An αIIbß3 monoclonal antibody traps a semiextended conformation and allosterically inhibits large ligand binding.

ABSTRACT: Monoclonal antibodies (mAbs) have provided valuable information regarding the structure and function of platelet αIIbß3. Protein disulfide isomerase (PDI) has been implicated in αIIbß3 activation and binds to thrombin-activated αIIbß3. Using human platelets as the immunogen, we identified a new mAb (R21D10) that inhibits the binding of PDI to platelets activated with thrombin receptor-activating peptide (T6). R21D10 also partially inhibited T6-induced fibrinogen and PAC-1 binding to platelets, as well as T6- and adenosine 5'-diphosphate-induced platelet aggregation. Mutual competition experiments showed that R21D10 does not inhibit the binding of mAbs 10E5 (anti-αIIb cap domain) or 7E3 (anti-ß3 ß-I domain), and immunoblot studies indicated that R21D10 binds to ß3. The dissociation of αIIbß3 by EDTA had a minimal effect on R21D10 binding. Cryogenic electron microscopy of the αIIbß3-R21D10 Fab complex revealed that R21D10 binds to the ß3 integrin-epidermal growth factor 1 (I-EGF1) domain and traps an intermediate conformation of αIIbß3 with semiextended leg domains. The binding of R21D10 produces a major structural change in the ß3 I-EGF2 domain associated with a new interaction between the ß3 I-EGF2 and αIIb thigh domains, which may prevent the swing-out motion of the ß3 hybrid domain required for high-affinity ligand binding and protect αIIbß3 from EDTA-induced dissociation. R21D10 partially reversed the ligand binding priming effect of eptifibatide, suggesting that it could convert the swung-out conformation into a semiextended conformation. We concluded that R21D10 inhibits ligand binding to αIIbß3 via a unique allosteric mechanism, which may or may not be related to its inhibition of PDI binding.

Expression, Purification and Characterization of Recombinant Disintegrin from Gloydius Brevicaudus Venom in Escherichia Coli.

Disintegrins, a family of snake venom protein, which are capable of modulating the activity of integrins that play a fundamental role in the regulation of many physiological and pathological processes. The main purpose of this study is to obtain the recombinant disintegrin (r-DI) and evaluate its biological activity. In this study, we explored a high-level expression prokaryotic system and purification strategy for r-DI. Then, r-DI was treated to assay effects on cell growth, migration, and invasion. The affinity for the interactions of r-DI with integrin was determined using Surface plasmon resonance (SPR) analyses. The r-DI can be expressed in Escherichia coli and purified by one-step chromatography. The r-DI can inhibit B16F10 cells proliferation, migration, and invasion. Also, we found that r-DI could interact with the integrin αIIbß3 (GPIIb/IIIa). The r-DI can be expressed, purified, characterized through functional assays, and can also maintain strong biological activities. Thus, this study showed potential therapeutic effects of r-DI for further functional and structural studies.

Not one, but many forms of thrombosis proteins.

The disulfide bond is a covalent bond formed between the sulfur atoms of two cysteine residues in proteins. Our understanding of the role of these ubiquitous bonds in protein function has changed dramatically over the past decade. Initially thought to be fully formed and inert in the native protein, we know now that both these assumptions are incorrect for many proteins. Here, we review recent evidence for production and function of multiple partially disulfide-bonded forms of plasma fibrinogen and platelet αIIbß3 integrin. The disulfide bonds are not cleaved in these mature proteins but rather a significant fraction of the bonds never form during maturation of the protein. The resulting different covalent states influence the functioning of the protein. These findings change our concept of the native, functional protein.

A Novel Frameshift Mutation in the ITGB3 Gene Leading to Glanzmann's Thrombasthenia in a Saudi Arabian Family.

