Small molecule inhibitors of integrin alpha2beta1.

Interactions between the integrin, alpha2beta1, and extracellular matrix (ECM), particularly collagen, play a pivotal role in platelet adhesion and thrombus formation. Platelets interact with collagen in the subendothelial matrix that is exposed by vascular damage. To evaluate the potential of alpha2beta1 inhibitors for anticancer and antithrombotic applications, we have developed a series of small molecule inhibitors of this integrin based on a prolyl-2,3-diaminopropionic acid (DAP) scaffold using solid-phase parallel synthesis. A benzenesulfonamide substituent at the N-terminus of the dipepetide and a benzyl urea at the DAP side chain resulted in tight and highly selective inhibition of alpha2beta1-mediated adhesion of human platelets and other cells to collagen.

Interactions of ADP-stimulated human platelets with PEGylated polystyrene substrates prepared by surface amidation.

A study primarily focused on the interactions between ADP-stimulated human platelets and PEGylated polystyrene substrates is described in this paper. The platelet-surface interactions were investigated using colorimetric acid phosphatase assay. Two types of amine-containing polymeric hydrogel materials based on poly(ethylene glycol) (PEG), H(2)N-PEG-OCH(3) and H(2)N-PEG-NH(2), were used to PEGylate polystyrene surfaces derivatized with maleic anhydride by amidation at alkaline pH. In addition, comparative studies using surfaces non-covalently adsorbed by bovine serum albumin (BSA) or fibrinogen (Fg) were also conducted. The assay results showed that no significant platelet adhesion was observed when PEGylated surfaces or BSA-coated surfaces were exposed to unstimulated gel-filtered platelets (GFP). However, upon ADP-stimulation, platelet adhesion to the surfaces under investigation in this study all increased to varying degrees. Most importantly, the results showed that polystyrene surfaces PEGylated using H(2)N-PEG-NH(2) were most effective in resisting platelet adhesion when assays were performed using ADP-stimulated GFP. By PEGylating the surfaces of polystyrene microtiter wells via the amidation reaction described in this paper, it is demonstrated that (i) higher degree of surface PEGylation is favored at more alkaline pH and (ii) polystyrene substrates capable of more effectively resisting the adhesion of ADP-stimulated GFP can be obtained by the PEGylation reaction carried out at pH 9.1 using H(2)N-PEG-NH(2).

Computational design of peptides that target transmembrane helices.

A variety of methods exist for the design or selection of antibodies and other proteins that recognize the water-soluble regions of proteins; however, companion methods for targeting transmembrane (TM) regions are not available. Here, we describe a method for the computational design of peptides that target TM helices in a sequence-specific manner. To illustrate the method, peptides were designed that specifically recognize the TM helices of two closely related integrins (alphaIIbbeta3 and alphavbeta3) in micelles, bacterial membranes, and mammalian cells. These data show that sequence-specific recognition of helices in TM proteins can be achieved through optimization of the geometric complementarity of the target-host complex.

Activation of platelet alphaIIbbeta3 by an exogenous peptide corresponding to the transmembrane domain of alphaIIb.

A transmembrane domain heterodimer, acting in concert with a membrane-proximal cytoplasmic domain clasp, is thought to maintain integrins in a low affinity state. To test whether helix-helix interactions between the alphaIIb and beta3 transmembrane domains regulate the activity of integrin alphaIIbbeta3, we synthesized a soluble peptide corresponding to the alphaIIb transmembrane domain, designated alphaIIb-TM, and we studied its ability to affect alphaIIbbeta3 activity in human platelets. alphaIIb-TM was alpha-helical in detergent micelles and phospholipid vesicles, readily inserted into membrane bilayers, bound to intact purified alphaIIbbeta3, and specifically associated with the transmembrane domain of alphaIIb, rather than the transmembrane domains of beta3, alpha2, and beta1, other integrin subunits present in platelets. When added to suspensions of gel-filtered platelets, alphaIIb-TM rapidly induced platelet aggregation that was not inhibited by preincubating platelets with the prostaglandin E(1) or the ADP scavenger apyrase but was prevented by the divalent cation chelator EDTA. Furthermore, alphaIIb-TM induced fibrinogen binding to platelets but not the binding of osteopontin, a specific ligand for platelet alphavbeta3. The peptide also induced fibrinogen binding to recombinant alphaIIbbeta3 expressed by Chinese hamster ovary cells, confirming that its effect was independent of platelet signal transduction. Finally, transmission electron microscopy of purified alphaIIbbeta3 revealed that alphaIIb-TM shifted the integrin from a closed configuration with its stalks touching to an open configuration with separated stalks. These observations demonstrate that transmembrane domain interactions regulate integrin function in situ and that it is possible to target intra-membranous protein-protein interactions in a way that can have functional consequences.

Arylamide derivatives as allosteric inhibitors of the integrin alpha2beta1/type I collagen interaction.

