Publications by authors named "Justin Hamilton"

46 Publications

Lessons Learned from a Collaborative to Develop a Sustainable Simulation-Based Training Program in Neonatal Resuscitation: Simulating Success.

Children (Basel) 2021 Jan 12;8(1). Epub 2021 Jan 12.

Division of Neonatology, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA.

Newborn resuscitation requires a multidisciplinary team effort to deliver safe, effective and efficient care. California Perinatal Quality Care Collaborative's Simulating Success program was designed to help hospitals implement on-site simulation-based neonatal resuscitation training programs. Partnering with the Center for Advanced Pediatric and Perinatal Education at Stanford, Simulating Success engaged hospitals over a 15 month period, including three months of preparatory training and 12 months of implementation. The experience of the first cohort (Children's Hospital of Orange County (CHOC), Sharp Mary Birch Hospital for Women and Newborns (SMB) and Valley Children's Hospital (VCH)), with their site-specific needs and aims, showed that a multidisciplinary approach with a sound understanding of simulation methodology can lead to a dynamic simulation program. All sites increased staff participation. CHOC reduced latent safety threats measured during team exercises from 4.5 to two per simulation while improving debriefing skills. SMB achieved 100% staff participation by identifying unit-specific hurdles within in situ simulation. VCH improved staff confidence level in responding to neonatal codes and proved feasibility of expanding simulation across their hospital system. A multidisciplinary approach to quality improvement in neonatal resuscitation fosters engagement, enables focus on patient safety rather than individual performance, and leads to identification of system issues.
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http://dx.doi.org/10.3390/children8010039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826853PMC
January 2021

Ionotropic glutamate receptors in platelets: opposing effects and a unifying hypothesis.

Platelets 2020 Dec 7:1-11. Epub 2020 Dec 7.

Red Blood Cell Research Group, Institute of Veterinary Physiology, Vetsuisse Faculty, University of Zurich, Zürich, Switzerland.

Ionotropic glutamate receptors include -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR), kainate receptors (KAR), and -methyl-D-aspartate receptors (NMDAR). All function as cation channels; AMPAR and KAR are more permeable to sodium and NMDAR to calcium ions. Compared to the brain, receptor assemblies in platelets are unusual, suggesting distinctive functionalities.There is convincing evidence that AMPAR and KAR amplify platelet function and thrombus formation and . Transgenic mice lacking GluA1 and GluK2 (AMPAR and KAR subunits, respectively) have longer bleeding times and prolonged time to thrombosis in an arterial model. In humans, rs465566 KAR gene polymorphism associates with altered platelet responses suggesting enhanced aspirin effect. The NMDAR contribution to platelet function is less well defined. NMDA at low concentrations (≤10 μM) inhibits platelet aggregation and high concentrations (≥100 μM) have no effect. However, open NMDAR channel blockers interfere with platelet activation and aggregation induced by other agonists ; anti-GluN1 antibodies interfere with thrombus formation under high shear rates ; and rats vaccinated with GluN1 develop iron deficiency anemia suggestive of mild chronic bleeding. In this review, we summarize data on glutamate receptors in platelets and propose a unifying model that reconciles some of the opposing effects observed.
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http://dx.doi.org/10.1080/09537104.2020.1852542DOI Listing
December 2020

Determination of PAR4 numbers on the surface of human platelets: no effect of the single nucleotide polymorphism rs773902.

Platelets 2021 Oct 21;32(7):988-991. Epub 2020 Aug 21.

Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.

The thrombin receptor, protease-activated receptor 4 (PAR4), is important for platelet activation and is the target of emerging anti-thrombotic drugs. A frequently occurring single nucleotide polymorphism (SNP; rs773902) causes a function-altering PAR4 sequence variant (NC_000019.10:p.Ala120Thr), whereby platelets from Thr120-expressing individuals are hyper-responsive to PAR4 agonists and hypo-responsive to some PAR4 antagonists than platelets from Ala120-expressing individuals. This altered pharmacology may impact PAR4 inhibitor development, yet the underlying mechanism(s) remain unknown. We tested whether PAR4 surface expression contributes to the altered receptor function. Quantitative flow cytometry was used to determine the absolute number of PAR4 on platelets from individuals subsequently genotyped at rs773902. We detected 539 ± 311 PAR4 per platelet (mean ± SD, ). This number was not different across rs773902 genotypes. This first determination of cellular PAR4 numbers indicates variations in platelet surface expression do not explain the altered pharmacology of the rs773902 PAR4 sequence variant.
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http://dx.doi.org/10.1080/09537104.2020.1810654DOI Listing
October 2021

Disrupting the platelet internal membrane via PI3KC2α inhibition impairs thrombosis independently of canonical platelet activation.

Sci Transl Med 2020 07;12(553)

Australian Centre for Blood Diseases, Monash University, Melbourne, VIC 3004, Australia.