Glanzmann's thrombasthenia (GT) is an autosomal recessive congenital bleeding disorder of platelet aggregation. Mutations in ITGA2B and ITGB3 genes result in quantitative and/or qualitative abnormalities of the glycoprotein receptor complex IIb/IIIa (integrin αIIbß3), which in turn impairs platelet aggregation and lead to GT. In this study, whole genome single nucleotide polymorphism (SNP) genotyping as well as whole exome sequencing was performed in a large family segregating GT. Analysis of the genotypes localized the disease region to chromosome 17q21.2-q21.3. Filtration of whole exome data and candidate variants prioritization identified a pathogenic variant in the ITGB3 gene. The single nucleotide deletion variant (c.2113delC) in exon 13 of the ITGB3 gene is predicted to cause a frameshift and absence of vital C-terminal domains including the transmembrane helix and the cytoplasmic domain. Clinical variability of the bleeding phenotype in affected individuals with the same mutation suggests that other genetic and nongenetic factors are responsible for determining GT features.

Structural determinants of the integrin transmembrane domain required for bidirectional signal transmission across the cell membrane.

Studying the tight activity regulation of platelet-specific integrin αIIbß3 is foundational and paramount to our understanding of integrin structure and activation. αIIbß3 is essential for the aggregation and adhesion function of platelets in hemostasis and thrombosis. Structural and mutagenesis studies have previously revealed the critical role of αIIbß3 transmembrane (TM) association in maintaining the inactive state. Gain-of-function TM mutations were identified and shown to destabilize the TM association leading to integrin activation. Studies using isolated TM peptides have suggested an altered membrane embedding of the ß3 TM α-helix coupled with αIIbß3 activation. However, controversies remain as to whether and how the TM α-helices change their topologies in the context of full-length integrin in native cell membrane. In this study, we utilized proline scanning mutagenesis and cysteine scanning accessibility assays to analyze the structure and function correlation of the αIIbß3 TM domain. Our identification of loss-of-function proline mutations in the TM domain suggests the requirement of a continuous TM α-helical structure in transmitting activation signals bidirectionally across the cell membrane, characterized by the inside-out activation for ligand binding and the outside-in signaling for cell spreading. Similar results were found for αLß2 and α5ß1 TM domains, suggesting a generalizable mechanism. We also detected a topology change of ß3 TM α-helix within the cell membrane, but only under conditions of cell adhesion and the absence of αIIb association. Our data demonstrate the importance of studying the structure and function of the integrin TM domain in the native cell membrane.

An alternate covalent form of platelet αIIbß3 integrin that resides in focal adhesions and has altered function.

The αIIbß3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in ∼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbß3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbß3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.

Conformation-Specific Blockade of αIIbß3 by a Non-RGD Peptide to Inhibit Platelet Activation without Causing Significant Bleeding and Thrombocytopenia.

Bleeding and thrombocytopenia to readministration are the most serious side effects of clinical integrin αIIbß3 antagonists such as RGD-containing peptides. Here we show that a non-RGD peptide ZDPI, identified from skin secretions of Amolops loloensis, inhibited platelet aggregation induced by agonists, such as adenosine diphosphate, collagen, arachidonic acid, PAR1AP, and integrin αIIbß3 allosteric activator, and reduces soluble fibrinogen binding to activated platelets without perturbing adhesion numbers on immobilized fibrinogen. Further study showed that ZDPI preferred to bind to the active conformation of integrin αIIbß3, and thus inhibited c-Src-mediated integrin signaling transduction. In contrast to currently used clinical blockers of integrin αIIbß3, which are all conformation-unspecific blockers, ZDPI conformation specifically binds to activated integrin αIIbß3, subsequently suppressing platelet spreading. In vivo study revealed that ZDPI inhibited carotid arterial thrombosis with limited bleeding and thrombocytopenia. A non-RGD peptide which targets the active conformation of integrin αIIbß3, such as ZDPI, might be an excellent candidate or template to develop antithrombotics without significant bleeding and thrombocytopenia side effects.

Drug-induced activation of integrin alpha IIb beta 3 leads to minor localized structural changes.

