It's the water! The open and shut case of drug-induced integrin activation.

Integrin receptors are established drug targets, but many of the drugs that have been developed act as partial agonists, inducing the receptor into a high-affinity, ligand-binding state. Lin et al. discovered a general mechanism to circumvent this problem-stabilizing a key water molecule that prevents receptor activation. Their findings are likely to impact future therapeutic development.

Pregnancy and childbirth in patients with Glanzmann Thrombasthenia.

Glanzmann thrombasthenia (GT) is a rare inherited platelet bleeding disorder caused by a quantitative and/or qualitative defect of the αIIbß3 integrin. Pregnancy and delivery pose special challenges as they entail increased risks of both maternal and foetal bleeding that may be life-threatening. Multidisciplinary management throughout the preconception, intrapartum and peripartum periods is vital to optimize pregnancy outcomes. This Nutshell review focuses on the challenging management of pregnancy and childbirth in patients with GT.

Prehospital treatment with zalunfiban (RUC-4) in patients with ST- elevation myocardial infarction undergoing primary percutaneous coronary intervention: Rationale and design of the CELEBRATE trial.

BACKGROUND: Early and complete restoration of target vessel patency in ST-elevation myocardial infarction (STEMI) is associated with improved outcomes. Oral P2Y12 inhibitors have failed to demonstrate either improved patency or reduced mortality when administered in the prehospital setting. Thus, there is a need for antiplatelet agents that achieve prompt and potent platelet inhibition, and that restore patency in the prehospital setting. Zalunfiban, a novel subcutaneously administered glycoprotein IIb/IIIa inhibitor designed for prehospital administration, has shown to achieve rapid, high-grade platelet inhibition that exceeds that of P2Y12 inhibitors. Whether prehospital administration of zalunfiban can improve clinical outcome is unknown. HYPOTHESIS: The present study is designed to assess the hypothesis that a single, prehospital injection of zalunfiban given in the ambulance, in addition to standard-of-care in patients with STEMI with intent to undergo primary percutaneous coronary intervention (PCI) will improve clinical outcome compared to standard-of-care with placebo. STUDY DESIGN: The ongoing CELEBRATE trial (NCT04825743) is a phase 3, randomized, double-blinded, placebo-controlled, international trial. Patients with STEMI intended to undergo primary PCI will receive treatment with a single subcutaneous injection containing either zalunfiban dose 1 (0.110 mg/kg), zalunfiban dose 2 (0.130 mg/kg) or placebo, and the study drug will be administered in the ambulance before transportation to the hospital. A target of 2499 patients will be randomly assigned to one of the treatment groups in a 1:1:1 ratio, ie, to have approximately 833 evaluable patients per group. The primary efficacy outcome is a ranked 7-point scale on clinical outcomes. The primary safety outcome is severe or life-threatening bleeding according to the Global Use of Strategies to Open Occluded Coronary Arteries (GUSTO) criteria. SUMMARY: The CELEBRATE trial will assess whether a single prehospital subcutaneous injection of zalunfiban in addition to standard-of-care in patients with STEMI with intent to undergo primary PCI will result in improved clinical outcome.

Prepercutaneous coronary intervention Zalunfiban dose-response relationship to target vessel blood flow at initial angiogram in st-elevation myocardial infarction - A post hoc analysis of the cel-02 phase IIa study.