Arterial thrombosis causes heart attacks and most strokes and is the most common cause of death in the world. Platelets are the cells that form arterial thrombi, and antiplatelet drugs are the mainstay of heart attack and stroke prevention. Yet, current drugs have limited efficacy, preventing fewer than 25% of lethal cardiovascular events without clinically relevant effects on bleeding. The key limitation on the ability of all current drugs to impair thrombosis without causing bleeding is that they block global platelet activation, thereby indiscriminately preventing platelet function in hemostasis and thrombosis. Here, we identify an approach with the potential to overcome this limitation by preventing platelet function independently of canonical platelet activation and in a manner that appears specifically relevant in the setting of thrombosis. Genetic or pharmacological targeting of the class II phosphoinositide 3-kinase (PI3KC2α) dilates the internal membrane reserve of platelets but does not affect activation-dependent platelet function in standard tests. Despite this, inhibition of PI3KC2α is potently antithrombotic in human blood ex vivo and mice in vivo and does not affect hemostasis. Mechanistic studies reveal this antithrombotic effect to be the result of impaired platelet adhesion driven by pronounced hemodynamic shear stress gradients. These findings demonstrate an important role for PI3KC2α in regulating platelet structure and function via a membrane-dependent mechanism and suggest that drugs targeting the platelet internal membrane may be a suitable approach for antithrombotic therapies with an improved therapeutic window.
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http://dx.doi.org/10.1126/scitranslmed.aar8430DOI Listing
July 2020

Analysis of the (PAR4) Single Nucleotide Polymorphism () in an Indigenous Australian Population.

Front Genet 2020 30;11:432. Epub 2020 Apr 30.

Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia.

The gene encoding protease activated receptor 4 (PAR4) contains a single nucleotide variant, , that is functional. The resulting PAR4 variants, Thr120, and Ala120, are known to differently affect platelet reactivity to thrombin. Significant population differences in the frequency of the allele indicate it may be an important determinant in the ethnic differences that exist in thrombosis and hemostasis, and for patient outcomes to PAR antagonist anti-platelet therapies. Here we determined the frequency of in an Indigenous Australian group comprising 467 individuals from the Tiwi Islands. These people experience high rates of renal disease that may be related to platelet and PAR4 function and are potential recipients of PAR-antagonist treatments. The minor allele frequency (Thr120) in the Tiwi Islanders was 0.32, which is similar to European and Asian groups and substantially lower than Melanesians and some African groups. Logistic regression and allele distortion testing revealed no significant associations between the variant and several markers of renal function, as well as blood glucose and blood pressure. These findings suggest that is not an important determinant for renal disease in this Indigenous Australian group. However, the relationships between genotype and platelet and drug responsiveness in the Tiwi, and the allele frequency in other Indigenous Australian groups should be evaluated.
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http://dx.doi.org/10.3389/fgene.2020.00432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7204273PMC
April 2020

Using PAR4 Inhibition as an Anti-Thrombotic Approach: Why, How, and When?

Int J Mol Sci 2019 Nov 11;20(22). Epub 2019 Nov 11.

Australian Centre for Blood Diseases, Monash University, Melbourne VIC 3800, Australia.

Protease-activated receptors (PARs) are a family of four GPCRs with a variety of cellular functions, yet the only advanced clinical endeavours to target these receptors for therapeutic gain to date relates to the impairment of platelet function for anti-thrombotic therapy. The only approved PAR antagonist is the PAR1 inhibitor, vorapaxar-the sole anti-platelet drug against a new target approved in the past 20 years. However, there are two PARs on human platelets, PAR1 and PAR4, and more recent efforts have focused on the development of the first PAR4 antagonists, with first-in-class agents recently beginning clinical trial. Here, we review the rationale for this approach, outline the various modes of PAR4 inhibition, and speculate on the specific therapeutic potential of targeting PAR4 for the prevention of thrombotic conditions.
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http://dx.doi.org/10.3390/ijms20225629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888008PMC
November 2019

Active Micropump-Mixer for Rapid Antiplatelet Drug Screening in Whole Blood.

Anal Chem 2019 08 6;91(16):10830-10839. Epub 2019 Aug 6.

The Australian Centre for Blood Diseases , Monash University , 99 Commercial Road , Melbourne , Victoria 3004 , Australia.

There is a need for scalable automated lab-on-chip systems incorporating precise hemodynamic control that can be applied to high-content screening of new more efficacious antiplatelet therapies. This paper reports on the development and characterization of a novel active micropump-mixer microfluidic to address this need. Using a novel reciprocating elastomeric micropump design, we take advantage of the flexible structural and actuation properties of this framework to manage the hemodynamics for on-chip platelet thrombosis assay on type 1 fibrillar collagen, using whole blood. By characterizing and harnessing the complex three-dimensional hemodynamics of the micropump operation in conjunction with a microvalve controlled reagent injection system we demonstrate that this prototype can act as a real-time assay of antiplatelet drug pharmacokinetics. In a proof-of-concept preclinical application, we utilize this system to investigate the way in which rapid dosing of human whole blood with isoform selective inhibitors of phosphatidylinositol 3-kinase dose dependently modulate platelet thrombus dynamics. This modular system exhibits utility as an automated multiplexable assay system with applications to high-content chemical library screening of new antiplatelet therapies.
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http://dx.doi.org/10.1021/acs.analchem.9b02486DOI Listing
August 2019

Shared roles for and in murine platelet production and function.

Blood 2019 09 12;134(10):826-835. Epub 2019 Jul 12.

Australian Centre for Blood Diseases, Central Clinical School, Monash University, Melbourne, Australia.

