Differential binding of ICln in platelets to integrin-derived activating and inhibitory peptides

The capacity of platelets to form a thrombus is mediated by integrin α_IIbβ₃. The cytoplasmic tail of α_IIb contains a highly conserved motif, ⁹⁸⁹KVGFFKR⁹⁹⁵, which plays a critical role in regulating integrin activation and acts as a recognition site for various intracellular proteins, e.g. CIB1, PP1, ICln and RN181. Previously, we demonstrated that a cell-permeable integrin-derived activating (IDA) peptide, KVGFFKR, induces platelet activation, whereas an integrin-derived inhibitory (IDI) peptide, KVGAAKR, is antithrombotic. To elucidate the molecular mechanism underlying these opposite effects we investigate the affinity of known integrin α_IIb binding proteins for the two immobilized peptides in dependence on the activation state of platelets by means of peptide-affinity chromatography, blotting techniques and protein:peptide docking studies.Our results provide a model for the inhibition of ICln interaction with the integrin in activated platelets by the IDI-peptide. Thus, ICln:IDI-peptide interaction profiles can have a pivotal purpose in the search for consensus pharmacophores specifically inhibiting ICln function in platelets potentially leading to the development of integrin-derived antithrombotic drugs.     

Protein interactions with the platelet integrin αIIb 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 α‐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 α_IIbβ₃. 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 α‐subunits. 68 recombinant human proteins expressed on the protein chip specifically bind with high affinity to biotin‐tagged α‐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.     

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.     

ICln, a novel integrin alphaIIbbeta3-associated protein, functionally regulates platelet activation

A critical role for the conserved alpha-integrin cytoplasmic motif, KVGFFKR, is recognized in the regulation of activation of the platelet integrin alpha(IIb)beta(3). To understand the molecular mechanisms of this regulation, we sought to determine the nature of the protein interactions with this cytoplasmic motif. We used a tagged synthetic peptide, biotin-KVGFFKR, to probe a high density protein expression array (37,200 recombinant human proteins) for high affinity interactions. A number of potential integrin-binding proteins were identified. One such protein, a chloride channel regulatory protein, ICln, was characterized further because its affinity for the integrin peptide was highest as was its expression in platelets. We verified the presence of ICln in human platelets by PCR, Western blots, immunohistochemistry, and its co-association with alpha(IIb)beta(3) by surface plasmon resonance. The affinity of this interaction was 82.2 +/- 24.4 nm in a cell free assay. ICln co-immunoprecipitates with alpha(IIb)beta(3) in platelet lysates demonstrating that this interaction is physiologically relevant. Furthermore, immobilized KVGFFKR peptides, but not control KAAAAAR peptides, specifically extract ICln from platelet lysates. Acyclovir (100 microm to 5 mm), a pharmacological inhibitor of the ICln chloride channel, specifically inhibits integrin activation (PAC-1 expression) and platelet aggregation without affecting CD62 P expression confirming a specific role for ICln in integrin activation. In parallel, a cell-permeable peptide corresponding to the potential integrin-recognition domain on ICln (AKFEEE, 10-100 microm) also inhibits platelet function. Thus, we have identified, verified, and characterized a novel functional interaction between the platelet integrin and ICln, in the platelet membrane.     

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI‐II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin‐binding agents. We generated yeast‐displayed libraries of EETI‐II by substituting its 6‐amino acid trypsin binding loop with 11‐amino acid loops containing the Arg‐Gly‐Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high‐throughput manner by fluorescence‐activated cell sorting to identify mutants that bound to α_vβ₃ integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half‐maximal inhibitory concentration values of 10–30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both α_vβ₃ and α_vβ₅ integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to α_vβ₃, α_vβ₅, and α₅β₁ integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI‐II as a scaffold for protein engineering, and highlight the development of unique integrin‐binding peptides with potential for translational applications in cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Recent advances in the understanding of the molecular mechanisms regulating platelet integrin αIIbβ3 activation