Integrins are transmembrane proteins involved in hemostasis, wound healing, immunity and cancer. In response to intracellular signals and ligand binding, integrins adopt different conformations: the bent (resting) form; the intermediate extended form; and the ligand-occupied active form. An integrin undergoing such conformational dynamics is the heterodimeric platelet receptor αIIbß3. Although the dramatic rearrangement of the overall structure of αIIbß3 during the activation process is potentially related to changes in the protein secondary structure, this has not been investigated so far in a membrane environment. Here we examine the Mn2+- and drug-induced activation of αIIbß3 and the impact on the structure of this protein reconstituted into liposomes. By quartz crystal microbalance with dissipation monitoring and activation assays we show that Mn2+ induces binding of the conformation-specific antibody PAC-1, which only recognizes the extended, active integrin. Circular dichroism spectroscopy reveals, however, that Mn2+-treatment does not induce major secondary structural changes of αIIbß3. Similarly, we found that treatment with clinically relevant drugs (e.g. quinine) led to the activation of αIIbß3 without significant changes in protein secondary structure. Molecular dynamics simulation studies revealed minor local changes in the beta-sheet probability of several extracellular domains of the integrin. Our experimental setup represents a new approach to study transmembrane proteins, especially integrins, in a membrane environment and opens a new way for testing drug binding to integrins under clinically relevant conditions.

Development of a new protein labeling system to map subunits and domains of macromolecular complexes for electron microscopy.

Several gene fusion technologies have been successfully applied to label particular subunits or domains within macromolecular complexes to enable positional mapping of electron microscopy (EM) density maps, but exogenous fusion of a protein domain into the target polypeptide can cause unwanted structural and functional outcomes. Fab fragments from antibodies can be used as labeling reagents during EM visualization without gene manipulation of the target protein, but this method requires a panel of high-affinity antibodies that recognize a wide variety of epitopes. Linear peptide tags and their anti-tag antibodies can be used but they have a limited mapping ability as their placement is usually limited to the terminal regions of a protein. The PA dodecapeptide epitope tag (GVAMPGAEDDVV), forms a tight ß-turn in the antigen binding pocket of its antibody (NZ-1). This capability allows for insertion of the PA tag into various surface-exposed loops within a multi-domain cell adhesion receptor, αIIbß3 integrin. We confirmed that the purified PA-tagged integrin ectodomain fragments can form a stable complex with NZ-1 Fab. Negative stain EM of the various integrin-NZ-1 complexes revealed that a majority of the particles exhibited a clear density corresponding to the NZ-1 Fab; and the positions of the bound Fab were in good agreement with the predicted location of the inserted PA tag. The high-affinity and insertion-compatibility of the PA tag system allowed us to develop a new EM labeling methodology applicable to proteins for which good antibodies are not available.

Pilot study of novel lab methodology and testing of platelet function in adolescent women with heavy menstrual bleeding.

BackgroundApproximately 40% of adolescent women experience heavy menstrual bleeding (HMB), and 10-62% of them have an underlying bleeding disorder (BD). Diagnosing a BD remains challenging because of limitations of available clinical platelet function assays. The aim of this study was to characterize platelet function in a population of adolescent women with HMB using small-volume whole-blood assays.MethodsAnticoagulated whole blood was used to assess platelet GPIIbIIIa activation, α-granule secretion, and aggregation in response to multiple agonists. Platelet adhesion on collagen or von Willebrand Factor (VWF) under static and shear flow was also assessed.ResultsFifteen participants with HMB were included in the study, of which eight were diagnosed with a clinically identifiable BD. Platelet activation was blunted in response to calcium ionophore in participants without a BD diagnosis compared with that in all other participants. Impaired GPIIbIIIa activation was observed in response to all GPCR agonists, except adenosine diphosphate (ADP), in participants with qualitative platelet disorders. Our assays detected platelet aggregation in the majority of participants with a BD in response to ADP, collagen-related peptide (CRP), thrombin receptor activator 6 (TRAP-6), or U46619. Platelet adhesion and aggregation on collagen and VWF was decreased for participants with VWD.ConclusionParticipants with and without BD exhibited aberrant platelet function in several assays in response to select agonists.

