Obesity in Adults: A 2022 Adapted Clinical Practice Guideline for Ireland.

BACKGROUND: This Clinical Practice Guideline (CPG) for the management of obesity in adults in Ireland, adapted from the Canadian CPG, defines obesity as a complex chronic disease characterised by excess or dysfunctional adiposity that impairs health. The guideline reflects substantial advances in the understanding of the determinants, pathophysiology, assessment, and treatment of obesity. SUMMARY: It shifts the focus of obesity management toward improving patient-centred health outcomes, functional outcomes, and social and economic participation, rather than weight loss alone. It gives recommendations for care that are underpinned by evidence-based principles of chronic disease management; validate patients' lived experiences; move beyond simplistic approaches of "eat less, move more" and address the root drivers of obesity. KEY MESSAGES: People living with obesity face substantial bias and stigma, which contribute to increased morbidity and mortality independent of body weight. Education is needed for all healthcare professionals in Ireland to address the gap in skills, increase knowledge of evidence-based practice, and eliminate bias and stigma in healthcare settings. We call for people living with obesity in Ireland to have access to evidence-informed care, including medical, medical nutrition therapy, physical activity and physical rehabilitation interventions, psychological interventions, pharmacotherapy, and bariatric surgery. This can be best achieved by resourcing and fully implementing the Model of Care for the Management of Adult Overweight and Obesity. To address health inequalities, we also call for the inclusion of obesity in the Structured Chronic Disease Management Programme and for pharmacotherapy reimbursement, to ensure equal access to treatment based on health-need rather than ability to pay.

COVID-19 induces a hyperactive phenotype in circulating platelets.

Coronavirus Disease 2019 (COVID-19), caused by the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has affected over 30 million globally to date. Although high rates of venous thromboembolism and evidence of COVID-19-induced endothelial dysfunction have been reported, the precise aetiology of the increased thrombotic risk associated with COVID-19 infection remains to be fully elucidated. Therefore, we assessed clinical platelet parameters and circulating platelet activity in patients with severe and nonsevere COVID-19. An assessment of clinical blood parameters in patients with severe COVID-19 disease (requiring intensive care), patients with nonsevere disease (not requiring intensive care), general medical in-patients without COVID-19, and healthy donors was undertaken. Platelet function and activity were also assessed by secretion and specific marker analysis. We demonstrated that routine clinical blood parameters including increased mean platelet volume (MPV) and decreased platelet:neutrophil ratio are associated with disease severity in COVID-19 upon hospitalisation and intensive care unit (ICU) admission. Strikingly, agonist-induced ADP release was 30- to 90-fold higher in COVID-19 patients compared with hospitalised controls and circulating levels of platelet factor 4 (PF4), soluble P-selectin (sP-selectin), and thrombopoietin (TPO) were also significantly elevated in COVID-19. This study shows that distinct differences exist in routine full blood count and other clinical laboratory parameters between patients with severe and nonsevere COVID-19. Moreover, we have determined all COVID-19 patients possess hyperactive circulating platelets. These data suggest abnormal platelet reactivity may contribute to hypercoagulability in COVID-19 and confirms the role that platelets/clotting has in determining the severity of the disease and the complexity of the recovery path.

Computational and experimental analysis of bioactive peptide linear motifs in the integrin adhesome.

Therapeutic modulation of protein interactions is challenging, but short linear motifs (SLiMs) represent potential targets. Focal adhesions play a central role in adhesion by linking cells to the extracellular matrix. Integrins are central to this process, and many other intracellular proteins are components of the integrin adhesome. We applied a peptide network targeting approach to explore the intracellular modulation of integrin function in platelets. Firstly, we computed a platelet-relevant integrin adhesome, inferred via homology of known platelet proteins to adhesome components. We then computationally selected peptides from the set of platelet integrin adhesome cytoplasmic and membrane adjacent protein-protein interfaces. Motifs of interest in the intracellular component of the platelet integrin adhesome were identified using a predictor of SLiMs based on analysis of protein primary amino acid sequences (SLiMPred), a predictor of strongly conserved motifs within disordered protein regions (SLiMPrints), and information from the literature regarding protein interactions in the complex. We then synthesized peptides incorporating these motifs combined with cell penetrating factors (tat peptide and palmitylation for cytoplasmic and membrane proteins respectively). We tested for the platelet activating effects of the peptides, as well as their abilities to inhibit activation. Bioactivity testing revealed a number of peptides that modulated platelet function, including those derived from α-actinin (ACTN1) and syndecan (SDC4), binding to vinculin and syntenin respectively. Both chimeric peptide experiments and peptide combination experiments failed to identify strong effects, perhaps characterizing the adhesome as relatively robust against within-adhesome synergistic perturbation. We investigated in more detail peptides targeting vinculin. Combined experimental and computational evidence suggested a model in which the positively charged tat-derived cell penetrating part of the peptide contributes to bioactivity via stabilizing charge interactions with a region of the ACTN1 negatively charged surface. We conclude that some interactions in the integrin adhesome appear to be capable of modulation by short peptides, and may aid in the identification and characterization of target sites within the complex that may be useful for therapeutic modulation.

