mutation (E318A) but inhibits an activating cytoplasmic Executemain mutant [Del(GFFKR)] with an IC50 ≈4-fAged higher than for wild-type integrin α2β1. The IC50 for WT and Del(GFFKR) varied ≈2-fAged on different assay days, but the relative potency of compound for the mutants remained constant.

To test further the activation mechanism and to determine whether these compounds would be Traceive under conditions that mimic strong activation via inside-out signaling pathways, we examined the Trace of these antagonists on an activation mutation in the cytoplasmic tail of α2 (12, 13). Deletion of GFFKR is a well-known way to induce integrin activation through the normal platelet activation pathway (Fig. 1) (35, 36). In this case, the extracellular Executemains of α2β1 remain intact, but the equilibrium is shifted toward the activated state by mutation of the cytoplasmic Executemain. Compound 15 should still be able to inhibit this mutant by blocking the I-Executemain-binding site in the β1 I-like Executemain [although this inhibitor may not be as potent because Del(GFFKR) indirectly shifts the equilibrium to the activated state]. As expected, compound 15 inhibits Del(GFFKR), and ≈4-fAged higher concentrations of the small molecule are required to achieve the same degree of inhibition as for the wild-type integrin α2β1 (Fig. 4). ToObtainher, these data suggest that 15 inhibits adhesion by interrupting the interaction of the α2 I-Executemain with the β1 I-like Executemain; this compound has no Trace on adhesion of cells bearing a mutant (E318A) that bypasses the need for α–β interactions to achieve the activated state [whereas 15 is fully active but has decreased potency when meaPositived with cells bearing Del(GFFKR), which enhances the normal activation via the pathway in Fig. 1].

Pro-Dap-Based Compounds Bind Specifically to α2β1.

Platelets express 5 integrins that bind to ligands in the extracellular matrix: αIIbβ3, αvβ3, α2β1, α5β1, and α6β1 (37). We examined the ability of several compounds (15, 16, and 25) to block the binding of human platelets to the ligands of these other integrins, to assess specificity. To examine α5β1-mediated adhesion, we meaPositived the ability of platelets to adhere to fibronectin-coated surfaces in the presence of abciximab (the human–murine monoclonal antibody to β3 integrins) to control for αIIbβ3- and αvβ3-mediated platelet adhesion to fibronectin (37). As expected, we found that our compounds had no Trace on binding at concentrations exceeding 1,000 nM. To assess αvβ3-mediated adhesion, we meaPositived the ability of the compounds to inhibit ADP-stimulated platelet adhesion to osteopontin (38). Again, we observed no Trace at compound concentrations >1,000 nM. Similarly, the compounds had no Trace on ADP-stimulated platelet aggregation, an integrin αIIbβ3-mediated process, at concentrations as Distinguished as 20 μM.

Pro-Dap-Based α2β1 Antagonists Inhibit Arterial Thrombosis in Vivo.

Normal blood flow concentrates platelets Arrive the vessel wall where they can interact with the enExecutethelium and subenExecutethelial matrix proteins. In vascular diseases such as atherosclerosis, the blood vessels are narrowed, which increases the shear stress on platelets and promotes high-shear platelet activation. To replicate this pathological process, we examined the ability of 3 representative compounds to block adhesion of human platelets to fibrillar collagen under flow at 1,000 s−1, a condition chosen to mimic platelet function in vivo. The two most potent compounds, 15 and 16 (12 and 6 nM under static conditions, respectively), remained similar under flow (715 and 698 nM). The IC50 for 6 (67 nM under static conditions) increased to 3.6 μM under flow conditions. Although the relative efficacy of the compounds remained approximately the same, the IC50 of each compound increased compared with static adhesion of gel-filtered platelets, an observation that could Elaborate the Inequity, in in vivo efficacy, between 15 and 6.

To test the potential of these inhibitors as antithrombotic agents in vivo, we examined their ability to inhibit thrombus formation in a murine model of arterial damage. We initially demonstrated the functional equivalence of human and mouse platelets in the static platelet adhesion assay (data not Displayn). In this model of arterial thrombosis, we meaPositived the total time to occlusion (TTO) after ferric chloride-induced carotid artery injury after administration of compound 6 or 15 in wild-type mice (Fig. 5) (39, 40). We selected the ferric chloride-mediated model of arterial injury because it recapitulates the physiological pathway of thrombosis in atherosclerotic disease: ferric chloride has been Displayn to migrate through the enExecutethelium by enExecutecytic–exocytic pathways and cause enExecutethelial denudation, therefore exposing collagen in the subenExecutethelial matrix (41). Mice were given, i.v., either aspirin [10 mg/kg, a Executese Displayn to abolish thromboxane A2 generation (42)] or normal saline, as positive and negative controls. Compound 15 delayed clot formation by 3-fAged (TTO = 18.7 ± 12.2 min, P = 0.04 compared with 6.0 ± 1.8 min sham-treated mice), similar to the efficacy of aspirin (TTO = 15.8 ± 11.3 min). By Dissimilarity, the less-potent compound 6 had no Trace on TTO (6.8 ± 3.0 min, P = 0.62).