BACKGROUND: Zalunfiban (RUC-4) is a novel, subcutaneously administered glycoprotein IIb/IIIa inhibitor (GPI) designed for prehospital treatment to initiate reperfusion in the infarct-related artery (IRA) before primary percutaneous coronary intervention in patients with ST-elevation myocardial infarction (STEMI). Since GPIs have been reported to rapidly reperfuse IRAs, we assessed whether there was a dose-dependent relationship between zalunfiban treatment and angiographic reperfusion indices and thrombus grade of the IRA at initial angiogram in patients with STEMI. METHODS: This was a post hoc analysis from the open-label Phase IIa study that investigated the pharmacodynamics, pharmacokinetics, and tolerability of three doses of zalunfiban - 0.075, 0.090 and 0.110 mg/kg - in STEMI patients. This analysis explored dose-dependent associations between zalunfiban and three angiographic indices of the IRA, namely coronary and myocardial blood flow and thrombus burden. Zalunfiban was administered in the cardiac catheterization laboratory prior to vascular access, ∼10 to 15 minutes before the initial angiogram. All angiographic data were analyzed by a blinded, independent, core laboratory. RESULTS: Twentyfour out of 27 STEMI patients were evaluable for angiographic analysis (0.075 mg/kg [n=7], 0.090 mg/kg [n=9], and 0.110 mg/kg [n=8]). TIMI flow grade 2 or 3 was seen in 1/7 patients receiving zalunfiban at 0.075 mg/kg, in 6/9 patients receiving 0.090 mg/kg, and in 7/8 patients receiving 0.110 mg/kg (ptrend = 0.004). A similar trend was observed based on TIMI flow grade 3. Myocardial perfusion was also related to zalunfiban dose (ptrend = 0.005) as reflected by more frequent TIMI myocardial perfusion grade 3. Consistent with the dose-dependent trends in greater coronary and myocardial perfusion, TIMI thrombus ≥4 grade was inversely related to zalunfiban dose (ptrend = 0.02). CONCLUSION: This post hoc analysis found that higher doses of zalunfiban administered in the cardiac catheterization lab prior to vascular access were associated with greater coronary and myocardial perfusion, and lower thrombus burden at initial angiogram in patients with STEMI undergoing primary percutaneous coronary intervention.

Structure-Based Discovery of a Novel Class of Small-Molecule Pure Antagonists of Integrin αVß3.

Inhibitors of integrin αVß3 have therapeutic promise for a variety of diseases. Most αVß3-targeting small molecules patterned after the RGD motif are partial agonists because they induce a high-affinity, ligand-binding conformation and prime the receptor to bind the ligand without an activating stimulus, in part via a charge-charge interaction between their aspartic acid carboxyl group and the metal ion in the metal-ion-dependent adhesion site (MIDAS). Building upon our previous studies on the related integrin αIIbß3, we searched for pure αVß3 antagonists that lack this typical aspartic acid carboxyl group and instead engage through direct binding to one of the coordinating residues of the MIDAS metal ion, specifically ß3 E220. By in silico screening of two large chemical libraries for compounds interacting with ß3 E220, we indeed discovered a novel molecule that does not contain an acidic carboxyl group and does not induce the high-affinity, ligand-binding state of the receptor. Functional and structural characterization of a chemically optimized version of this compound led to the discovery of a novel small-molecule pure αVß3 antagonist that (i) does not prime the receptor to bind the ligand and does not induce hybrid domain swing-out or receptor extension as judged by antibody binding and negative-stain electron microscopy, (ii) binds at the RGD-binding site as predicted by metadynamics rescoring of induced-fit docking poses and confirmed by a cryo-electron microscopy structure of the compound-bound integrin, and (iii) coordinates the MIDAS metal ion via a quinoline moiety instead of an acidic carboxyl group.

Ethics of Human Genome Editing.

Advances in human genome editing, in particular the development of the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 method, have led to increasing concerns about the ethics of editing the human genome. In response, the US National Academy of Sciences and the National Academy of Medicine constituted a multidisciplinary, international committee to review the current status and make recommendations. I was a member of that committee, and the core of this review reflects the committee's conclusions. The committee's report, issued in February 2017, recommends the application of current ethical and regulatory standards for gene therapy to somatic (nonheritable) human genome editing. It also recommends allowing experimental germline genome editing to proceed if ( a) it is restricted to preventing transmission of a serious disease or condition, ( b) the edit is a modification to a common DNA sequence known not to be associated with disease, and ( c) the research is conducted under a stringent set of ethical and regulatory requirements. Crossing the so-called red line of germline genome editing raises important bioethical issues, most importantly, serious concern about the potential negative impact on individuals with disabilities. This review highlights some of the major ethical considerations in human genome editing in light of the report's recommendations.