Integrins are allosteric cell adhesion receptors that cycle from a low to a high affinity ligand binding state, a complex process of receptor activation that is of particular importance in blood cells such as platelets or leukocytes. Here we highlight recent progress in the understanding of the molecular pathways that regulate integrin activation in platelets and leukocytes, with a special focus on the structural changes in platelet integrin αIIbβ3 brought about by key intracellular proteins, namely talin and kindlins, that are of crucial importance in the regulation of integrin function. Evidence that the small GTPase Rap1 and its guanine exchange factor CalDAG-GEF1, together with RIAM, a Rap1GTP adaptor protein, promote the interaction of talin with the integrin β subunit, has greatly contributed to fill the gap in our understanding of the signaling pathway from G-coupled agonist receptors and their phospholipase C-dependant second messengers, to integrin activation. Studies of patients with the rare blood cell disorder LAD-III have contributed to the identification of kindlins as new co-regulators of the talin-dependent integrin activation process in platelets and leukocytes, underlining the relevance for the in-depth investigation of patients with rare genetic blood cell disorders.     

Mitochondrially Mediated Integrin αIIbβ3 Protein Inactivation Limits Thrombus Growth

When platelets are strongly stimulated, a procoagulant platelet subpopulation is formed that is characterized by phosphatidylserine (PS) exposure and epitope modulation of integrin α_IIbβ₃ or a loss of binding of activation-dependent antibodies. Mitochondrial permeability transition pore (mPTP) formation, which is essential for the formation of procoagulant platelets, is impaired in the absence of cyclophilin D (CypD). Here we investigate the mechanisms responsible for these procoagulant platelet-associated changes in integrin α_IIbβ₃ and the physiologic role of procoagulant platelet formation in the regulation of platelet aggregation. Among strongly stimulated adherent platelets, integrin α_IIbβ₃ epitope changes, mPTP formation, PS exposure, and platelet rounding were closely associated. Furthermore, platelet mPTP formation resulted in a decreased ability to recruit additional platelets. In the absence of CypD, integrin α_IIbβ₃ function was accentuated in both static and flow conditions, and, in vivo, a prothrombotic phenotype occurred in mice with a platelet-specific deficiency of CypD. CypD-dependent proteolytic events, including cleavage of the integrin β₃ cytoplasmic domain, coincided closely with integrin α_IIbβ₃ inactivation. Calpain inhibition blocked integrin β₃ cleavage and inactivation but not mPTP formation or PS exposure, indicating that integrin inactivation and PS exposure are mediated by distinct pathways subsequent to mPTP formation. mPTP-dependent alkalinization occurred in procoagulant platelets, suggesting a possible alternative mechanism for enhancement of calpain activity in procoagulant platelets. Together, these results indicate that, in strongly stimulated platelets, mPTP formation initiates the calpain-dependent cleavage of integrin β₃ and associated regulatory proteins, resulting in integrin α_IIbβ₃ inactivation, and demonstrate a novel CypD-dependent negative feedback mechanism that limits platelet aggregation and thrombotic occlusion.     

Dok-2 Adaptor Protein Regulates the Shear-dependent Adhesive Function of Platelet Integrin αIIbβ3 in Mice

The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin α_IIbβ₃, raising the possibility that it participates in integrin α_IIbβ₃ outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin α_IIbβ₃-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P₂ accumulation. Although agonist-induced integrin α_IIbβ₃ affinity regulation was unaltered in Dok-2^−/− platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin α_IIbβ₃ adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays.     