The stem cell leukemia (Scl or Tal1) protein forms part of a multimeric transcription factor complex required for normal megakaryopoiesis. However, unlike other members of this complex such as Gata1, Fli1, and Runx1, mutations of have not been observed as a cause of inherited thrombocytopenia. We postulated that functional redundancy with its closely related family member, lymphoblastic leukemia 1 (Lyl1) might explain this observation. To determine whether Lyl1 can substitute for Scl in megakaryopoiesis, we examined the platelet phenotype of mice lacking 1 or both factors in megakaryocytes. Conditional knockout (KO) mice crossed with transgenic mice expressing Cre recombinase under the control of the mouse platelet factor 4 () promoter generated megakaryocytes with markedly reduced but not absent These -KO mice had mild thrombocytopenia and subtle defects in platelet aggregation. However, -KO mice generated on an -null background (double knockout [DKO] mice) had severe macrothrombocytopenia, abnormal megakaryocyte morphology, defective pro-platelet formation, and markedly impaired platelet aggregation. DKO megakaryocytes, but not single-knockout megakaryocytes, had reduced expression of , , , and many other genes that cause inherited thrombocytopenia. These gene expression changes were significantly associated with shared and binding sites that were also enriched for , , and motifs. Thus, Scl and Lyl1 share functional roles in platelet production by regulating expression of partner proteins including Gata1. We propose that this functional redundancy provides one explanation for the absence of and mutations in inherited thrombocytopenia.
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http://dx.doi.org/10.1182/blood.2019896175DOI Listing
September 2019

The mode of anesthesia influences outcome in mouse models of arterial thrombosis.

Res Pract Thromb Haemost 2019 Apr 15;3(2):197-206. Epub 2019 Feb 15.

Australian Centre for Blood Diseases Central Clinical School Monash University Alfred Hospital Melbourne Vic. Australia.

Background: Arterial thrombosis models are important for preclinical evaluation of antithrombotics but how anesthetic protocol can influence experimental results is not studied.

Objectives: We studied how three most commonly used rodent anesthetics affect the induction of thrombosis and thrombus resolution with antiplatelet agent integrilin (Eptifibatide).

Methods: The Folts, electrolytic, and FeCl models of carotid artery thrombosis were evaluated. The extent of blood flow reduction required to elicit cyclic flow reductions (CFR) was examined in the Folts model. The occlusion time and stability following electrolytic or FeCl injury was assessed. The efficacy of Eptifibatide was studied in each cohort and clot composition following FeCl application was assessed histologically.

Results: Isoflurane and ketamine-xylazine (ket-x) elicited higher basal blood flow velocities. For reliable CFR in the Folts model, a higher degree of blood flow reduction was required under ket-x and isoflurane. For the FeCl and electrolytic models, injury severity had to be increased in mice under ket-x anesthesia to achieve rapid occlusion. FeCl-injured artery sections from ket-x and isoflurane-treated mice showed vessel dilatation and clots that were more fibrin/red-cell rich compared to pentobarbitone. Integrilin led to cycle abolishment for all three Folts-injury cohorts but for the electrolytic model a 2.5-fold higher dose was required to restore blood flow under pentobarbitone. Integrilin after FeCl arterial injury was partially ineffective in isoflurane-treated mice.

Conclusions: Anesthesia impacts rodent carotid artery occlusion experiments and alters integrilin efficacy. It is important to consider anesthetic protocols in animal experiments involving pharmacological agents for treatment of atherothrombosis.
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http://dx.doi.org/10.1002/rth2.12184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6462741PMC
April 2019

The PI 3-kinase PI3KC2α regulates mouse platelet membrane structure and function independently of membrane lipid composition.

FEBS Lett 2019 01 24;593(1):88-96. Epub 2018 Nov 24.

Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.

PI3KC2α is a phosphoinositide 3-kinase with a recently reported function in platelets; PI3KC2α-deficient mouse platelets have altered membrane structure and impaired function. Yet, how these membrane changes cause platelet dysfunction remains unknown. Here, focused ion beam-scanning electron microscopy of PI3KC2α-deficient platelet ultrastructure reveals a specific effect on the internal membrane structure, while liquid chromatography-tandem mass spectrometry profiling of 294 lipid species shows unaltered lipid composition. Functionally, PI3KC2α-deficient platelets exhibit impaired thrombosis specifically under conditions involving membrane tethering. These studies indicate that the structural changes in PI3KC2α-deficient platelets are limited to the membrane, occur without major changes in lipid composition, and selectively impair cell function during thrombus formation. These findings illustrate a unique mechanism that may be targetable for anti-thrombotic benefit.
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http://dx.doi.org/10.1002/1873-3468.13295DOI Listing
January 2019

A function-blocking PAR4 antibody is markedly antithrombotic in the face of a hyperreactive PAR4 variant.

Blood Adv 2018 06;2(11):1283-1293

Australian Centre for Blood Diseases.

Thrombin activates human platelets via 2 protease-activated receptors (PARs), PAR1 and PAR4, both of which are antithrombotic drug targets: a PAR1 inhibitor is approved for clinical use, and a PAR4 inhibitor is in trial. However, a common sequence variant in human PAR4 (rs773902, encoding Thr120 in place of Ala120) renders the receptor more sensitive to agonists and less sensitive to antagonists. Here, we develop the first human monoclonal function-blocking antibody to human PAR4 and show it provides equivalent efficacy against the Ala120 and Thr120 PAR4 variants. This candidate was generated from a panel of anti-PAR4 antibodies, was found to bind PAR4 with affinity (K ≈ 0.4 nM) and selectivity (no detectable binding to any of PAR1, PAR2, or PAR3), and is capable of near-complete inhibition of thrombin cleavage of either the Ala120 or Thr120 PAR4 variant. Platelets from individuals expressing the Thr120 PAR4 variant exhibit increased thrombin-induced aggregation and phosphatidylserine exposure vs those with the Ala120 PAR4 variant, yet the PAR4 antibody inhibited these responses equivalently (50% inhibitory concentration, 4.3 vs 3.2 µg/mL against Ala120 and Thr120, respectively). Further, the antibody significantly impairs platelet procoagulant activity in an ex vivo thrombosis assay, with equivalent inhibition of fibrin formation and overall thrombus size in blood from individuals expressing the Ala120 or Thr120 PAR4 variant. These findings reveal antibody-mediated inhibition of PAR4 cleavage and activation provides robust antithrombotic activity independent of the rs773902 PAR4 sequence variant and provides rationale for such an approach for antithrombotic therapy targeting this receptor.
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http://dx.doi.org/10.1182/bloodadvances.2017015552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5998926PMC
June 2018

Structure and function of the open canalicular system - the platelet's specialized internal membrane network.