Fibrinogen Motif Discriminates Platelet and Cell Capture in Peptide-Modified Gold Micropore Arrays.

Human blood platelets and SK-N-AS neuroblastoma cancer-cell capture at spontaneously adsorbed monolayers of fibrinogen-binding motifs, GRGDS (generic integrin adhesion), HHLGGAKQAGDV (exclusive to platelet integrin αIIbß3), or octanethiol (adhesion inhibitor) at planar gold and ordered 1.6 µm diameter spherical cap gold cavity arrays were compared. In all cases, arginine/glycine/aspartic acid (RGD) promoted capture, whereas alkanethiol monolayers inhibited adhesion. Conversely only platelets adhered to alanine/glycine/aspartic acid (AGD)-modified surfaces, indicating that the AGD motif is recognized preferentially by the platelet-specific integrin, αIIbß3. Microstructuring of the surface effectively eliminated nonspecific platelet/cell adsorption and dramatically enhanced capture compared to RGD/AGD-modified planar surfaces. In all cases, adhesion was reversible. Platelets and cells underwent morphological change on capture, the extent of which depended on the topography of the underlying substrate. This work demonstrates that both the nature of the modified interface and its underlying topography influence the capture of cancer cells and platelets. These insights may be useful in developing cell-based cancer diagnostics as well as in identifying strategies for the disruption of platelet cloaks around circulating tumor cells.

The Role of Platelet-Derived ADP and ATP in Promoting Pancreatic Cancer Cell Survival and Gemcitabine Resistance.

Platelets have been demonstrated to be vital in cancer epithelial-mesenchymal transition (EMT), an important step in metastasis. Markers of EMT are associated with chemotherapy resistance. However, the association between the development of chemoresistance, EMT, and the contribution of platelets to the process, is still unclear. Here we report that platelets regulate the expression of (1) human equilibrative nucleoside transporter 1 (hENT1) and (2) cytidine deaminase (CDD), markers of gemcitabine resistance in pancreatic cancer. Human ENT1 (hENT1) is known to enable cellular uptake of gemcitabine while CDD deactivates gemcitabine. Knockdown experiments demonstrate that Slug, a mesenchymal transcriptional factor known to be upregulated during EMT, regulates the expression of hENT1 and CDD. Furthermore, we demonstrate that platelet-derived ADP and ATP regulate Slug and CDD expression in pancreatic cancer cells. Finally, we demonstrate that pancreatic cancer cells express the purinergic receptor P2Y12, an ADP receptor found mainly on platelets. Thus ticagrelor, a P2Y12 inhibitor, was used to examine the potential therapeutic effect of an ADP receptor antagonist on cancer cells. Our data indicate that ticagrelor negated the survival signals initiated in cancer cells by platelet-derived ADP and ATP. In conclusion, our results demonstrate a novel role of platelets in modulating chemoresistance in pancreatic cancer. Moreover, we propose ADP/ATP receptors as additional potential drug targets for treatment of pancreatic cancer.

Peptide-Mediated Platelet Capture at Gold Micropore Arrays.