Reconstructing and deconstructing agonist-induced activation of integrin alphaIIbbeta3

BACKGROUND: Integrin receptors, composed of transmembrane alpha and beta subunits, are essential for the development and functioning of multicellular animals. Agonist stimulation leads cells to regulate integrin affinity ("activation"), thus controlling cell adhesion and migration, controlling extracellular-matrix assembly, and contributing to angiogenesis, tumor cell metastasis, inflammation, the immune response, and hemostasis. A final step in integrin activation is the binding of talin, a cytoskeletal protein, to integrin beta cytoplasmic domains. Many different signaling molecules that regulate integrin affinity have been described, but a pathway that connects agonist stimulation to talin binding and activation has not been mapped. RESULTS: We used forward, reverse, and synthetic genetics to engineer and order an integrin activation pathway in cells expressing a prototype activatable integrin, platelet alphaIIbbeta3. Phorbol myristate acetate (PMA) activated alphaIIbbeta3 only after the increased expression of both recombinant protein kinase Calpha (PKCalpha) and talin to levels approximating those in platelets. Inhibition of Rap1 GTPase reduced alphaIIbbeta3 activation, whereas activated Rap1A(G12V) bypassed the requirement for PKC, establishing that Rap1 is downstream of PKC. Talin binding to integrins mediates Rap1-induced activation because Rap1A(G12V) failed to activate alphaIIbbeta3 in cells expressing integrin binding-defective talin (W359A). Rap1 activated integrins by forming an integrin-associated complex containing talin in combination with the Rap effector, RIAM. Furthermore, siRNA-mediated knockdown of RIAM blocked integrin activation. CONCLUSIONS: We have, for the first time, ordered a pathway from agonist stimulation to integrin activation and established the Rap1-induced formation of an "integrin activation complex," containing RIAM and talin, that binds to and activates the integrin.     

Beta 3-endonexin, a novel polypeptide that interacts specifically with the cytoplasmic tail of the integrin beta 3 subunit

The adhesive and signaling functions of integrins are regulated through their cytoplasmic domains. We identified a novel 111 residue polypeptide, designated beta 3-endonexin, that interacted with the cytoplasmic tail of the beta 3 integrin subunit in a yeast two-hybrid system. This interaction is structurally specific, since it was reduced by 64% by a point mutation in the beta 3 cytoplasmic tail (S752-->P) that disrupts integrin signaling. Moreover, this interaction is integrin subunit specific since it was not observed with the cytoplasmic tails of the alpha IIb, beta 1, or beta 2 subunits. beta 3- Endonexin fusion proteins bound selectively to detergent-solubilized beta 3 from platelets and human umbilical vein endothelial cells, and beta 3-endonexin mRNA and protein were detected in platelets and other tissues. A related mRNA encoded a larger polypeptide that failed to bind to beta integrin tails. The apparent specificity of beta 3- endonexin for the beta 3 integrin subunit suggests potential mechanisms for selective modulation of integrin functions.     

Norcantharidin,a clinical used chemotherapeutic agent,acts as a powerful inhibitor by interfering with fibrinogen–integrin αIIbβ3 binding in human platelets

During platelet activation, fibrinogen binds to its specific platelet receptor, integrin α_IIbβ₃, thus completing the final common pathway for platelet aggregation. Norcantharidin (NCTD) is a promising anticancer agent in China from medicinal insect blister beetle. In this study, we provided the evidence to demonstrate NCTD (0.1–1.0 μM) possesses very powerful antiplatelet activity in human platelets; nevertheless, it had no effects on surface P‐selectin expression and only slight inhibition on ATP‐release reaction in activated platelets. Moreover, NCTD markedly hindered integrin α_IIbβ₃ activation by interfering with the binding of FITC‐labelled PAC‐1. It also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as clot retraction. Additionally, NCTD attenuated phosphorylation of proteins such as integrin β₃, Src and FAK in platelets spreading on immobilized fibrinogen. These results indicate that NCTD restricts integrin α_IIbβ₃‐mediated outside‐in signalling in human platelets. Besides, NCTD substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation and prolonged the bleeding time in mice. In conclusion, NCTD has dual activities, it can be a chemotherapeutic agent for cancer treatment, and the other side it possesses powerful antiplatelet activity for treating thromboembolic disorders.     