Synergistic effect of peptide inhibitors derived from the extracellular and intracellular domain of αIIb subunit of integrin αIIbß3 on platelet activation and aggregation.

αIIbß3, the major platelet integrin, plays a central role in hemostasis and thrombosis. Upon platelet activation, conformation of αIIbß3 changes and allows fibrinogen binding and, subsequently, platelet aggregation. It was previously shown that a lipid-modified platelet permeable peptide, which corresponds to the intracellular acidic membrane distal sequence 1000LEEDDEEGE1008 of αIIb (pal-K-LEEDDEEGE or pal-K-1000-1008), inhibits thrombin-induced human platelet aggregation, by inhibiting talin association with the integrin. YMESRADR, a peptide corresponding to the extracellular sequence 313-320 of αIIb, is also a potent platelet aggregation inhibitor by mimicking the effect of a clasp between the head domains of αIIb and ß3. The aim of the present study was to investigate the synergistic effect of the intra- and extracellular- peptide inhibitors on platelet aggregation, as well as on the phosphorylation of two signaling proteins, focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Platelet preincubation with Pal-K-LEEDDEGE followed by YMESRADR showed a synergistic inhibitory activity on platelet aggregation. Platelet incubation with threshold inhibitory concentrations of both peptides provoked almost the total inhibition of aggregation, PAC-1 binding, and fibrinogen binding, but not P-selectin exposure on activated platelets' surface. Like RGDS peptide, this mixture inhibits FAK phosphorylation whose phosphorylation is well known to be consecutive to the aggregation (postoccupancy events). However, in contrast to RGDS peptide that enhances ERK phosphorylation and activation, the mixture of threshold inhibitory concentrations of Pal-K-LEEDDEEGE and YMESRADR inhibits ERK phosphorylation. We suggest that the use of the intracellular in combination with the extracellular peptide inhibitor, acting with a non-RGD-like mechanism, may provide an alternative way to antagonize integrin αIIbß3 activation.

An αIIb ß3 antagonist prevents thrombosis without causing Fc receptor γ-chain IIa-mediated thrombocytopenia.

Essentials FcγRIIa-mediated thrombocytopenia is associated with drug-dependent antibodies (DDAbs). We investigated the correlation between αIIb ß3 binding epitopes and induction of DDAbs. An FcγRIIa-transgenic mouse model was used to evaluate thrombocytopenia among anti-thrombotics. An antithrombotic with binding motif toward αIIb ß-propeller domain has less bleeding tendency. SUMMARY: Background Thrombocytopenia, a common side effect of Arg-Gly-Asp-mimetic antiplatelet drugs, is associated with drug-dependent antibodies (DDAbs) that recognize conformation-altered integrin αIIb ß3 . Objective To explore the correlation between αIIb ß3 binding epitopes and induction of DDAb binding to conformation-altered αIIb ß3 , we examined whether two purified disintegrins, TMV-2 and TMV-7, with distinct binding motifs have different effects on induction of αIIb ß3 conformational change and platelet aggregation in the presence of AP2, an IgG1 inhibitory mAb raised against αIIb ß3 . Methods We investigated the possible mechanisms of intrinsic platelet activation of TMV-2 and TMV-7 in the presence of AP2 by examining the signal cascade, tail bleeding time and immune thrombocytopenia in Fc receptor γ-chain IIa (FcγRIIa) transgenic mice. Results TMV-7 has a binding motif that recognizes the αIIb ß-propeller domain of αIIb ß3 , unlike that of TMV-2. TMV-7 neither primed the platelets to bind ligand, nor caused a conformational change of αIIb ß3 as identified with the ligand-induced binding site mAb AP5. In contrast to eptifibatide and TMV-2, cotreatment of TMV-7 with AP2 did not induce FcγRIIa-mediated platelet aggregation and the downstream activation cascade. Both TMV-2 and TMV-7 efficaciously prevented occlusive thrombosis in vivo. Notably, both eptifibatide and TMV-2 caused severe thrombocytopenia mediated by FcγRIIa, prolonged tail bleeding time in vivo, and repressed human whole blood coagulation indexes, whereas TMV-7 did not impair hemostatic capacity. Conclusions TMV-7 shows antiplatelet and antithrombotic activities resulting from a mechanism different from that of all other tested αIIb ß3 antagonists, and may offer advantages as a therapeutic agent with a better safety profile.