Ordered spherical cap gold cavity arrays with 5.4, 1.6, and 0.98 µm diameter apertures were explored as capture surfaces for human blood platelets to investigate the impact of surface geometry and chemical modification on platelet capture efficiency and their potential as platforms for surface enhanced Raman spectroscopy of single platelets. The substrates were chemically modified with single-constituent self-assembled monolayers (SAM) or mixed SAMs comprised of thiol-functionalized arginine-glycine-aspartic acid (RGD, a platelet integrin target) with or without 1-octanethiol (adhesion inhibitor). As expected, platelet adhesion was promoted and inhibited at RGD and alkanethiol modified surfaces, respectively. Platelet adhesion was reversible, and binding efficiency at the peptide modified substrates correlated inversely with pore diameter. Captured platelets underwent morphological change on capture, the extent of which depended on the topology of the underlying substrate. Regioselective capture of the platelets enabled study for the first time of the surface enhanced Raman spectroscopy of single blood platelets, yielding high quality Raman spectroscopy of individual platelets at 1.6 µm diameter pore arrays. Given the medical importance of blood platelets across a range of diseases from cancer to psychiatric illness, such approaches to platelet capture may provide a useful route to Raman spectroscopy for platelet related diagnostics.

Cell Adhesion Molecules: Therapeutic Targets for Inhibition of Inflammatory States.

The diversity of integrins and their complex role in many diseases suggests great potential for this superfamily as drug targets. The initial successes of anti-integrin therapeutics in the treatment of thrombotic disorders suggested that similar anti-integrin agents could be developed for the treatment of inflammatory disorders. While initially a promising strategy, inhibition of the integrins proved to be elusive despite the discovery of highly potent inhibitors. This is due to several reasons, including redundancy among the integrins and the importance of integrins in key physiological systems. Further exploration of the selective role for distinct leukocytic integrins indicated that homing of inflammatory cells to select disease sites depends on a highly regulated expression of discrete integrins and their ligands in limited locations. Selective control of integrin function is also regulated by local chemokines permitting exquisite homing of populations of inflammatory cells to disease sites. A more complete understanding of the regulation of integrin activation in disease states will permit the development of more effective and specific anti-integrin therapeutic agents.

Poly(Ethylene glycol)-based backbones with high peptide loading capacities.

Polymer-peptide conjugates are a promising class of compounds, where polymers can be used to overcome some of the limitations associated with peptides intended for therapeutic and/or diagnostic applications. Linear polymers such as poly(ethylene glycol) can be conjugated through terminal moieties and have therefore limited loading capacities. In this research, functionalised linear poly(ethylene glycol)s are utilised for peptide conjugation, to increase their potential loading capacities. These poly(ethylene glycol) derivatives are conjugated to peptide sequences containing representative side-chain functionalised amino acids, using different conjugation chemistries, including copper-catalysed azide-alkyne cycloaddition, amide coupling and thiol-ene reactions. Conjugation of a sequence containing the RGD motif to poly(allyl glycidyl ether) by the thiol-ene reaction, provided a conjugate which could be used in platelet adhesion studies.

Cadherin juxtamembrane region derived peptides inhibit TGFß1 induced gene expression.

Bioactive peptides in the juxtamembrane regions of proteins are involved in many signaling events. The juxtamembrane regions of cadherins were examined for the identification of bioactive regions. Several peptides spanning the cytoplasmic juxtamembrane regions of E- and N-cadherin were synthesized and assessed for the ability to influence TGFß responses in epithelial cells at the gene expression and protein levels. Peptides from regions closer to the membrane appeared more potent inhibitors of TGFß signaling, blocking Smad3 phosphorylation. Thus inhibiting nuclear translocation of phosphorylated Smad complexes and subsequent transcriptional activation of TGFß signal propagating genes. The peptides demonstrated a peptide-specific potential to inhibit other TGFß superfamily members, such as BMP4.

RGD labeled Ru(II) polypyridyl conjugates for platelet integrin αIIbß3 recognition and as reporters of integrin conformation.