Cryo-Electron Microscopy Structure of the αIIbß3-Abciximab Complex.

OBJECTIVE: The αIIbß3 antagonist antiplatelet drug abciximab is the chimeric antigen-binding fragment comprising the variable regions of murine monoclonal antibody 7E3 and the constant domains of human IgG1 and light chain κ. Previous mutagenesis studies suggested that abciximab binds to the ß3 C177-C184 specificity-determining loop (SDL) and Trp129 on the adjacent ß1-α1 helix. These studies could not, however, assess whether 7E3 or abciximab prevents fibrinogen binding by steric interference, disruption of either the αIIbß3-binding pocket for fibrinogen or the ß3 SDL (which is not part of the binding pocket but affects fibrinogen binding), or some combination of these effects. To address this gap, we used cryo-electron microscopy to determine the structure of the αIIbß3-abciximab complex at 2.8 Å resolution. Approach and Results: The interacting surface of abciximab is comprised of residues from all 3 complementarity-determining regions of both the light and heavy chains, with high representation of aromatic residues. Binding is primarily to the ß3 SDL and neighboring residues, the ß1-α1 helix, and ß3 residues Ser211, Val212 and Met335. Unexpectedly, the structure also indicated several interactions with αIIb. As judged by the cryo-electron microscopy model, molecular-dynamics simulations, and mutagenesis, the binding of abciximab does not appear to rely on the interaction with the αIIb residues and does not result in disruption of the fibrinogen-binding pocket; it does, however, compress and reduce the flexibility of the SDL. CONCLUSIONS: We deduce that abciximab prevents ligand binding by steric interference, with a potential contribution via displacement of the SDL and limitation of the flexibility of the SDL residues.

THE GORDON WILSON LECTURE: THE ETHICS OF HUMAN GENOME EDITING.

Human genome editing has undergone major technological advances, raising the possibility of treating or preventing many illnesses. Somatic (nonheritable) genome editing, both in vitro and in vivo, is already being employed under a robust regulatory and ethical framework developed for human gene therapy. In contrast, the prospect of germline (heritable) genome editing is much more contentious, and there is currently no consensus on the proper path forward. The 2017 National Academy of Sciences (NAS) and National Academy of Medicine (NAM) report proposed a series of requirements designed to minimize ethical objections while allowing couples to accept the risks of genome editing in order to have a biologically related child without passing on a known genetic disorder. It is vital to prevent gene editing from resulting in unintended negative consequences for individuals with genetic variants. The utilization of genome editing to enhance human function is highly contentious; it may be better to focus on whether an edit creates an "unfair advantage" rather than an enhancement.

αIIbß3 variants defined by next-generation sequencing: predicting variants likely to cause Glanzmann thrombasthenia.

Next-generation sequencing is transforming our understanding of human genetic variation but assessing the functional impact of novel variants presents challenges. We analyzed missense variants in the integrin αIIbß3 receptor subunit genes ITGA2B and ITGB3 identified by whole-exome or -genome sequencing in the ThromboGenomics project, comprising ∼32,000 alleles from 16,108 individuals. We analyzed the results in comparison with 111 missense variants in these genes previously reported as being associated with Glanzmann thrombasthenia (GT), 20 associated with alloimmune thrombocytopenia, and 5 associated with aniso/macrothrombocytopenia. We identified 114 novel missense variants in ITGA2B (affecting ∼11% of the amino acids) and 68 novel missense variants in ITGB3 (affecting ∼9% of the amino acids). Of the variants, 96% had minor allele frequencies (MAF) < 0.1%, indicating their rarity. Based on sequence conservation, MAF, and location on a complete model of αIIbß3, we selected three novel variants that affect amino acids previously associated with GT for expression in HEK293 cells. αIIb P176H and ß3 C547G severely reduced αIIbß3 expression, whereas αIIb P943A partially reduced αIIbß3 expression and had no effect on fibrinogen binding. We used receiver operating characteristic curves of combined annotation-dependent depletion, Polyphen 2-HDIV, and sorting intolerant from tolerant to estimate the percentage of novel variants likely to be deleterious. At optimal cut-off values, which had 69-98% sensitivity in detecting GT mutations, between 27% and 71% of the novel αIIb or ß3 missense variants were predicted to be deleterious. Our data have implications for understanding the evolutionary pressure on αIIbß3 and highlight the challenges in predicting the clinical significance of novel missense variants.