Platelets 2018 Jun 14;29(4):319-325. Epub 2018 Feb 14.

a Australian Centre for Blood Diseases, Monash University , Melbourne , Australia.

The open canalicular system (OCS) is an internal membrane structure found in platelets. First identified 50 years ago, the OCS comprises a tunneling network of surface-connected channels that appear to play an important role in platelet function. Yet, our understanding of how the OCS forms, how it functions, and what might regulate its structure and behavior remains fairly rudimentary. Structural abnormalities of the OCS are observed in some human platelet disorders. Yet, because platelets from these patients display multiple defects, the specific contribution of any OCS dysregulation to the impaired platelet function is unclear. However, recent studies have begun to shed light on mechanisms that regulate the OCS structure and to understand what influence the OCS has on overall platelet function. Advances in cellular imaging techniques have allowed whole-cell visualization of the OCS, providing the opportunity for a more detailed structural examination. Furthermore, recent work indicates that the modulation of the OCS structure may be sufficient to impact in vivo platelet function, opening up the intriguing possibility of manipulating the OCS structure as an anti-thrombotic approach. On the 50 anniversary of its discovery, we review here what is known about OCS structure and function, and outline some of the key microscopy tools for studying this intriguing internal membrane system.
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http://dx.doi.org/10.1080/09537104.2018.1431388DOI Listing
June 2018

Drugs targeting protease-activated receptor-4 improve the anti-thrombotic therapeutic window.

Ann Transl Med 2017 Dec;5(23):464

Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.

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http://dx.doi.org/10.21037/atm.2017.09.10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733327PMC
December 2017

Perinatal lethality of Par4 mice delivered by primiparous dams reveals spontaneous bleeding in mice without platelet thrombin receptor function.

Platelets 2018 Mar 29;29(2):196-198. Epub 2017 Sep 29.

a Australian Centre for Blood Diseases, Monash University , Melbourne , Australia.

Protease-activated receptor 4 (PAR4) is a cell surface G protein-coupled receptor for serine proteases, such as thrombin. Par4 mice have platelets that are unresponsive to thrombin and thereby allow examination of the importance of thrombin-induced platelet activation in (patho)physiology. Par4 mice are protected against arterial thrombosis but show no evidence of spontaneous bleeding. This contrasts with the bleeding experienced by mice with marked thrombocytopenia, such as those with genetic deficiency of the transcription factor, nuclear factor erythroid 2 (Nfe2), that have high rates of perinatal death due to hemorrhage. Given this discrepancy in spontaneous perinatal bleeding between mice without platelets and those without thrombin-induced platelet activation mechanisms, we examined in detail the immediate postnatal survival of Par4 pups. We observed significant postpartum loss of Par4 pups derived from Par4 intercrosses that was restricted to a dam's first litter; only 9% of surviving pups genotyped as Par4 in first litters and this normalized from the second litter onward (26%). A similar perinatal lethality in pups delivered by primiparous dams occurred in mice lacking platelets (Nfe2; 10%) but not in those lacking fibrinogen (Fga; 26%). These data,, provide the first evidence of spontaneous bleeding in Par4 mice, suggest that a dam's first litter provides a greater hemostatic challenge than subsequent litters, and uncovers an important role for platelets-and more specifically thrombin-induced platelet activation-in hemostasis during these more traumatic births.
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http://dx.doi.org/10.1080/09537104.2017.1349310DOI Listing
March 2018

Synthesis and biological evaluation of 8-aryl-2-morpholino-7-O-substituted benzo[e][1,3]oxazin-4-ones against DNA-PK, PI3K, PDE3A enzymes and platelet aggregation.

Bioorg Med Chem 2017 10 15;25(20):5531-5536. Epub 2017 Aug 15.

Pharmacy and Applied Science, La Trobe Institute for Molecular Science, La Trobe University, P.O. Box 199 Bendigo, VIC 3552, Australia. Electronic address:

A series of 40 7-(O-substituted)-2-morpholino-8-aryl-4H-benzo[e][1,3]oxazin-4-one derivatives was synthesized. They were prepared via synthesis of a key precursor, 8-bromo-7-hydroxy-2-morpholino-4H-benzo[e][1,3]oxazin-4-one 13 which was amenable to ether synthesis at the 7-position and Suzuki coupling at the 8-position. The 2 protons of 7-OCH in compounds 18g, 18h, 18i, 18l and 18m prove to be magnetically non-equivalent, atropisomerism (axial chirality), as result of sterically hindered rotation of the bulky 8-aryl-substituent. The products were evaluated for their activities against PI3K isoforms, DNA-PK and PDE3. The results showed that this substitution pattern has a deleterious effect on PI3K activities, which may arise from steric hindrance in the active site. PI3Kδ was somewhat more tolerant of this substitution particularly where 8-(4-methoxylphenyl) substituents were present (ICs∼2-3μM). Good activities against PDE3 were also obtained for compounds, with particular members of the 7-(2-pyridinyl) methoxy series 19 showing good inhibition (ICs∼2-3μM), comparable to previously described analogues. A piperazinyl derivative 26a effectively inhibited ADP-induced platelet aggregation with an IC of 8μM.
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http://dx.doi.org/10.1016/j.bmc.2017.08.022DOI Listing
October 2017

Inhibition of NMDA receptor function with an anti-GluN1-S2 antibody impairs human platelet function and thrombosis.

