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Two novel polyarginine labelled ruthenium polypyridyl dyes are reported, one conjugated to five, (Ru-Ahx-R 5 ), and one to eight arginine residues, (Ru-Ahx-R 8 ).Both complexes exhibit long-lived, intense, and oxygen sensitive luminescence. Luminescent dye molecules capable of passive cell delivery may be used as molecular probes for example in cellular imaging, cell biology, molecular biology, microbiology, and flow cytometry applications.The majority of probes used in cellular imaging are fluorescent and based on organic, typically polyaromatic, chromophores. The short luminescence lifetimes of such species, typically <10 ns, limits their environmental sensitivity, e.g., towards molecular oxygen, and their application for fluorescent lifetime imaging (FLIM).

Integrins are cysteine-rich heterodimeric cell-surface adhesion molecules that alter their affinity for ligands in response to cellular activation. The molecular mechanisms involved in this activation of integrins are not understood. Treatment with the thiol-reducing agent, dithiothreitol, can induce an activation-like state in many integrins suggesting that cysteine-cysteine dithiol bonds are important for the receptor's tertiary structure and may be involved in activation-induced conformational changes. Here we demonstrate that the platelet-specific integrin, ␣ IIb ␤ 3 , contains an endogenous thiol isomerase activity, predicted from the presence of the tetrapeptide motif, CXXC, in each of the cysteinerich repeats of the ␤ 3 polypeptide. This motif comprises the active site in enzymes involved in disulfide exchange reactions, including protein-disulfide isomerase (EC 5.3.4.1) and thioredoxin. Intrinsic thiol isomerase activity is also observed in the related integrin, ␣ v ␤ 3 , which shares a common ␤-subunit. Thiol isomerase activity within ␣ IIb ␤ 3 is time-dependent and saturable, and is inhibited by the protein-disulfide isomerase inhibitor, bacitracin. Furthermore, this activity is calcium-sensitive and is regulated in the EDTA-stabilized conformation of the integrin. This novel demonstration of an enzymatic activity associated with an integrin subunit suggests that altered thiol bonding within the integrin or its substrates may be locally modified during ␣ IIb ␤ 3 activation.Integrins are cell-surface, calcium-dependent, heterodimeric adhesion molecules that play a critical role in cell-cell and cell-substrate adhesion. In cells at rest, integrins are present in a latent or resting conformation. Following cellular activation, they undergo conformational changes to become high affinity receptors for their specific ligand(s). The "switch" mechanism whereby integrins are converted from their resting conformation is critically important to their cellular function. However, the mechanisms underlying these conformational changes have not yet been deduced.The conformational changes in the platelet-specific integrin, ␣ IIb ␤ 3 , are the composite result of at least two processes. First, intracellular signals converge on the cytoplasmic tails of the integrin conveying the intention to activate. Second, the extracellular domains, which constitute Ͼ95% of the molecules, respond with an increased affinity for ligand and an altered display of antibody epitopes suggestive of altered protein folding. We have shown that the conserved ␣-subunit cytoplasmic sequence, KVGFFKR, is critical for the intracellular-mediated activation of the platelet integrin (1). The precise role played by this peptide sequence remains uncharacterized. However, Vinogradova et al. (2), have recently proposed a structural basis for this effect which proposes a protein-protein interaction with the integrin cytoplasmic tails. Deletion or mutation of this cytoplasmic sequence from the ␣ IIb subunit were found to increase the ligand binding affinity...

Platelet activation causes conformational changes of integrin GPIIb/IIIa (alpha(IIb)beta3), resulting in the exposure of its ligand-binding pocket. This provides the unique possibility to design agents that specifically block activated platelets only. We used phage display of single-chain antibody (scFv) libraries in combination with several rounds of depletion/selection to obtain human scFvs that bind specifically to the activated conformation of GPIIb/IIIa. Functional evaluation of these scFv clones revealed that fibrinogen binding to human platelets and platelet aggregation can be effectively inhibited by activation-specific scFvs. In contrast to clinically used GPIIb/IIIa blockers, which are all conformation unspecific, activation-specific GPIIb/IIIa blockers do not induce conformational changes in GPIIb/IIIa or outside-in signaling, as evaluated by ligand-induced binding-site (LIBS) exposure in flow cytometry or P-selectin expression in immunofluorescence microscopy, respectively. In contrast to the conformation-unspecific blocker abciximab, activation-specific scFvs permit cell adhesion and spreading on immobilized fibrinogen, which is mediated by nonactivated GPIIb/IIIa. Mutagenesis studies and computer modeling indicate that exclusive binding of activation-specific scFv is mediated by RXD motifs in the heavy-chain complementary-determining region (CDR) 3 of the antibodies, which in comparison with other antibodies forms an exceptionally extended loop. In vivo experiments in a ferric-chloride thrombosis model of the mouse carotid artery demonstrate similar antithrombotic potency of activation-specific scFv, when compared with the conformation-unspecific blockers tirofiban and eptifibatide. However, in contrast to tirofiban and eptifibatide, bleeding times are not prolonged with the activation-specific scFvs, suggesting lower bleeding risks. In conclusion, activation-specific GPIIb/IIIa blockade via human single-chain antibodies represents a promising novel strategy for antiplatelet therapy.