Blood at 70: its roots in the history of hematology and its birth.

This year we celebrate Blood's 70th year of publication. Created from the partnership of the book publisher Henry M. Stratton and the prominent hematologist Dr William Dameshek of Tufts School of Medicine, Blood has published many papers describing major advances in the science and clinical practice of hematology. Blood's founding antedated that of the American Society of Hematology (ASH) by more than 11 years and Stratton and Dameshek helped galvanize support for the creation of ASH. In this review, I place the birth of Blood in the context of the history of hematology before 1946, emphasizing the American experience from which it emerged, and focusing on research conducted during World War II. I also provide a few milestones along Blood's 70 years of publication, including: the growth in Blood's publications, the evolution of its appearance, the countries of submission of Blood papers, current subscriptions to Blood, and the evolution of topics reported in Blood's papers. The latter provides a snapshot of the evolution of hematology as a scientific and clinical discipline and the introduction of new technology to study blood and bone marrow. Detailed descriptions of the landmark discoveries reported in Blood will appear in later papers celebrating Blood's birthday authored by past Editors-in-Chief.

Planning for the future workforce in hematology research.

The medical research and training enterprise in the United States is complex in both its scope and implementation. Accordingly, adaptations to the associated workforce needs present particular challenges. This is particularly true for maintaining or expanding national needs for physician-scientists where training resource requirements and competitive transitional milestones are substantial. For the individual, these phenomena can produce financial burden, prolong the career trajectory, and significantly influence career pathways. Hence, when national data suggest that future medical research needs in a scientific area may be met in a less than optimal manner, strategies to expand research and training capacity must follow. This article defines such an exigency for research and training in nonneoplastic hematology and presents potential strategies for addressing these critical workforce needs. The considerations presented herein reflect a summary of the discussions presented at 2 workshops cosponsored by the National Heart, Lung, and Blood Institute and the American Society of Hematology.

Increased Smad2/3 phosphorylation in circulating leukocytes and platelet-leukocyte aggregates in a mouse model of aortic valve stenosis: Evidence of systemic activation of platelet-derived TGF-ß1 and correlation with cardiac dysfunction.

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1) has been implicated in the pathogenesis of aortic valve stenosis (AS). There is, however, little direct evidence for a role of active TGF-ß1 in AS due to the sensitivity of current assays. We searched for evidence of plasma TGF-ß1 activation by assaying Smad2/3 phosphorylation in circulating leukocytes and platelet-leukocyte aggregates (PLAs) in a mouse model of AS (Reversa). METHODS: Echocardiography was used to measure AS and cardiac function. Intracellular phospho-flow cytometry in combination with optical fluorescence microscopy was used to detect PLAs and p-Smad2/3 levels. RESULTS: Reversa mice on a western diet developed AS, had significantly increased numbers of PLAs and more intense staining for p-Smad2/3 in both PLAs and single leukocytes (all p<0.05). p-Smad2/3 staining was more intense in PLAs than in single leukocytes in both diet groups (p<0.05) and correlated with plasma total TGF-ß1 levels (r=0.38, p=0.05 for PLAs and r=0.37, p=0.06 for single leukocytes) and reductions in ejection fraction (r=-0.42, p=0.03 for PLAs and r=-0.37, p=0.06 for single leukocytes). CONCLUSIONS: p-Smad2/3 staining is more intense in leukocytes of hypercholesterolemic mice that developed AS, suggesting increased circulating active TGF-ß1 levels. Leukocyte p-Smad2/3 may be a valuable surrogate indicator of circulating active TGF-ß1.