Modulation of Integrin Activity is Vital for Morphogenesis

Cells can vary their adhesive properties by modulating the affinity of integrin receptors. The activation and inactivation of integrins by inside-out mechanisms acting on the cytoplasmic domains of the integrin subunits has been demonstrated in platelets, lymphocytes, and keratinocytes. We show that in the embryo, normal morphogenesis requires the α subunit cytoplasmic domain to control integrin adhesion at the right times and places. PS2 integrin (α_PS2β_PS) adhesion is normally restricted to the muscle termini, where it is required for attaching the muscles to the ends of other muscles and to specialized epidermal cells. Replacing the wild-type α_PS2 with mutant forms containing cytoplasmic domain deletions results in the rescue of the majority of defects associated with the absence of the α_PS2 subunit, however, the mutant PS2 integrins are excessively active. Muscles containing these mutant integrins make extra muscle attachments at aberrant positions on the muscle surface, disrupting the muscle pattern and causing embryonic lethality. A gain- of-function phenotype is not observed in the visceral mesoderm, showing that regulation of integrin activity is tissue-specific. These results suggest that the α_PS2 subunit cytoplasmic domain is required for inside-out regulation of integrin affinity, as has been seen with the integrin α_IIbβ₃.     

Regulation of adaptor protein GIT1 in platelets, leading to the interaction between GIT1 and integrin alpha(IIb)beta3

GIT1 is an adaptor protein, which links signaling proteins to focal adhesion, thereby regulating cytoskeletal reorganization. Platelets undergo dynamic cytoskeletal reorganization during platelet activation, for which a large number of adaptor proteins are required. However, there has been no report of GIT1 in platelets. We found that GIT1 was abundantly expressed in platelets and underwent tyrosine phosphorylation downstream of integrin α_IIbβ₃, which was inhibited by the Src kinase inhibitor PP2. Furthermore, GIT1 constitutively associated with βPIX, a guanine nucleotide exchange factor (GEF) for Rac. The GIT1/βPIX complex associated with α_IIbβ₃, concomitantly with GIT1 tyrosine phosphorylation. Moreover, both GIT1 and α_IIbβ₃ rapidly translocated to the cytoskeletal fraction during platelet aggregation, which was not observed in the absence of aggregation. These results suggest that tyrosine phosphorylation of GIT1 by Src kinases may regulate cytoskeletal reorganization downstream of α_IIbβ₃ by bringing the Rac GEF βPIX to the vicinity of the integrin.     

Relevance of N-terminal residues for amyloid-β binding to platelet integrin αIIbβ3, integrin outside-in signaling and amyloid-β fibril formation

A pathological hallmark of Alzheimer's disease (AD) is the aggregation of amyloid-β peptides (Aβ) into fibrils, leading to deposits in cerebral parenchyma and vessels known as cerebral amyloid angiopathy (CAA). Platelets are major players of hemostasis but are also implicated in AD. Recently we provided strong evidence for a direct contribution of platelets to AD pathology. We found that monomeric Aβ40 binds through its RHDS sequence to integrin α_IIbβ₃, and promotes the formation of fibrillar Aβ aggregates by the secretion of adenosine diphosphate (ADP) and the chaperone protein clusterin (CLU) from platelets. Here we investigated the molecular mechanisms of Aβ binding to integrin α_IIbβ₃ by using Aβ11 and Aβ16 peptides. These peptides include the RHDS binding motif important for integrin binding but lack the central hydrophobic core and the C-terminal sequence of Aβ. We observed platelet adhesion to truncated N-terminal Aβ11 and Aβ16 peptides that was not mediated by integrin α_IIbβ₃. Thus, no integrin outside-in signaling and reduced CLU release was detected. Accordingly, platelet mediated Aβ fibril formation was not observed. Taken together, the RHDS motif of Aβ is not sufficient for Aβ binding to platelet integrin α_IIbβ₃ and platelet mediated Aβ fibril formation but requires other recognition or binding motifs important for platelet mediated processes in CAA. Thus, increased understanding of the molecular mechanisms of Aβ binding to platelet integrin α_IIbβ₃ is important to understand the role of platelets in amyloid pathology.     