The ability of two novel ruthenium(II) polypyridyl-Arg-Gly-Asp (RGD) peptide conjugates to act as molecular probes for reporting on the presence and conformation of integrin αIIbß3 in solution and in live cells was described. The compounds are [Ru(bpy)2PIC-RGD](2+), bpy-RGD, and [Ru(dpp)2PIC-RGD](2+), dpp-RGD, where dpp is 4,7-diphenyl-1,10-phenanthroline, bpy is 2,2'-bipyridine, and PIC is 2-(4-carboxyphenyl)imidazo[4,5-f][1,10]phenanthroline. Bpy-RGD is hydrophilic, whereas dpp-RGD is comparatively hydrophobic. Both probes exhibited good affinity and high specificity for purified αIIbß3 in solution. Binding of either complex to the resting integrin resulted in an approximately 8-fold increase of emission intensity from the metal center with dissociation constants (Kd) in the micromolar range for each complex. The Kd for each conjugate/αIIbß3 assembly were compared following treatment of the integrin with the activating agents, Mn(2+) and dithiothreitol (DTT), which are commonly used to induce active-like conformational changes in the integrin. For bpy-RGD/αIIbß3 Kd showed relatively little variation with integrin activation, presenting the following trend: denatured αIIbß3 > resting αIIbß3 = pretreated DTT = pretreated Mn(2+). Kd for dpp-RGD/ αIIbß3 showed greater variation with integrin activation and the following trend was followed: denatured αIIbß3 > resting αIIbß3 > pretreated Mn(2+) = pretreated DTT. Time resolved luminescence anisotropy was carried out to obtain the rotational correlation time of bpy-RGD and dpp-RGD bound to resting or nominally activated integrin. The rotational correlation times of bpy-RGD and dpp-RGD, too fast to measure unbound, decreased to 1.50 ± 0.03 µs and 2.58 ± 0.04 µs, respectively, when the complexes were bound to resting integrin. Addition of Mn(2+) to bpy-RGD/αIIbß3 or dpp-RGD/αIIbß3 reduced the rotational correlation time of the ruthenium center to 1.29 ± 0.03 µs and to 1.72 ± 0.03 µs, respectively. Following treatment, the rotational correlation time decreased to 1.04 ± 0.01 µs and 1.29 ± 0.03 µs for bpy-RGD/αIIbß3, and dpp-RGD/αIIbß3, respectively. The large relative changes in rotational correlation times observed for Mn(2+) or DTT activated integrin indicates significant change in protein conformation compared with the resting integrin. The results also indicated that the metal complex itself affects the final conformational and/or aggregation status of the protein obtained. Furthermore, the extent of conformational change was influenced by whether the probe was bound to the integrin before or after activator treatment. Finally, in vitro studies indicated that both probes selectively bind to CHO cells expressing the resting form of αIIbß3. In each case the probe colocalized with αIIb specific SZ22 antibody. Overall, this work indicates that bpy-RGD and dpp-RGD may be useful peptide-probes for rapid assessment of integrin structural status and localization in solution and cells.

A novel and essential role for FcγRIIa in cancer cell-induced platelet activation.

Platelets play a role in cancer by acting as a dynamic reservoir of effectors that facilitate tumor vascularization, growth, and metastasis. However, little information is available about the mechanism of tumor cell-induced platelet secretion (TCIPS) or the molecular machinery by which effector molecules are released from platelets. Here we demonstrate that tumor cells directly induce platelet secretion. Preincubation of platelets with human colon cancer (Caco-2), prostate cancer (PC3M-luc), or breast cancer cells (MDA-MB-231;MCF-7) resulted in a marked dose-dependent secretion of dense granules. Importantly, TCIPS preceded aggregation which always displayed a characteristic lag time. We investigated the role of platelet receptors and downstream molecules in TCIPS. The most potent modulators of TCIPS were the pharmacologic antagonists of Syk kinase, phospholipase C and protein kinase C, all downstream mediators of the immunoreceptor tyrosine-based activation motif (ITAM) cascade in platelets. Supporting this, we demonstrated a central role for the immune Fcγ receptor IIa (FcγRIIa) in mediating platelet-tumor cell cross-talk. In conclusion, we demonstrate that cancer cells can promote platelet dense-granule secretion, which is required to augment platelet aggregation. In addition, we show a novel essential role for FcγRIIa in prostate cancer cell-induced platelet activation opening the opportunity to develop novel antimetastatic therapies.

Regulation of platelet activity in a changing redox environment.