We herein report a group of allosteric inhibitors of integrin alpha(2)beta(1) based on an arylamide scaffold. Compound 4 showed an IC(50) of 4.80 microM in disrupting integrin I-domain/collagen binding in an ELISA. These arylamide compounds are able to block collagen binding to integrin alpha(2)beta(1) on the platelet surface. Further we find that compound 4 recognizes a hydrophobic cleft on the side of the alpha(2) I-domain, suggesting an alternative targeting site for drug development.

Activation of individual alphaIIbbeta3 integrin molecules by disruption of transmembrane domain interactions in the absence of clustering.

We used laser tweezers-based force spectroscopy to measure the binding strength between fibrinogen molecules covalently bound to latex beads and either wild-type alphaIIbbeta3 molecules or alphaIIbbeta3 molecules containing the transmembrane domain mutations beta3 G708N or alphaIIb G972N expressed on Chinese hamster ovary cells. As we demonstrated previously for alphaIIbbeta3 on agonist-stimulated platelets and for purified alphaIIbbeta3 molecules incubated with Mn(2+), two regimes of rupture forces were present when wild-type alphaIIbbeta3 was activated by the monoclonal antibody PT25-2: rupture forces of 20-60 pN with an exponentially decreasing probability of detection and rupture forces in the range of 60-150 pN with a maximum at approximately 70-80 pN. Both rupture force regimes were specific for fibrinogen binding to the activated conformation of alphaIIbbeta3 because they were inhibited by alphaIIbbeta3-specific antagonists. Identical rupture force regimes were present constitutively when cells expressing the alphaIIb and beta3 transmembrane domain mutants were studied, confirming that these mutations induced an active alphaIIbbeta3 conformation. Moreover, there were no significant differences in the yield strength of the low-to-moderate and strong force regimes when alphaIIbbeta3 was activated by PT25-2 or the transmembrane domain mutations, implying that there was no fundamental difference in the way these forms of activated alphaIIbbeta3 interacted with fibrinogen. Thus, the two-step pathway of the interaction of alphaIIbbeta3 with fibrinogen we have identified appears to be a fundamental property of the high-affinity state of alphaIIbbeta3 and is identical regardless of whether this affinity state is achieved by intracellular, extracellular, or membrane-associated events.

Iron acquisition from transferrin by Candida albicans depends on the reductive pathway.

Host-pathogen interactions that alter virulence are influenced by critical nutrients such as iron. In humans, free iron is unavailable, being present only in high-affinity iron binding proteins such as transferrin. The fungal pathogen Candida albicans grows as a saprophyte on mucosal surfaces. Occasionally it invades systemically, and in this circumstance it will encounter transferrin iron. Here we report that C. albicans is able to acquire iron from transferrin. Iron-loaded transferrin restored growth to cultures arrested by iron deprivation, whereas apotransferrin was unable to promote growth. By using congenic strains, we have been able to show that iron uptake by C. albicans from transferrin was mediated by the reductive pathway (via FTR1). The genetically separate siderophore and heme uptake systems were not involved. FRE10 was required for a surface reductase activity and for efficient transferrin iron uptake activity in unbuffered medium. Other reductase genes were apparently up-regulated in medium buffered at pH 6.3 to 6.4, and the fre10(-/-) mutant had no effect under these conditions. Experiments in which transferrin was sequestered in a dialysis bag demonstrated that cell contact with the substrate was required for iron reduction and release. The requirement of FTR1 for virulence in a systemic infection model and its role in transferrin iron uptake raise the possibility that transferrin is a source of iron during systemic C. albicans infections.

Functional and structural correlations of individual alphaIIbbeta3 molecules.

The divalent cation Mn(2+) and the reducing agent dithiothreitol directly shift integrins from their inactive to their active states. We used transmission electron microscopy and laser tweezers-based force spectroscopy to determine whether structural rearrangements induced by these agents in the integrin alphaIIbbeta3 correlate with its ability to bind fibrinogen. Mn(2+) increased the probability of specific fibrinogen-alphaIIbbeta3 interactions nearly 20-fold in platelets, and both Mn(2+) and dithiothreitol increased the probability more than 2-fold using purified proteins. Of 3 alphaIIbbeta3 conformations, closed with stalks touching, open with stalks separated, and globular without visible stalks, Mn(2+) and dithiothreitol induced a significant increase in the proportion of open structures, as well as structural changes in the alphaIIbbeta3 headpiece. Mn(2+) also increased the number of complexes between fibrinogen and purified alphaIIbbeta3 molecules, all of which were in the open conformation. Finally, Mn(2+) induced the formation of alphaIIbbeta3 clusters that resulted from interactions exclusively involving the distal ends of the stalks. These results indicate that there is a direct correlation between alphaIIbbeta3 activation and the overall conformation of the molecule. Further, they are consistent with the presence of a linked equilibrium between single inactive and single active alphaIIbbeta3 molecules and active alphaIIbbeta3 clusters.

Pro-thrombotic state induced by post-translational modification of fibrinogen by reactive nitrogen species.