Platelets 2017 Dec 21;28(8):799-811. Epub 2017 Feb 21.

a Department of Molecular Medicine and Pathology , University of Auckland , Auckland , New Zealand.

GluN1 is a mandatory component of N-methyl-D-aspartate receptors (NMDARs) best known for their roles in the brain, but with increasing evidence for relevance in peripheral tissues, including platelets. Certain anti-GluN1 antibodies reduce brain infarcts in rodent models of ischaemic stroke. There is also evidence that human anti-GluN1 autoantibodies reduce neuronal damage in stroke patients, but the underlying mechanism is unclear. This study investigated whether anti-GluN1-mediated neuroprotection involves inhibition of platelet function. Four commercial anti-GluN1 antibodies were screened for their abilities to inhibit human platelet aggregation. Haematological parameters were examined in rats vaccinated with GluN1. Platelet effects of a mouse monoclonal antibody targeting the glycine-binding region of GluN1 (GluN1-S2) were tested in assays of platelet activation, aggregation and thrombus formation. The epitope of anti-GluN1-S2 was mapped and the mechanism of antibody action modelled using crystal structures of GluN1. Our work found that rats vaccinated with GluN1 had a mildly prolonged bleeding time and carried antibodies targeting mostly GluN1-S2. The monoclonal anti-GluN1-S2 antibody (from BD Biosciences) inhibited activation and aggregation of human platelets in the presence of adrenaline, adenosine diphosphate, collagen, thrombin and a protease-activated receptor 1-activating peptide. When human blood was flowed over collagen-coated surfaces, anti-GluN1-S2 impaired thrombus growth and stability. The epitope of anti-GluN1-S2 was mapped to α-helix H located within the glycine-binding clamshell of GluN1, where the antibody binding was computationally predicted to impair opening of the NMDAR channel. Our results indicate that anti-GluN1-S2 inhibits function of human platelets, including dense granule release and thrombus growth. Findings add to the evidence that platelet NMDARs regulate thrombus formation and suggest a novel mechanism by which anti-GluN1 autoantibodies limit stroke-induced neuronal damage.
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http://dx.doi.org/10.1080/09537104.2017.1280149DOI Listing
December 2017

Humanizing the Protease-Activated Receptor (PAR) Expression Profile in Mouse Platelets by Knocking PAR1 into the Par3 Locus Reveals PAR1 Expression Is Not Tolerated in Mouse Platelets.

PLoS One 2016 27;11(10):e0165565. Epub 2016 Oct 27.

Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.

Anti-platelet drugs are the mainstay of pharmacotherapy for heart attack and stroke prevention, yet improvements are continually sought. Thrombin is the most potent activator of platelets and targeting platelet thrombin receptors (protease-activated receptors; PARs) is an emerging anti-thrombotic approach. Humans express two PARs on their platelets-PAR1 and PAR4. The first PAR1 antagonist was recently approved for clinical use and PAR4 antagonists are in early clinical development. However, pre-clinical studies examining platelet PAR function are challenging because the platelets of non-primates do not accurately reflect the PAR expression profile of human platelets. Mice, for example, express Par3 and Par4. To address this limitation, we aimed to develop a genetically modified mouse that would express the same repertoire of platelet PARs as humans. Here, human PAR1 preceded by a lox-stop-lox was knocked into the mouse Par3 locus, and then expressed in a platelet-specific manner (hPAR1-KI mice). Despite correct targeting and the predicted loss of Par3 expression and function in platelets from hPAR1-KI mice, no PAR1 expression or function was detected. Specifically, PAR1 was not detected on the platelet surface nor internally by flow cytometry nor in whole cell lysates by Western blot, while a PAR1-activating peptide failed to induce platelet activation assessed by either aggregation or surface P-selectin expression. Platelets from hPAR1-KI mice did display significantly diminished responsiveness to thrombin stimulation in both assays, consistent with a Par3-/- phenotype. In contrast to the observations in hPAR1-KI mouse platelets, the PAR1 construct used here was successfully expressed in HEK293T cells. Together, these data suggest ectopic PAR1 expression is not tolerated in mouse platelets and indicate a different approach is required to develop a small animal model for the purpose of any future preclinical testing of PAR antagonists as anti-platelet drugs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0165565PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5082849PMC
June 2017

Class II Phosphoinositide 3-Kinases as Novel Drug Targets.

J Med Chem 2017 01 21;60(1):47-65. Epub 2016 Oct 21.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus) , 381 Royal Parade, Parkville, Victoria 3052, Australia.

The phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases central to regulating a wide range of important intracellular processes. Despite the vast knowledge around class I PI3Ks, the class II PI3Ks have been neglected, seemingly only due to the chronology of their discovery. Here we focus on the cellular functions of the three class II PI3K isoforms, PI3KC2α, PI3KC2β, and PI3KC2γ, in different cell systems and underline the emerging importance of these enzymes in different physiological and pathological contexts. We provide an overview on the current development of class II PI3 kinase inhibitors and outline the potential use for such inhibitors. The field is in its infancy as compared to their class I counterparts. Nevertheless, recent advances in understanding the roles of class II PI3 kinases in different pathological contexts is leading to an increased interest in the development of specific inhibitors that can provide potential novel pharmacological tools.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00963DOI Listing
January 2017

Challenges and Opportunities in Protease-Activated Receptor Drug Development.

Annu Rev Pharmacol Toxicol 2017 01 9;57:349-373. Epub 2016 Sep 9.

Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California 92093; email:

Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors (GPCRs) that transduce cellular responses to extracellular proteases. PARs have important functions in the vasculature, inflammation, and cancer and are important drug targets. A unique feature of PARs is their irreversible proteolytic mechanism of activation that results in the generation of a tethered ligand that cannot diffuse away. Despite the fact that GPCRs have proved to be the most successful class of druggable targets, the development of agents that target PARs specifically has been challenging. As a consequence, researchers have taken a remarkable diversity of approaches to develop pharmacological entities that modulate PAR function. Here, we present an overview of the diversity of therapeutic agents that have been developed against PARs. We further discuss PAR biased signaling and the influence of receptor compartmentalization, posttranslational modifications, and dimerization, which are important considerations for drug development.
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http://dx.doi.org/10.1146/annurev-pharmtox-011613-140016DOI Listing
January 2017

Platelet and Erythrocyte Sources of S1P Are Redundant for Vascular Development and Homeostasis, but Both Rendered Essential After Plasma S1P Depletion in Anaphylactic Shock.

Circ Res 2016 Sep 31;119(8):e110-26. Epub 2016 Aug 31.

From the INSERM U970, Paris Cardiovascular Research Centre, 75015 Paris, France (S.L.G., B.M., L.C., V.B., S.M.L.G., N.Z., J.R., P.-L.T., E.C.); Université Sorbonne Paris Cité, Paris, France (S.L.G., B.M., B. Decouture, L.C., P.B., V.B., S.M.L.G., B. Dizier, N.Z., J.R., P.G., P.-L.T., C.B.-L., B.H.-T.-N., E.C.); AP-HP, Hôpital Bicêtre, Service de Biochimie, 94275 Le Kremlin Bicêtre, France (P.T.); Lip(Sys)2-Biochimie appliquée, Université Paris-Sud, Université Paris-Saclay, 92290 Châtenay-Malabry, France (P.T.); INSERM U1140, Faculté de Pharmacie, 75006 Paris, France (B. Decouture, B. Dizier, P.G., C.B.-L.); Center for Vascular Biology, Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, Cornell University, New York (Y.X., T.H.); AP-HP, Hôpital Lariboisière, Physiologie Clinique-Explorations-Fonctionnelles, INSERM U965, 75010, Paris, France (P.B.); Australian Centre for Blood Diseases & Department of Clinical Haematology, Monash University, Melbourne, Australia (J.R.H.); AP-HP, Hôpital Européen Georges Pompidou, Service d'Hématologie Biologique, Paris, France (P.G.); Department of Molecular and Cellular Neuroscience, Dorris Neuroscience Center, The Scripps Research Institute, La Jolla, CA (J.C.); Cardiovascular Research Institute, University of California, San Francisco (S.R.C.); and INSERM U698, 75018 Paris, France (B.H.-T.-N.).

Rationale: Sphingosine-1-phosphate (S1P) signaling is essential for vascular development and postnatal vascular homeostasis. The relative importance of S1P sources sustaining these processes remains unclear.

Objective: To address the level of redundancy in bioactive S1P provision to the developing and mature vasculature.

Methods And Results: S1P production was selectively impaired in mouse platelets, erythrocytes, endothelium, or smooth muscle cells by targeted deletion of genes encoding sphingosine kinases -1 and -2. S1P deficiency impaired aggregation and spreading of washed platelets and profoundly reduced their capacity to promote endothelial barrier function ex vivo. However, and in contrast to recent reports, neither platelets nor any other source of S1P was essential for vascular development, vascular integrity, or hemostasis/thrombosis. Yet rapid and profound depletion of plasma S1P during systemic anaphylaxis rendered both platelet- and erythrocyte-derived S1P essential for survival, with a contribution from blood endothelium observed only in the absence of circulating sources. Recovery was sensitive to aspirin in mice with but not without platelet S1P, suggesting that platelet activation and stimulus-response coupling is needed. S1P deficiency aggravated vasoplegia in this model, arguing a vital role for S1P in maintaining vascular resistance during recovery from circulatory shock. Accordingly, the S1P2 receptor mediated most of the survival benefit of S1P, whereas the endothelial S1P1 receptor was dispensable for survival despite its importance for maintaining vascular integrity.

Conclusions: Although source redundancy normally secures essential S1P signaling in developing and mature blood vessels, profound depletion of plasma S1P renders both erythrocyte and platelet S1P pools necessary for recovery and high basal plasma S1P levels protective during anaphylactic shock.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064286PMC
http://dx.doi.org/10.1161/CIRCRESAHA.116.308929DOI Listing
September 2016

Discovery and antiplatelet activity of a selective PI3Kβ inhibitor (MIPS-9922).