Molecular Types of Methicillin-Resistant Staphylococcus aureus and Methicillin-Sensitive S. aureus Strains Causing Skin and Soft Tissue Infections and Nasal Colonization, Identified in Community Health Centers in New York City.

In November 2011, The Rockefeller University Center for Clinical and Translational Science (CCTS), the Laboratory of Microbiology and Infectious Diseases, and Clinical Directors Network (CDN) launched a research and learning collaborative project with six community health centers in the New York City metropolitan area to determine the nature (clonal type) of community-acquired Staphylococcus aureus strains causing skin and soft tissue infections (SSTIs). Between November 2011 and March 2013, wound and nasal samples from 129 patients with active SSTIs suspicious for S. aureus were collected and characterized by molecular typing techniques. In 63 of 129 patients, the skin wounds were infected by S. aureus: methicillin-resistant S. aureus (MRSA) was recovered from 39 wounds and methicillin-sensitive S. aureus (MSSA) was recovered from 24. Most-46 of the 63-wound isolates belonged to the CC8/Panton-Valentine leukocidin-positive (PVL(+)) group of S. aureus clone USA300: 34 of these strains were MRSA and 12 were MSSA. Of the 63 patients with S. aureus infections, 30 were also colonized by S. aureus in the nares: 16 of the colonizing isolates were MRSA, and 14 were MSSA, and the majority of the colonizing isolates belonged to the USA300 clonal group. In most cases (70%), the colonizing isolate belonged to the same clonal type as the strain involved with the infection. In three of the patients, the identity of invasive and colonizing MRSA isolates was further documented by whole-genome sequencing.

Three-dimensional reconstruction of intact human integrin αIIbß3: new implications for activation-dependent ligand binding.

Integrin αIIbß3 plays a central role in hemostasis and thrombosis. We provide the first 3-dimensional reconstruction of intact purified αIIbß3 in a nanodisc lipid bilayer. Unlike previous models, it shows that the ligand-binding head domain is on top, pointing away from the membrane. Moreover, unlike the crystal structure of the recombinant ectodomain, the lower legs are not parallel, straight, and adjacent. Rather, the αIIb lower leg is bent between the calf-1 and calf-2 domains and the ß3 Integrin-Epidermal Growth Factor (I-EGF) 2 to 4 domains are freely coiled rather than in a cleft between the ß3 headpiece and the αIIb lower leg. Our data indicate an important role for the region that links the distal calf-2 and ß-tail domains to their respective transmembrane (TM) domains in transmitting the conformational changes in the TM domains associated with inside-out activation.

Association between shear stress and platelet-derived transforming growth factor-ß1 release and activation in animal models of aortic valve stenosis.