SIGNIFICANCE: The regulation of platelet function is finely tuned by a balance between the vasculature's redox environment and the oxidative processes that occur in it. The activation of platelets at sites of vascular damage is essential for the maintenance of normal hemostasis. In the extracellular milieu, a normal redox environment is maintained by thiol/disulfide redox couples, which include reduced and oxidized glutathione (GSH/GSSG) and cysteine (Cys/CySS). Oxidative changes in either of the plasma redox potentials are directly linked with risk factors for cardiovascular disease. RECENT ADVANCES: Many proteins found on the surface of platelets contain cysteine residues that are targets for oxidation. These include platelet-specific integrins and thiol isomerase enzymes that respond to changes in the extracellular redox environment, thus influencing normal platelet responses. CRITICAL ISSUES: The post-translational modification of critical cysteine thiol groups is linked to alterations in redox potentials and occurs both intracellularly and extracellularly in normal platelet activation. Platelet integrins, in particular, are prime targets for redox modification due to their high cysteine content. Although the role of thiol/disulfide bond exchange in platelet activation is established, the effects of a changing redox environment on platelet reactivity are unclear. FUTURE DIRECTIONS: A thorough understanding of these mechanisms and how they interact with other platelet signaling events is of the utmost importance for the development of novel therapeutic targets so that we can protect against inappropriate thrombus formation.

Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase A oncogene.

BACKGROUND: Protein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. RESULTS: As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA's primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. CONCLUSIONS: PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

Platelet signalling networks: pathway perturbation demonstrates differential sensitivity of ADP secretion and fibrinogen binding.

Platelet signalling responses to single agonists have been identified previously. However, a model of the total platelet signalling network is still lacking. In order to gain insights into this network, we explored the effects of a range of platelet-function inhibitors in two independent assays of platelet function, namely fibrinogen binding and ADP secretion. In this study, we targeted the intracellular signalling molecules targeted intracellular signalling molecules, Syk and PI3K and targeted intracellular signalling molecules, Syk and PI3K, the prostaglandin synthesis enzyme cyclooxygenase, surface receptors for TxA(2) and ADP (P2Y1 and P2Y12) and the integrin cell adhesion molecule, αIIbß3. We demonstrate that the platelet responses of fibrinogen binding and secretion can be differentially affected by the individual inhibitors permitting the generation of a model delineating novel regulatory links in the platelet signalling network. Importantly, the model illustrates the interconnections among portions that are traditionally studied as separate modules, promoting a more integrated view of the platelet.

Integrin α(IIb)ß3 exists in an activated state in subjects with elevated plasma homocysteine levels.

Elevated levels of plasma homocysteine (Hcy) are an independent risk factor for cardiovascular disease and thrombosis. The molecular basis for this phenomenon is not known but may relate to modification of cell surface thiols. The platelet specific integrin α(IIb)ß3 is a cysteine-rich cell adhesion molecule that plays a critical role in platelet aggregation and adhesion in haemostasis and thrombosis. In this study, we looked for evidence of a homocysteine-induced modification of α(IIb)ß3 using a fluorescently labeled PAC-1 antibody that recognizes the activated conformation of the integrin on the platelet surface. We show that exogenous Hcy (10-100 µM) and homocysteine thiolactone (HcyTL) (10-100 µM) increased PAC-1 binding to platelets in a concentration dependent manner in vitro. In parallel, we show subjects with clinical hyperhomocysteinemia exhibit a greater degree of activation of α(IIb)ß3 compared to age-matched controls. These findings demonstrate that circulating Hcy can modulate the activation state of the platelet integrin α(IIb)ß3, a key player in platelet aggregation and thrombosis.

Integrins as therapeutic targets: lessons and opportunities.

The integrins are a large family of cell adhesion molecules that are essential for the regulation of cell growth and function. The identification of key roles for integrins in a diverse range of diseases, including cancer, infection, thrombosis and autoimmune disorders, has revealed their substantial potential as therapeutic targets. However, so far, pharmacological inhibitors for only three integrins have received marketing approval. This article discusses the structure and function of integrins, their roles in disease and the chequered history of the approved integrin antagonists. Recent advances in the understanding of integrin function, ligand interaction and signalling pathways suggest novel strategies for inhibiting integrin function that could help harness their full potential as therapeutic targets.

Protein interactions with the platelet integrin alpha(IIb) regulatory motif.