High-Affinity "Click" RGD Peptidomimetics as Radiolabeled Probes for Imaging αv ß3 Integrin.

Nonpeptidic Arg-Gly-Asp (RGD)-mimic ligands were designed and synthesized by click chemistry between an arginine-azide mimic and an aspartic acid-alkyne mimic. Some of these molecules combine excellent in vitro properties (high αv ß3 affinity, selectivity, drug-like logD, high metabolic stability) with a variety of radiolabeling options (e.g., tritium and fluorine-18, plus compatibility with radio-iodination), not requiring the use of chelators or prosthetic groups. The binding mode of the resulting triazole RGD mimics to αv ß3 or αIIb ß3 receptors was investigated by molecular modeling simulations. Lead compound 12 was successfully radiofluorinated and used for in vivo positron emission tomography/computed tomography (PET/CT) studies in U87 tumor models, which showed only modest tumor uptake and retention, owing to rapid excretion. These results demonstrate that the novel click RGD mimics are excellent radiolabeled probes for in vitro and cell-based studies on αv ß3 integrin, whereas further optimization of their pharmacokinetic and dynamic profiles is necessary for successful use in in vivo imaging.

Epigallocatechin gallate has pleiotropic effects on transmembrane signaling by altering the embedding of transmembrane domains.

Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrinαIIbß3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbß3 activation, but it activates αIIbß3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbß3 is tightly regulated by the topology of ß3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin ß3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin α-ß TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs.

Radiolabeled tirofiban - a potential radiopharmaceutical for detection of deep venous thrombosis.

AIM: The aim of this study was to investigate the possibility of using 99mtechnetium (99mTc)-labeled tirofiban (a reversible antagonist of glycoprotein IIb/IIIa) for detection of deep venous thrombosis (DVT) in rats without causing an antiplatelet effect. METHODS: The ability of in vitro tirofiban to inhibit adenosine 5'-diphosphate (ADP)-induced platelet aggregation was evaluated using optical aggregometer. Binding of 99mTc-tirofiban to platelets was evaluated. Serum levels of unlabeled (a validated high performance liquid chromatography method) and 99mTc-tirofiban after single intravenous injection were evaluated in male Wistar rats with or without induced DVT (femoral vein ligation model), and the rats were also subjected to whole body scintigraphy. RESULTS: Tirofiban in vitro inhibits ADP-induced aggregation of human platelets in a dose- and concentration-dependent manner (10 nM to 2 µM), but only if it is added before ADP and not after ADP. 99mTc labeling did not affect the ability of tirofiban to bind to either human or rat platelets, nor did it affect tirofiban pharmacokinetics in intact rats or in animals with induced DVT. When 99mTc-tirofiban was injected to rats after induction of DVT, at a molar dose lower than the one showing only a weak antiaggregatory effect in vitro, whole body scintigraphy indicated localization of 99mTc-tirofiban around the place of the induced DVT. CONCLUSION: 99mTc labeling of tirofiban does not affect its ability to bind to glycoprotein IIb/IIIa or its in vivo pharmacokinetics in rats, either intact or with DVT. A low, nonantiaggregatory dose of 99mTc-tirofiban may be used to visualize DVT at an early stage.