All integrin ␣ subunits contain a highly conserved KXGFFKR motif in their cytoplasmic domains that plays a crucial role in the regulation of integrin affinity for their ligands. We show that a lipid-modified peptide corresponding to the cytoplasmic region, 989 -995, of the platelet integrin subunit glycoprotein GpIIb (␣IIb), palmitoyl-KVGFFKR (Ppep; 10 M), but not a similarly modified scrambled peptide (palmitoyl-FKFVRGK), can specifically induce platelet activation and aggregation equivalent to that of strong agonists such as thrombin. Ppep-induced aggregation is also associated with indices of platelet activation including thromboxane A 2 (TXA 2 ) synthesis (EC 50 ‫؍‬ 45 ؎ 5 M), secretion of ␣-granules detected as enhanced surface expression of P-selectin (EC 50 ‫؍‬ 52 ؎ 8 M), and conformational changes in GpIIb/IIIa measured by the monoclonal antibody, PAC-1 (EC 50 ‫؍‬ 3.7 ؎ 1 M). The TXA 2 receptor antagonist, SQ29548, PGE 1 , and the ADP scavenger, apyrase, differentially inhibit the aggregation response and TXA 2 synthesis in response to Ppep. Similarly, GpIIb/IIIa antagonists (RO-449883 and integrelin), which inhibit aggregation by greater than 90%, have little effect on peptide-induced TXA 2 synthesis, suggesting that this event is independent of fibrinogen binding to GpIIb/IIIa. Alanine-stepping of the Ppep sequence identifies GFFK(991-994) as the critical residues in all peptidemediated events. We conclude that this peptide can imitate the cytoplasmic domain of GpIIb and initiate parallel but independent signaling pathways, one leading to ligand binding and platelet aggregation and the other to intracellular signaling events such as TXA 2 synthesis and secretion.Integrins are a family of cell adhesion molecules composed of two subunits, ␣ and ␤, which form a complex on the cell surface. Ligand recognition by integrins may be modulated by intracellular signals that interact with the cytoplasmic tails of the subunits. This has been demonstrated most clearly for the platelet glycoprotein (Gp) 1 IIb/IIIa (␣IIb␤3), the most abundant platelet integrin, which acts as a receptor for fibrinogen, fibronectin, and other RGD-containing macromolecules (1). Under resting conditions, this receptor has a low affinity for its ligands (2). However, when platelets are stimulated by agonists such as thrombin or ADP, GpIIb/IIIa undergoes a conformational change (3) detected by the appearance of neoepitopes for monoclonal antibodies such as PAC-1 (4) and acquires a high affinity binding for its ligands, principally fibrinogen (5, 6). The binding of fibrinogen results in platelet aggregation, an early step in the generation of a thrombus. Deletion or mutation of the cytoplasmic domains of the integrin subunits can produce a constitutively active or inactive receptor (7-9), suggesting that signals resulting from cell activation interact with the intracellular components of GpIIb/IIIa to modify ligand recognition (inside-out signaling).The cytoplasmic domains are also important for events occurring as a consequence of ligand...

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.

Pregnancy-specific glycoproteins (PSGs) are immunoglobulin superfamily members encoded by multigene families in rodents and primates. In human pregnancy, PSGs are secreted by the syncytiotrophoblast, a fetal tissue, and reach a concentration of up to 400 ug/ml in the maternal bloodstream at term. Human and mouse PSGs induce release of anti-inflammatory cytokines such as IL-10 and TGFβ1 from monocytes, macrophages, and other cell types, suggesting an immunoregulatory function. RGD tri-peptide motifs in the majority of human PSGs suggest that they may function like snake venom disintegrins, which bind integrins and inhibit interactions with ligands. We noted that human PSG1 has a KGD, rather than an RGD motif. The presence of a KGD in barbourin, a platelet integrin αIIbβ3 antagonist found in snake venom, suggested that PSG1 may be a selective αIIbβ3 ligand. Here we show that human PSG1 binds αIIbβ3 and inhibits the platelet – fibrinogen interaction. Unexpectedly, however, the KGD is not critical as multiple PSG1 domains independently bind and inhibit αIIbβ3 function. Human PSG9 and mouse Psg23 are also inhibitory suggesting conservation of this function across primate and rodent PSG families. Our results suggest that in species with haemochorial placentation, in which maternal blood is in direct contact with fetal trophoblast, the high expression level of PSGs reflects a requirement to antagonise abundant (3 mg/ml) fibrinogen in the maternal circulation, which may be necessary to prevent platelet aggregation and thrombosis in the prothrombotic maternal environment of pregnancy.

A critical role for the conserved ␣-integrin cytoplasmic motif, KVGFFKR, is recognized in the regulation of activation of the platelet integrin ␣ IIb ␤ 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 ␣ IIb ␤ 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 ␣ IIb ␤ 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 M 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 M) 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.