Proteomic and Phospho-Proteomic Profile of Human Platelets in Basal,Resting State: Insights into Integrin Signaling

During atherogenesis and vascular inflammation quiescent platelets are activated to increase the surface expression and ligand affinity of the integrin αIIbβ3 via inside-out signaling. Diverse signals such as thrombin, ADP and epinephrine transduce signals through their respective GPCRs to activate protein kinases that ultimately lead to the phosphorylation of the cytoplasmic tail of the integrin αIIbβ3 and augment its function. The signaling pathways that transmit signals from the GPCR to the cytosolic domain of the integrin are not well defined. In an effort to better understand these pathways, we employed a combination of proteomic profiling and computational analyses of isolated human platelets. We analyzed ten independent human samples and identified a total of 1507 unique proteins in platelets. This is the most comprehensive platelet proteome assembled to date and includes 190 membrane-associated and 262 phosphorylated proteins, which were identified via independent proteomic and phospho-proteomic profiling. We used this proteomic dataset to create a platelet protein-protein interaction (PPI) network and applied novel contextual information about the phosphorylation step to introduce limited directionality in the PPI graph. This newly developed contextual PPI network computationally recapitulated an integrin signaling pathway. Most importantly, our approach not only provided insights into the mechanism of integrin αIIbβ3 activation in resting platelets but also provides an improved model for analysis and discovery of PPI dynamics and signaling pathways in the future.     

Integrin ��IIb��3 Activation in Chinese Hamster Ovary Cells and Platelets Increases Clustering Rather than Affinity

Integrin αIIbβ3 affinity regulation by talin binding to the cytoplasmic tail of β3 is a generally accepted model for explaining activation of this integrin in Chinese hamster ovary cells and human platelets. Most of the evidence for this model comes from the use of multivalent ligands. This raises the possibility that the activation being measured is that of increased clustering of the integrin rather than affinity. Using a newly developed assay that probes integrins on the surface of cells with only monovalent ligands prior to fixation, I do not find increases in affinity of αIIbβ3 integrins by talin head fragments in Chinese hamster ovary cells, nor do I observe affinity increases in human platelets stimulated with thrombin. Binding to a multivalent ligand does increase in both of these cases. This assay does report affinity increases induced by either Mn²⁺, a cytoplasmic domain mutant (D723R) in the cytoplasmic domain of β3, or preincubation with a peptide ligand. These results reconcile the previously observed differences between talin effects on integrin activation in Drosophila and vertebrate systems and suggest new models for talin regulation of integrin activity in human platelets.     

Phosphoproteomic Analysis of Platelets Activated by Pro-Thrombotic Oxidized Phospholipids and Thrombin

Specific oxidized phospholipids (oxPC_CD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPC_CD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPC_CD36 as well as by the strong physiological agonist thrombin. oxPC_CD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPC_CD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPC_CD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPC_CD36.     

Kindlin-3 is essential for integrin activation and platelet aggregation

Integrin-mediated platelet adhesion and aggregation are essential for sealing injured blood vessels and preventing blood loss, and excessive platelet aggregation can initiate arterial thrombosis, causing heart attacks and stroke. To ensure that platelets aggregate only at injury sites, integrins on circulating platelets exist in a low-affinity state and shift to a high-affinity state (in a process known as integrin activation or priming) after contacting a wounded vessel. The shift is mediated through binding of the cytoskeletal protein Talin to the beta subunit cytoplasmic tail. Here we show that platelets lacking the adhesion plaque protein Kindlin-3 cannot activate integrins despite normal Talin expression. As a direct consequence, Kindlin-3 deficiency results in severe bleeding and resistance to arterial thrombosis. Mechanistically, Kindlin-3 can directly bind to regions of beta-integrin tails distinct from those of Talin and trigger integrin activation. We have therefore identified Kindlin-3 as a novel and essential element for platelet integrin activation in hemostasis and thrombosis.     