OBJECTIVE: Aortic valve stenosis (AS) is characterized by fibrosis and calcification of valves leading to aortic valve narrowing, resulting in high wall shear stress across the valves. We previously demonstrated that high shear stress can activate platelet-derived transforming growth factor-ß1 (TGF-ß1), a cytokine that induces fibrosis and calcification. The aim of this study was to investigate the role of shear-induced platelet release of TGF-ß1 and its activation in AS. APPROACH AND RESULTS: We studied hypercholesterolemic Ldlr(-/-)Apob(100/100)/Mttp(fl/fl)/Mx1Cre(+/+) (Reversa) mice that develop AS on Western diet and a surgical ascending aortic constriction mouse model that acutely simulates the hemodynamics of AS to study shear-induced platelet TGF-ß1 release and activation. Reversa mice on Western diet for 6 months had thickening of the aortic valves, increased wall shear stress, and increased plasma TGF-ß1 levels. There were weak and moderate correlations between wall shear stress and TGF-ß1 levels in the progression and reversed Reversa groups and a stronger correlation in the ascending aortic constriction model in wild-type mice but not in mice with a targeted deletion of megakaryocyte and platelet TGF-ß1 (Tgfb1(flox)). Plasma total TGF-ß1 levels correlated with collagen deposition in the stenotic valves in Reversa mice. Although active TGF-ß1 levels were too low to be measured directly, we found (1) canonical TGF-ß1 (phosphorylated small mothers against decapentaplegic 2/3) signaling in the leukocytes and canonical and noncanonical (phosphorylated extracellular signal-regulated kinases 1/2) TGF-ß1 signaling in aortic valves of Reversa mice on a Western diet, and (2) TGF-ß1 signaling of both pathways in the ascending aortic constriction stenotic area in wild-type but not Tgfb1(flox) mice. CONCLUSIONS: Shear-induced, platelet-derived TGF-ß1 activation may contribute to AS.

RUC-4: a novel αIIbß3 antagonist for prehospital therapy of myocardial infarction.

OBJECTIVE: Treatment of myocardial infarction within the first 1 to 2 hours with a thrombolytic agent, percutaneous coronary intervention, or an αIIbß3 antagonist decreases mortality and the later development of heart failure. We previously reported on a novel small molecule αIIbß3 antagonist, RUC-2, that has a unique mechanism of action. We have now developed a more potent and more soluble congener of RUC-2, RUC-4, designed to be easily administered intramuscularly by autoinjector to facilitate its use in the prehospital setting. Here, we report the properties of RUC-4 and the antiplatelet and antithrombotic effects of RUC-2 and RUC-4 in animal models. APPROACH AND RESULTS: RUC-4 was ≈ 20% more potent than RUC-2 in inhibiting human ADP-induced platelet aggregation and much more soluble in aqueous solutions (60-80 mg/mL). It shared RUC-2's specificity for αIIbß3 versus αVß3, did not prime the receptor to bind fibrinogen, or induce changes in ß3 identified by a conformation-specific monoclonal antibody. Both RUC-2 and RUC-4 prevented FeCl3-induced thrombotic occlusion of the carotid artery in mice and decreased microvascular thrombi in response to laser injury produced by human platelets infused into transgenic mice containing a mutated von Willebrand factor that reacts with human but not mouse platelets. Intramuscular injection of RUC-4 in nonhuman primates at 1.9 and 3.85 mg/kg led to complete inhibition of platelet aggregation within 15 minutes, with dose-dependent return of platelet aggregation after 4.5 to 24 hours. CONCLUSIONS: RUC-4 has favorable biochemical, pharmacokinetic, pharmacodynamic, antithrombotic, and solubility properties as a prehospital therapy of myocardial infarction, but the possibility of increased bleeding with therapeutic doses remains to be evaluated.

Identification of the thiol isomerase-binding peptide, mastoparan, as a novel inhibitor of shear-induced transforming growth factor ß1 (TGF-ß1) activation.

TGF-ß1 is a disulfide-bonded homodimeric protein produced by platelets and other cells that plays a role in many physiologic and pathologic processes. TGF-ß1 is secreted as an inactive large latent complex (LLC) comprised of TGF-ß1, latency-associated peptide, and latent TGF-ß binding protein 1. We previously demonstrated that shear force can activate LLC and that thiol-disulfide exchange contributes to the process. We have now investigated the role of thiol isomerases in the activation of LLC in platelet releasates (PR) and recombinant LLC. The wasp venom peptide mastoparan, which inhibits the chaperone activity of PDI, inhibited stirring- and shear-induced activation of latent TGF-ß1 by 90 and 75% respectively. To identify the proteins that bind to mastoparan either directly or indirectly, PR were chromatographed on a mastoparan affinity column. Latent TGF-ß binding protein 1, latency-associated peptide, TGF-ß1, clusterin, von Willebrand factor, multimerin-1, protein disulfide isomerase (PDI), ERp5, ERp57, and ERp72 eluted specifically from the column. Anti-PDI RL90 attenuated the inhibitory effect of mastoparan on LLC activation. Furthermore, reduced PDI inhibited activation of PR LLC, whereas oxidized PDI had no effect. We conclude that thiol isomerases and thiol-disulfide exchange contribute to TGF-ß1 activation and identify a number of molecules that may participate in the process.