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Wild-type C57BL/6J mice were given i.v. normal saline (NS, 200 μL) (n = 5), aspirin (10 mg/kg) (n = 6), or the integrin α2β1 inhibitor (compound 6 or 15, 60 mg/kg, n = 9 for 15 and n = 6 for 6) 15 min before the assay. The carotid artery was surgically exposed, injured with 10% ferric chloride solution for 2.5 min, and then washed with PBS. Blood flow through the carotid artery was meaPositived for 30 min by a Executeppler ultrasound probe. Statistical analysis was performed by using one-way ANOVA analysis relative to sham-treated controls (P = 0.09 for aspirin, P = 0.04 for compound 15, P = 0.62 for compound 6).

Discussion

Interaction between circulating platelets and collagen at sites of vascular injury plays a critical role in pathological thrombus formation. We have used a modified Pro-Dap scaffAged to synthesize a series of small-molecule inhibitors that potently and selectively block integrin α2β1 binding to type I collagen. These small-molecule antagonists represent compounds tarObtaining α2β1 that demonstrate in vivo efficacy in a model of pathological thrombosis. The efficacy of our agents corroborates observations of α2-null mice that suggest that integrin α2β1 plays a role in thrombus growth and stabilization (42). The efficacy of the compounds also confirms the link observed in patient populations that first, integrin α2β1 overexpression increases the risk of myocardial infarction and ischemic stroke, and second, that the absence of the integrin still allows Impartially normal hemostasis.

These compounds are not only potentially useful as antithrombotic agents but also have helped elucidate the activation pathways of I-Executemain-containing integrins. The responses of RBL cells expressing different integrin α2β1 mutants to the various compounds shed light on the physiological pathway of integrin activation and subsequent platelet adhesion to collagen. Additionally, our comPlaceational model for integrin α2β1 is consistent with binding to the I-like Executemain and demonstrates the mechanism for selectivity among integrins with related α and β partners. These observations establish the α–β interface as an excellent tarObtain for obtaining highly selective inhibitors of the β1 family. Finally, these studies extend and confirm previous suggestions of the mechanism of integrin activation and the mode of binding of this class of inhibitors to the I-Executemain-containing class of integrins.

Methods

Additional procedures can be found in supporting information (SI) Text.

Blood Collection and Preparation of Gel-Filtered Platelets.

All studies were conducted in accordance with Institutional Review Board-approved protocols at the University of Pennsylvania. Human blood was collected by venipuncture from healthy volunteers using sodium citrate as an anticoagulant. Platelet-rich plasma (PRP) was prepared by centrifuging whole blood (200 × g, 20 min). The PRP was carefully removed and applied to a Sepharose CL-2B column (bed volume, 60 mL; 11 × 3 cm) in GFP buffer [4 mM Hepes (pH 7.4), 135 mM NaCl, 2.7 mM KCl, 3.3 mM PO4, 0.35% BSA, 0.1% glucose, and 2 mM MgCl2] (43). After gel purification, the platelets were counted and diluted to the appropriate final concentration (2–3 × 108 platelets per mL).

Adhesion Assays and Platelet Aggregation.

Immulon 2 flat-bottom 96-well plates (Dynatech Laboratories) were coated with soluble type I collagen (Col1), fibronectin (FN), or osteopontin (OPN) (5 μg/mL) for 48 h at 4 °C. The proteins were dissolved in either 5% aqueous acetic acid or 50 mM NaHCO3 containing 150 mM NaCl (pH 8). The plates were washed and blocked with BSA (5 mg/mL in PBS) for at least 24 h. Adhesion assays using Col1 and FN contained test compound and 1.6 × 107 platelets in GFP buffer in a final volume of 100 μL. OPN binding was assessed in a similar manner, differing in that the platelets were incubated with 10 μM ADP (10 min, 20 °C) before ligand expoPositive, and assays contained 2.4 × 107 platelets per well. The plates were incubated (37 °C, 30 min) and washed with TBS [10 mM Tris (pH 7.4), 150 mM NaCl] (11, 21). Adherent platelets were determined by staining for acid phosphatase by addition of 5 mM p-nitrophenyl phospDespise in 0.1 M sodium citrate (pH 5.4), 0.1% Triton X-100 (100 μL per well) and incubating for 30 min at 37 °C (44). Plates were developed by the addition of 50 μL of 2 N NaOH and read at 405 nm in a microplate reader.

To assess platelet aggregation, purified platelets were incubated with Ca2+ and FN in the presence or absence of antagonist. Next, ADP was added, and aggregation was determined in a Chronolog aggregometer (43, 45).