The macromolecular architecture of platelet-derived microparticles.

Platelets are essential for hemostasis and wound healing. They are involved in fundamental processes of vascular biology such as angiogenesis, tissue regeneration, and tumor metastasis. Upon activation, platelets shed small plasma membrane vesicles termed platelet-derived microparticles (PMPs). PMPs include functional cell adhesion machinery that comprises transmembrane receptors (most abundant are the αIIbß3 integrins), cytoskeletal systems and a large variety of adapter and signaling molecules. Glanzmann thrombasthenia (GT) is a condition characterized by platelets that are deficient of the integrin αIIbß3 heterodimer. Here, we use cryo-electron tomography (cryo-ET) to study the structural organization of PMPs (in both healthy and GT patients), especially the cytoskeleton organization and receptor architecture. PMPs purified from GT patients show a significantly altered cytoskeletal organization, characterized by a reduced number of filaments present, compared to the healthy control. Furthermore, our results show that incubating healthy PMPs with manganese ions (Mn(2+)), in the presence of fibrinogen, induces a major conformational change of integrin receptors, whereas thrombin activation yields a moderate response. These results provide the first insights into the native molecular organization of PMPs.

Quantification of the Blood Platelet Reactivity in the ADP-Induced Model of Non-Lethal Pulmonary Thromboembolism in Mice with the Use of Laser Doppler Flowmetry.

INTRODUCTION: The paper describes an alternative method for quantification of in vivo ADP-induced thromboembolism. The aim of the studies was to develop a method of quantification which would not require either extravasation or labelling of platelets. Our proposed approach is based on the monitoring of changes of blood flow with the use of laser Doppler flowmetry. MATERIALS AND METHODS: Mice of C57Bl strain were used in the study. ADP was injected to the vena cava and blood flow was monitored with the use of a laser Doppler flowmeter in the mesentery. Measurements in platelet-depleted mice, mice pretreated with cangrelor, an ADP receptor antagonist, and eptifibatide, a blocker of fibrinogen binding to GPIIbIIIa, were conducted as the proof-of-concept in the performed experiments. Intravital microscopy and ex vivo imaging of organs was performed to identify the sites of aggregate formation resulting from ADP injection. RESULTS: The injection of ADP resulted in a dose-dependent reduction of the blood flow in the mesentery. These responses were fully attributable to blood platelet aggregation, as shown by the lack of the effect in platelet-depleted mice, and significantly reduced responses in mice pretreated with cangrelor and eptifibatide. No platelet aggregate formation in mesenteric vessels was revealed by intravital microscopy, while ex vivo imaging showed accumulation of fluorescent labelled platelets in the lung. CONCLUSIONS: Injection of ADP to the venous system results in the formation of platelet aggregates predominantly in the lung. This results in reversible blood flow cessation in peripheral blood vessels. The measurement of this blood flow cessation in the mesentery allows indirect measurement of ADP-induced pulmonary thromboembolism. We suggest that this approach can be useful for in vivo screening for antiplatelet drug candidates.

Assessment of neonatal platelet adhesion, activation, and aggregation.

BACKGROUND: Acquired and inherited bleeding disorders may present in the neonatal period with devastating lifelong effects. Diagnosing bleeding disorders in the neonatal population could aid in preventing and treating the associated complications. However, currently available platelet function testing is limited in neonates, owing to difficulties in obtaining an adequate blood volume, a lack of normal reference ranges, and an incomplete understanding of the neonatal platelet functional phenotype. OBJECTIVE: To develop small-volume, whole blood platelet function assays in order to quantify and compare neonatal and adult platelet function. METHODS AND RESULTS: Peripheral blood was obtained from healthy, full-term neonates at 24 h of life. Platelet activation, secretion and aggregation were measured via flow cytometry. Platelet adhesion and aggregation were assessed under static and flow conditions. As compared with adult platelets, peripheral neonatal platelet P-selectin expression and integrin glycoprotein IIbIIIa activation were significantly reduced in response to the G-protein-coupled receptor (GPCR) agonists thrombin receptor activator peptide-6 (TRAP-6), ADP, and U46619, and the immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway agonists collagen-related peptide (CRP) and rhodocytin. Neonatal platelet aggregation was markedly reduced in response to TRAP-6, ADP, U46619, CRP and rhodocytin as compared with adult platelets. The extents of neonatal and adult platelet adhesion and aggregate formation under static and shear conditions on collagen and von Willebrand factor were similar. CONCLUSIONS: As compared with adult platelets, we found that neonatal platelet activation and secretion were blunted in response to GPCR or ITAM agonists, whereas the extent of neonatal platelet adhesion and aggregate formation was similar to that of adult platelets.