Red cell ICAM-4 is a novel ligand for platelet-activated alpha IIbbeta 3 integrin

ICAM-4 (LW blood group glycoprotein) is an erythroid-specific membrane component that belongs to the family of intercellular adhesion molecules and interacts in vitro with different members of the integrin family, suggesting a potential role in adhesion or cell interaction events, including hemostasis and thrombosis. To evaluate the capacity of ICAM-4 to interact with platelets, we have immobilized red blood cells (RBCs), platelets, and ICAM-Fc fusion proteins to a plastic surface and analyzed their interaction in cell adhesion assays with RBCs and platelets from normal individuals and patients, as well as with cell transfectants expressing the alpha(IIb)beta(3) integrin. The platelet fibrinogen receptor alpha(IIb)beta(3) (platelet GPIIb-IIIa) in a high affinity state following GRGDSP peptide activation was identified for the first time as the receptor for RBC ICAM-4. The specificity of the interaction was demonstrated by showing that: (i) activated platelets adhered less efficiently to immobilized ICAM-4-negative than to ICAM-4-positive RBCs, (ii) monoclonal antibodies specific for the beta(3)-chain alone and for a complex-specific epitope of the alpha(IIb)beta(3) integrin, and specific for ICAM-4 to a lesser extent, inhibited platelet adhesion, whereas monoclonal antibodies to GPIb, CD36, and CD47 did not, (iii) activated platelets from two unrelated type-I glanzmann's thrombasthenia patients did not bind to coated ICAM-4. Further support to RBC-platelet interaction was provided by showing that dithiothreitol-activated alpha(IIb)beta(3)-Chinese hamster ovary transfectants strongly adhere to coated ICAM-4-Fc protein but not to ICAM-1-Fc and was inhibitable by specific antibodies. Deletion of individual Ig domains of ICAM-4 and inhibition by synthetic peptides showed that the alpha(IIb)beta(3) integrin binding site encompassed the first and second Ig domains and that the G65-V74 sequence of domain D1 might play a role in this interaction. Although normal RBCs are considered passively entrapped in fibrin polymers during thrombus, these studies identify ICAM-4 as the first RBC protein ligand of platelets that may have relevant physiological significance.     

Flow cytometric detection of activated mouse integrin alphaIIbbeta3 with a novel monoclonal antibody

BACKGROUND: Integrin alphaIIbbeta3 mediates platelet adhesion and aggregation and plays a crucial role in thrombosis and hemostasis. alphaIIbbeta3 is expressed in a low affinity state on resting platelets. Upon platelet activation, alphaIIbbeta3 shifts to a high affinity conformation that efficiently binds its ligands. On human platelets, the high affinity conformation of alphaIIbbeta3 is detected by the monoclonal antibody (mAb), PAC-1. However, a reagent with binding specificity to high affinity mouse alphaIIbbeta3 has not been described so far. METHODS: A novel rat mAb directed against mouse alphaIIbbeta3 (JON/A) was generated and characterized. JON/A was conjugated with fluorescein isothiocyanate (JON/A(FITC)) or with R-phycoerythrin (JON/A(PE)) and used for flow cytometric analysis of mouse platelets. RESULTS: Although JON/A(FITC) bound to resting and activated platelets, virtually no binding of the larger JON/A(PE) to resting platelets was detectable. However, strong binding of JON/A(PE) occurred on platelet activation in a dose-dependent manner. Binding of JON/A(PE) required extracellular free calcium and was irreversible, thereby stabilizing the high affinity conformation of alphaIIbbeta3. CONCLUSION: JON/A(PE) is the first tool for direct assessment of integrin alphaIIbbeta3 activation in mice. Furthermore, JON/A(FITC) and JON/A(PE) provide the first examples of fluorescent antibody derivatives with identical antigenic specificity that allow the discrimination between the resting and the activated state of an integrin.