Talin-driven inside-out activation mechanism of platelet αIIbß3 integrin probed by multimicrosecond, all-atom molecular dynamics simulations.

Platelet aggregation is the consequence of the binding of extracellular bivalent ligands such as fibrinogen and von Willebrand factor to the high affinity, active state of integrin αIIbß3. This state is achieved through a so-called "inside-out" mechanism characterized by the membrane-assisted formation of a complex between the F2 and F3 subdomains of intracellular protein talin and the integrin ß3 tail. Here, we present the results of multi-microsecond, all-atom molecular dynamics simulations carried on the complete transmembrane (TM) and C-terminal (CT) domains of αIIbß3 integrin in an explicit lipid-water environment, and in the presence or absence of the talin-1 F2 and F3 subdomains. These large-scale simulations provide unprecedented molecular-level insights into the talin-driven inside-out activation of αIIbß3 integrin. Specifically, they suggest a preferred conformation of the complete αIIbß3 TM/CT domains in a lipid-water environment, and testable hypotheses of key intermolecular interactions between αIIbß3 integrin and the F2/F3 domains of talin-1. Notably, not only do these simulations give support to a stable left-handed reverse turn conformation of the αIIb juxtamembrane motif rather than a helical turn, but they raise the question as to whether TM helix separation is required for talin-driven integrin activation.

Platelet TGF-ß1 contributions to plasma TGF-ß1, cardiac fibrosis, and systolic dysfunction in a mouse model of pressure overload.

Circulating platelets contain high concentrations of TGF-ß1 in their α-granules and release it on platelet adhesion/activation. We hypothesized that uncontrolled in vitro release of platelet TGF-ß1 may confound measurement of plasma TGF-ß1 in mice and that in vivo release and activation may contribute to cardiac pathology in response to constriction of the transverse aorta, which produces both high shear and cardiac pressure overload. Plasma TGF-ß1 levels in blood collected from C57Bl/6 mice by the standard retro-bulbar technique were much higher than those obtained when prostaglandin E1 was added to inhibit release or when blood was collected percutaneously from the left ventricle under ultrasound guidance. Even with optimal blood drawing, plasma TGF-ß1 was lower in mice rendered profoundly thrombocytopenic or mice with selectively low levels of platelet TGF-ß1 because of megakaryocyte-specific disruption of their TGF-ß1 gene (Tgfb1(flox)). Tgfb1(flox) mice were also partially protected from developing cardiac hypertrophy, fibrosis, and systolic dysfunction in response to transverse aortic constriction. These studies demonstrate that plasma TGF-ß1 levels can be assessed accurately, but it requires special precautions; that platelet TGF-ß1 contributes to plasma levels of TGF-ß1; and that platelet TGF-ß1 contributes to the pathologic cardiac changes that occur in response to aortic constriction.

A novel class of ion displacement ligands as antagonists of the αIIbß3 receptor that limit conformational reorganization of the receptor.

A collection of αIIbß3 integrin receptor antagonists possessing a unique MIDAS metal ion displacement mechanism of action is presented. Insight into these agents' structure-activity relationships, binding modality, and pharmacokinetic and pharmacodynamic profiles highlight the potential of these small molecule ion displacement ligands as attractive candidates for clinical development.