The structure of hookworm platelet inhibitor (HPI), a CAP superfamily member from Ancylostoma caninum.

Secreted protein components of hookworm species include a number of representatives of the cysteine-rich/antigen 5/pathogenesis-related 1 (CAP) protein family known as Ancylostoma-secreted proteins (ASPs). Some of these have been considered as candidate antigens for the development of vaccines against hookworms. The functions of most CAP superfamily members are poorly understood, but one form, the hookworm platelet inhibitor (HPI), has been isolated as a putative antagonist of the platelet integrins αIIbß3 and α2ß1. Here, the crystal structure of HPI is described and its structural features are examined in relation to its possible function. The HPI structure is similar to those of other ASPs and shows incomplete conservation of the sequence motifs CAP1 and CAP2 that are considered to be diagnostic of CAP superfamily members. The asymmetric unit of the HPI crystal contains a dimer with an extensive interaction interface, but chromatographic measurements indicate that it is primarily monomeric in solution. In the dimeric structure, the putative active-site cleft areas from both monomers are united into a single negatively charged depression. A potential Lys-Gly-Asp disintegrin-like motif was identified in the sequence of HPI, but is not positioned at the apex of a tight turn, making it unlikely that it interacts with the integrin. Recombinant HPI produced in Escherichia coli was found not to inhibit the adhesion of human platelets to collagen or fibrinogen, despite having a native structure as shown by X-ray diffraction. This result corroborates previous analyses of recombinant HPI and suggests that it might require post-translational modification or have a different biological function.

The Tyrosine Kinase c-Src Specifically Binds to the Active Integrin αIIbß3 to Initiate Outside-in Signaling in Platelets.

It is currently believed that inactive tyrosine kinase c-Src in platelets binds to the cytoplasmic tail of the ß3 integrin subunit via its SH3 domain. Although a recent NMR study supports this contention, it is likely that such binding would be precluded in inactive c-Src because an auto-inhibitory linker physically occludes the ß3 tail binding site. Accordingly, we have re-examined c-Src binding to ß3 by immunoprecipitation as well as NMR spectroscopy. In unstimulated platelets, we detected little to no interaction between c-Src and ß3. Following platelet activation, however, c-Src was co-immunoprecipitated with ß3 in a time-dependent manner and underwent progressive activation as well. We then measured chemical shift perturbations in the (15)N-labeled SH3 domain induced by the C-terminal ß3 tail peptide NITYRGT and found that the peptide interacted with the SH3 domain RT-loop and surrounding residues. A control peptide whose last three residues where replaced with those of the ß1 cytoplasmic tail induced only small chemical shift perturbations on the opposite face of the SH3 domain. Next, to mimic inactive c-Src, we found that the canonical polyproline peptide RPLPPLP prevented binding of the ß3 peptide to the RT- loop. Under these conditions, the ß3 peptide induced chemical shift perturbations similar to the negative control. We conclude that the primary interaction of c-Src with the ß3 tail occurs in its activated state and at a site that overlaps with PPII binding site in its SH3 domain. Interactions of inactive c-Src with ß3 are weak and insensitive to ß3 tail mutations.