Integrins are transmembrane proteins regulating cellular shape, mobility and the cell cycle. A highly conserved signature motif in the cytoplasmic tail of the integrin alpha-subunit, KXGFFKR, plays a critical role in regulating integrin function. To date, six proteins have been identified that target this motif of the platelet-specific integrin alpha(IIb)beta(3). We employ peptide-affinity chromatography followed-up with LC-MS/MS analysis as well as protein chips to identify new potential regulators of integrin function in platelets and put them into their biological context using information from protein:protein interaction (PPI) databases. Totally, 44 platelet proteins bind with high affinity to an immobilized LAMWKVGFFKR-peptide. Of these, seven have been reported in the PPI literature as interactors with integrin alpha-subunits. 68 recombinant human proteins expressed on the protein chip specifically bind with high affinity to biotin-tagged alpha-integrin cytoplasmic peptides. Two of these proteins are also identified in the peptide-affinity experiments, one is also found in the PPI databases and a further one is present in the data to all three approaches. Finally, novel short linear interaction motifs are common to a number of proteins identified.

Ligand switching in cell-permeable peptides: manipulation of the alpha-integrin signature motif.

A synthetic cell-permeable peptide corresponding to the highly conserved alpha-integrin signature motif, Palmityl-K(989)VGFFKR(995) (Pal-FF), induces integrin activation and aggregation in human platelets. Systematic replacement of the F(992)-F(993) with amino acids of greater or lesser hydrophobicity to create Pal-KVGxxKR peptides demonstrate that hydrophobic amino acids (isoleucine, phenylalanine, tyrosine, tryptophan) are essential for agonist potency. In marked contrast, substitution with small and/or hydrophilic amino acids (glycine, alanine, serine) causes a switch in the biological activity resulting in inhibition of platelet aggregation, adhesion, ADP secretion, and thromboxane synthesis. These substituted, hydrophilic peptides are not true pharmacological antagonists, as they actively induce a phosphotyrosine signaling cascade in platelets. Singly substituted peptides (Pal-AF and Pal-FA) cause preferential retention of pro- or anti-thrombotic properties, respectively. Because the alpha-integrin signature motif is an established docking site for a number of diverse cytoplasmic proteins, we conclude that eliminating critical protein-protein interactions mediated through the hydrophobic amino acids, especially F(993), favors an anti-thrombotic pathway in platelets. Agents derived from the inhibitory peptides described in this study may represent a new therapeutic strategy for anti-platelet or anti-integrin drug development.

Ruthenium polypyridyl peptide conjugates: membrane permeable probes for cellular imaging.

Two novel polyarginine labelled ruthenium polypyridyl dyes are reported, one conjugated to five, (Ru-Ahx-R5), and one to eight arginine residues, (Ru-Ahx-R8); both complexes exhibit long-lived, intense, and oxygen-sensitive luminescence; (Ru-R8) is passively, efficiently and very rapidly transported across the cell membrane into the cytoplasm without requirement for its permeablisation.

A peptide affinity column for the identification of integrin alpha IIb-binding proteins.

To understand the regulation of integrin alpha(IIb)beta(3), a critical platelet adhesion molecule, we have developed a peptide affinity chromatography method using the known integrin regulatory motif, LAMWKVGFFKR. Using standard Fmoc chemistry, this peptide was synthesized onto a Toyopearl AF-Amino-650 M resin on a 6-aminohexanoic acid (Ahx) linker. Peptide density was controlled by acetylation of 83% of the Ahx amino groups. Four recombinant human proteins (CIB1, PP1, ICln and RN181), previously identified as binding to this integrin regulatory motif, were specifically retained by the column containing the integrin peptide but not by a column presenting an irrelevant peptide. Hemoglobin, creatine kinase, bovine serum albumin, fibrinogen and alpha-tubulin failed to bind under the chosen conditions. Immunodetection methods confirmed the binding of endogenous platelet proteins, including CIB1, PP1, ICln RN181, AUP-1 and beta3-integrin, from a detergent-free platelet lysate. Thus, we describe a reproducible method that facilitates the reliable extraction of specific integrin-binding proteins from complex biological matrices. This methodology may enable the sensitive and specific identification of proteins that interact with linear, membrane-proximal peptide motifs such as the integrin regulatory motif LAMWKVGFFKR.