Formation of nitric oxide-derived oxidants has been linked to development of atherosclerosis and associated thrombotic complications. Although systemic levels of protein nitrotyrosine predict risk for coronary artery disease, neither specific proteins targeted for modification nor functional consequences that might contribute to disease pathogenesis have been defined. Here we report a selective increase in circulating levels of nitrated fibrinogen in patients with coronary artery disease. Exposure of fibrinogen to nitrating oxidants, including those produced by the myeloperoxidase-hydrogen peroxide-nitrite system, significantly accelerates clot formation and factor XIII cross-linking, whereas exposure of fibrinogen to non-nitrating oxidants decelerates clot formation. Clots formed with fibrinogen exposed to nitrating oxidants are composed of large bundles made from twisted thin fibrin fibers with increased permeation and a decrease in storage modulus G' value, suggesting that these clots could be easily deformed by mechanical stresses. In contrast, clots formed with fibrinogen exposed to non-nitrating oxidants showed decreased permeation with normal architecture. Fibrinogen modified by exposure to physiologic nitration systems demonstrated no difference in the rate of plasmin-induced clot lysis, platelet aggregation, or binding. Thus, increased levels of fibrinogen nitration may lead to a pro-thrombotic state via acceleration in formation of fibrin clots. The present results may account, in part, for the association between nitrative stress and risk for coronary artery disease.

Quantitative analysis of platelet alpha v beta 3 binding to osteopontin using laser tweezers.

To determine whether platelet adhesion to surfaces coated with the matrix protein osteopontin requires an agonist-induced increase in the affinity of the integrin alpha v beta 3 for this ligand, we used laser tweezers to measure the rupture force between single alpha v beta 3 molecules on the platelet surface and osteopontin-coated beads. Virtually all platelets stimulated with 10 microM ADP bound strongly to osteopontin, producing rupture forces as great as 100 piconewtons (pN) with a peak at 45-50 pN. By contrast, 90% of unstimulated, resting non-reactive platelets bound weakly to osteopontin, with rupture forces rarely exceeding 30-35 pN. However, approximately 10% of unstimulated platelets, resting reactive platelets, exhibited rupture force distributions similar to stimulated platelets. Moreover, ADP stimulation resulted in a 12-fold increase in the probability of detecting rupture forces >30 pN compared with resting non-reactive platelets. Pre-incubating stimulated platelets with the inhibitory prostaglandin E1, a cyclic RGD peptide, the monoclonal antibody abciximab, or the alpha v beta 3-specific cyclic peptide XJ735 returned force histograms to those of non-reactive platelets. These experiments demonstrate that ADP stimulation increases the strength of the interaction between platelet alpha v beta 3 and osteopontin. Furthermore, they indicate that platelet adhesion to osteopontin-coated surfaces requires an agonist-induced exposure of alpha v beta 3-binding sites for this ligand.

Activation of integrin alphaIIbbeta3 by modulation of transmembrane helix associations.

Transmembrane helices of integrin alpha and beta subunits have been implicated in the regulation of integrin activity. Two mutations, glycine-708 to asparagine-708 (G708N)and methionine-701 to asparagine-701, in the transmembrane helix of the beta3 subunit enabled integrin alphaIIbbeta3 to constitutively bind soluble fibrinogen. Further characterization of the G708N mutant revealed that it induced alphaIIbbeta3 clustering and constitutive phosphorylation of focal adhesion kinase. This mutation also enhanced the tendency of the transmembrane helix to form homotrimers. These results suggest that homomeric associations involving transmembrane domains provide a driving force for integrin activation. They also suggest a structural basis for the coincidence of integrin activation and clustering.

Platelet-derived microparticles stimulate proliferation, survival, adhesion, and chemotaxis of hematopoietic cells.

OBJECTIVE: Peripheral blood platelet-derived microparticles (PMPs) circulate in blood and may interact directly with target cells affecting their various biological functions. METHODS: To investigate the effect of human PMPs on hematopoiesis, we first phenotyped them for expression of various surface molecules and subsequently studied various biological responses of normal stem/progenitor (CD34(+)) and more differentiated precursor cells as well as several leukemic cell lines to PMPs. RESULTS: We found that, in addition to platelet-endothelium attachment receptors (CD41, CD61 and CD62), PMPs express G-protein-coupled seven transmembrane-span receptors such as CXCR4 and PAR-1; cytokine receptors including TNF-RI, TNF-RII, and CD95; and ligands such as CD40L and PF-4. Moreover, we found that several of these receptors could be transferred by PMPs to the membranes of normal as well as malignant cells and observed that PMPs: 1) chemoattract these cells, 2) increase their adhesion, proliferation, and survival, and 3) activate in these cells various intracellular signaling cascades including MAPK p42/44, PI-3K-AKT, and STAT proteins. The biological effects of PMPs were only partly reduced by heat inactivation or trypsin digest, indicating that, in addition to the protein components of PMPs, lipid components are also responsible for their biological activity. CONCLUSIONS: We conclude that PMPs modulate biological functions of hematopoietic cells and postulate that they play an important but as yet not fully understood role in intercellular cross-talk in hematopoiesis. Further studies, however, are needed to identify the PMP components that exert specific biological effects.