Eur J Med Chem 2016 Oct 10;122:339-351. Epub 2016 Jun 10.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia. Electronic address:

A series of amino-substituted triazines were developed and examined for PI3Kβ inhibition and anti-platelet function. Structural adaptations of a morpholine ring of the prototype pan-PI3K inhibitor ZSTK474 yielded PI3Kβ selective compounds, where the selectivity largely derives from an interaction with the non-conserved Asp862 residue, as shown by site directed mutagenesis. The most PI3Kβ selective inhibitor from the series was studied in detail through a series of in vitro and in vivo functional studies. MIPS-9922, 10 potently inhibited ADP-induced washed platelet aggregation. It also inhibited integrin αIIbβ3 activation and αIIbβ3 dependent platelet adhesion to immobilized vWF under high shear. It prevented arterial thrombus formation in the in vivo electrolytic mouse model of thrombosis without inducing prolonged bleeding or excess blood loss.
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http://dx.doi.org/10.1016/j.ejmech.2016.06.010DOI Listing
October 2016

Combined deficiency of PI3KC2α and PI3KC2β reveals a nonredundant role for PI3KC2α in regulating mouse platelet structure and thrombus stability.

Platelets 2016 Jul 4;27(5):402-9. Epub 2016 Mar 4.

a Australian Centre for Blood Diseases , Monash University , Melbourne , VIC , Australia.

The physiological functions and cellular signaling of Class II phosphoinositide 3-kinases (PI3Ks) remain largely unknown. Platelets express two Class II PI3Ks: PI3KC2α and PI3KC2β. PI3KC2α deficiency was recently reported to cause disruption of the internal membrane reserve structure of platelets (open canalicular system, OCS) that results in dysregulated platelet adhesion and impaired arterial thrombosis in vivo. Notably, these effects on platelets occurred despite normal agonist-induced 3-phosphorylated phosphoinositide (3-PPI) production and cellular activation in PI3KC2α-deficient platelets. However, the potential compensatory actions of PI3KC2β in platelets have not yet been investigated. Here, we report the first mice deficient in both PI3KC2α and PI3KC2β (no Class II PI3Ks in platelets) and reveal a nonredundant role for PI3KC2α in mouse platelet structure and function. Specifically, we show that the disrupted OCS and impaired thrombus stability observed in PI3KC2α-deficient platelets does not occur in PI3KC2β-deficient platelets and is not exaggerated in platelets taken from mice deficient in both enzymes. Furthermore, detailed examination of 3-PPI production in platelets from this series of mice revealed no changes in either unactivated or activated platelets, including those with a complete lack of Class II PI3Ks. These findings indicate a nonredundant role for PI3KC2α in regulating platelet structure and function, and suggest that Class II PI3Ks do not significantly contribute to the acute agonist-induced production of 3-PPIs in these cells.
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http://dx.doi.org/10.3109/09537104.2016.1145202DOI Listing
July 2016

Protease-activated receptor 4: from structure to function and back again.

Br J Pharmacol 2016 10 10;173(20):2952-65. Epub 2016 Mar 10.

Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.

Protease-activated receptors are a family of four GPCRs (PAR1-PAR4) with a number of unique attributes. Nearly two and a half decades after the discovery of the first PAR, an antagonist targeting this receptor has been approved for human use. The first-in-class PAR1 antagonist, vorapaxar, was approved for use in the USA in 2014 for the prevention of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. These recent developments indicate the clinical potential of manipulating PAR function. While much work has been aimed at uncovering the function of PAR1 and, to a lesser extent, PAR2, comparatively little is known regarding the pharmacology and physiology of PAR3 and PAR4. Recent studies have begun to develop the pharmacological and genetic tools required to study PAR4 function in detail, and there is now emerging evidence for the function of PAR4 in disease settings. In this review, we detail the discovery, structure, pharmacology, physiological significance and therapeutic potential of PAR4. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.
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http://dx.doi.org/10.1111/bph.13455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341247PMC
October 2016

Thrombin-induced reactive oxygen species generation in platelets: A novel role for protease-activated receptor 4 and GPIbα.

Redox Biol 2015 Dec 28;6:640-647. Epub 2015 Oct 28.

Department of Experimental Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland; Faculty of Health Sciences, Curtin University, Perth, Australia; Curtin Health Innovation Research Institute, Curtin University, Perth, Australia. Electronic address:

Background: Platelets are essential for maintaining haemostasis and play a key role in the pathogenesis of cardiovascular disease. Upon ligation of platelet receptors through subendothelial matrix proteins, intracellular reactive oxygen species (ROS) are generated, further amplifying the platelet activation response. Thrombin, a potent platelet activator, can signal through GPIbα and protease-activated receptor (PAR) 1 and PAR4 on human platelets, and recently has been implicated in the generation of ROS. While ROS are known to have key roles in intra-platelet signalling and subsequent platelet activation, the precise receptors and signalling pathways involved in thrombin-induced ROS generation have yet to be fully elucidated.

Objective: To investigate the relative contribution of platelet GPIbα and PARs to thrombin-induced reactive oxygen species (ROS) generation.

Methods And Results: Highly specific antagonists targeting PAR1 and PAR4, and the GPIbα-cleaving enzyme, Naja kaouthia (Nk) protease, were used in quantitative flow cytometry assays of thrombin-induced ROS production. Antagonists of PAR4 but not PAR1, inhibited thrombin-derived ROS generation. Removal of the GPIbα ligand binding region attenuated PAR4-induced and completely inhibited thrombin-induced ROS formation. Similarly, PAR4 deficiency in mice abolished thrombin-induced ROS generation. Additionally, GPIbα and PAR4-dependent ROS formation were shown to be mediated through focal adhesion kinase (FAK) and NADPH oxidase 1 (NOX1) proteins.