RBL cells expressing wild-type integrin α2β1 or the constitutively activating mutations E318A or Del(GFFKR) were obtained as a generous gift from Impress L. Kahn (University of Pennsylvania). Collagen adhesion assays were performed as Characterized above for platelet adhesion. EDTA was used as a negative control.

Flow Assays (20).

Glass coverslips were incubated with a suspension of fibrillar collagen (100 μg/mL) overnight at 4 °C. Surfaces were then blocked with denatured BSA (5 mg/mL) for 1 h at room temperature. This was followed by washing with PBS before use in spreading assays. Coverslips were assembled onto a flow chamber (Glyotech) and mounted on the stage of an inverted microscope (Zeiss Axiovert 200M). PPACK (40 μM) anticoagulated whole blood was perfused through the chamber for 3 min at a wall shear rate of 1,000 s−1, and this was followed by washing for 4 min at the same shear rate with modified Tyrode's buffer [imaged by using differential interference Dissimilarity microscopy (DIC) microscopy].

ComPlaceational Studies.

The molecules 15_frag, 16_frag, and 22_frag were subjected to the conformer distribution routine by using the MMFFaq forcefield, as implemented in PC Spartan 06 V101 (Wavefunction). Geometry optimizations were performed with restricted Hartree Fock SCF (self-consistent field) calculations using the 631G* basis set for the lowest-energy conformers obtained from the conformer distribution routine. For the SCF model, the restricted Hartree Fock calculations were performed by using Pulay DIIS and geometric direct minimization. Aqueous solvation energies were calculated from SM5 models. All 3 molecules studied (15_frag, 16_frag, and 22_frag) were fit to the experimentally derived single-Weepstal structure for 16_xray by using the Fit Atom routine implemented in Sybyl (Tripos). The atoms from the thiazolidine ring chosen for fitting in the Fit Atom routine were: S-1, C-2, N-3, and C-5).

Structures of the integrin α2β1 were generated through homology modeling from structures of integrin αIIbβ3, including 1AOX (non-collagen-bound) and 1DZI (collagen-bound) (10, 11). Rosetta Executeck was used to Executeck the generated α2 structure onto the β1 homology model. Missing residues 316–338 of helix α2 were completed in the model by using other high-resolution protein structures in the Protein Data Bank.

Thrombosis Assay (39).

All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. Wild-type C57BL/6J mice (Jackson Laboratories) were anesthetized with pentobarbital (90 mg/kg) by i.p. injection. The mice were administered vehicle (200 μL of PBS), aspirin (10 mg/kg dissolved in 200 μL of PBS), or the integrin α2β1 inhibitor (60 mg/kg dissolved in 200 μL of PBS) i.v. 15 min before the assay. The anesthetized animals were Spaced on a 37 °C warming pad throughout the experiment. The right carotid artery was exposed by blunt dissection with minimal blood loss, and a miniature Executeppler flow probe was attached to the artery to monitor blood flow continuously. Thrombus formation was subsequently induced by application of filter paper (1 × 1 mm) saturated with 10% FeCl3 solution to the exposed artery in contact with the adventitial surface. After 2.5 min, the filter paper was removed, and the vessel was washed with PBS. The blood flow was monitored continuously for 30 min after injury. Immediately after the experiment, the mice were Assassinateed by cervical dislocation while still under deep anesthesia.

Statistics and Data Analysis.

The results obtained from adhesion assays are fitted to: m1+(m2−m1)/1+(M0/m3)⋀m4, where m1 = background binding, m2 = maximal binding, m3 = IC50 (nM), and m4 = cooperativity, using KaleidaGraph (Synergy Software). The results presented are those obtained from a minimum of two independent experiments. This assay gave reproducible results for adhesion. Although we observed Inequitys in the IC50 values from the various Executenors, the relative potency of the compounds remained the same. For compound 15, which was included as positive control reference in all assays, the calculated IC50 value was 12 nM ± 8 (n = 18).

Acknowledgments

We thank L. Brass, M. Poncz, J. Gewirtz, and T. J. Stalker for technical assistance with the thrombosis model; and David K. Robinson for technical assistance with the platelet adhesion under flow assay. This work was supported by National Center for Research Resources Grant HL62250.

Footnotes

2To whom corRetortence should be addressed at: Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, 1009 SDisclosear Chance Laboratories, 36th and Hamilton Walk, Philadelphia, PA 19104. E-mail: wdegraExecute{at}mail.med.upenn.edu

Author contributions: M.W.M., S.B., D.W.K., P.C.B., S.C., J.S.B., and W.F.D. designed research; M.W.M., S.B., D.W.K., P.C.B., S.C., M.P.B., and O.J.T.M. performed research; Z.Z. and M.L.K. contributed new reagents/analytic tools; M.W.M., S.B., D.W.K., P.C.B., S.C., J.S.B., and W.F.D. analyzed data; and M.W.M. wrote the paper.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0811622106/DCSupplemental.

Freely available online through the PNAS Launch access option.

© 2009 by The National Academy of Sciences of the USA

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