Conclusions: Both GPIbα and PAR4 are required for thrombin-induced ROS formation, suggesting a novel functional cooperation between GPIbα and PAR4. Our study identifies a novel role for PAR4 in mediating thrombin-induced ROS production that was not shared by PAR1. This suggests an independent signalling pathway in platelet activation that may be targeted therapeutically.
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http://dx.doi.org/10.1016/j.redox.2015.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4656914PMC
December 2015

The class II PI 3-kinase, PI3KC2α, links platelet internal membrane structure to shear-dependent adhesive function.

Nat Commun 2015 Mar 17;6:6535. Epub 2015 Mar 17.

1] Australian Centre for Blood Diseases, Monash University, Level 6, 89 Commercial Road, Melbourne, Victoria 3004, Australia [2] The Heart Research Institute and Charles Perkins Centre, The University of Sydney, Newtown 2050, Australia [3] Department of Molecular and Experimental Medicine, The Scripps Research Institute, San Diego, CA 92037, USA.

PI3KC2α is a broadly expressed lipid kinase with critical functions during embryonic development but poorly defined roles in adult physiology. Here we utilize multiple mouse genetic models to uncover a role for PI3KC2α in regulating the internal membrane reserve structure of megakaryocytes (demarcation membrane system) and platelets (open canalicular system) that results in dysregulated platelet adhesion under haemodynamic shear stress. Structural alterations in the platelet internal membrane lead to enhanced membrane tether formation that is associated with accelerated, yet highly unstable, thrombus formation in vitro and in vivo. Notably, agonist-induced 3-phosphorylated phosphoinositide production and cellular activation are normal in PI3KC2α-deficient platelets. These findings demonstrate an important role for PI3KC2α in regulating shear-dependent platelet adhesion via regulation of membrane structure, rather than acute signalling. These studies provide a link between the open canalicular system and platelet adhesive function that has relevance to the primary haemostatic and prothrombotic function of platelets.
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http://dx.doi.org/10.1038/ncomms7535DOI Listing
March 2015

Class II but Not Second Class-Prospects for the Development of Class II PI3K Inhibitors.

ACS Med Chem Lett 2015 Jan 24;6(1):3-6. Epub 2014 Sep 24.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus) , 381 Royal Parade, Parkville, Victoria 3052, Australia.

The Class II PI3 kinases are emerging from the shadows of their Class I cousins. The data emerging from PIK3C2 genetic modification studies and from siRNA knockdown suggest important roles in physiology and pathology. With some well-studied Class I isoform inhibitors showing strong Class II activity and a wealth of crystallographic information available, the structural similarity of these isoforms to Class I provides both the opportunity and the challenge in design of selective pharmacological inhibitors.
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http://dx.doi.org/10.1021/ml500354eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4291705PMC
January 2015

Approval of the first protease-activated receptor antagonist: Rationale, development, significance, and considerations of a novel anti-platelet agent.

Blood Rev 2015 May 6;29(3):179-89. Epub 2014 Nov 6.

Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria, Australia; Department of Clinical Haematology, Monash University, Melbourne, Victoria, Australia. Electronic address:

Twenty-three years after the discovery of the first thrombin receptor, now known as protease-activated receptor 1 (PAR1), the first drug targeting this receptor is available for human use. The PAR1 inhibitor, vorapaxar (Zontivity, MSD), was recently approved by the FDA for use in the USA for the prevention of thrombotic cardiovascular events in patients with a history of myocardial infarction or peripheral artery disease. In this review, we detail the rationale, development, as well as the clinical significance and considerations of vorapaxar, the original PAR antagonist and the latest anti-platelet agent in the pharmaco-armoury against arterial thrombosis.
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http://dx.doi.org/10.1016/j.blre.2014.10.006DOI Listing
May 2015

Differential signaling by protease-activated receptors: implications for therapeutic targeting.

Int J Mol Sci 2014 Apr 11;15(4):6169-83. Epub 2014 Apr 11.

Australian Centre for Blood Diseases & Department of Clinical Haematology, Monash University, Melbourne 3004, Australia.

Protease-activated receptors (PARs) are a family of four G protein-coupled receptors that exhibit increasingly appreciated differences in signaling and regulation both within and between the receptor class. By nature of their proteolytic self-activation mechanism, PARs have unique processes of receptor activation, "ligand" binding, and desensitization/resensitization. These distinctive aspects have presented both challenges and opportunities in the targeting of PARs for therapeutic benefit-the most notable example of which is inhibition of PAR1 on platelets for the prevention of arterial thrombosis. However, more recent studies have uncovered further distinguishing features of PAR-mediated signaling, revealing mechanisms by which identical proteases elicit distinct effects in the same cell, as well as how distinct proteases produce different cellular consequences via the same receptor. Here we review this differential signaling by PARs, highlight how important distinctions between PAR1 and PAR4 are impacting on the progress of a new class of anti-thrombotic drugs, and discuss how these more recent insights into PAR signaling may present further opportunities for manipulating PAR activation and signaling in the development of novel therapies.
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http://dx.doi.org/10.3390/ijms15046169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4013622PMC
April 2014

Mondor's Disease of the Penis.

West J Emerg Med 2013 Mar;14(2):180

Madigan Army Medical Center, Department of Emergency Medicine, Tacoma, Washington.

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http://dx.doi.org/10.5811/westjem.2012.8.13276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3628478PMC
March 2013
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