Species differences in small molecule binding to αIIbβ3 are the result of sequence differences in 2 loops of the αIIb β propeller

Basani, Ramesh B.; Zhu, Hua; Thornton, Michael A.; Soto, Cinque; DeGrado, William F.; Kowalska, M. Anna; Bennett, Joel S.; Poncz, Mortimer

doi:10.1182/blood-2008-09-177337

Cited by 14 publications

(20 citation statements)

References 47 publications

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“…Species differences in the ligand binding to the GPIIb/IIIa receptors are reported in the literature (13,16,25,26), and rodents were shown to be not appropriate for the in vivo evaluation of this class of new receptor ligands. Species-dependent effects are also reported for elarofiban, necessitating the use of human blood or thrombi for binding studies and autoradiography as well as establishing a nonhuman primate model for PET imaging of thrombi.…”

Section: Discussionmentioning

confidence: 99%

¹⁸F-GP1, a Novel PET Tracer Designed for High-Sensitivity, Low-Background Detection of Thrombi

et al. 2017

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Thromboembolic diseases such as myocardial infarction, stroke, transient ischemic attacks, and pulmonary embolism are major causes of morbidity and mortality worldwide. Glycoprotein IIb/IIIa (GPIIb/IIIa) is the key receptor involved in platelet aggregation and is a validated target for therapeutic approaches and diagnostic imaging. The aim of this study was to develop and characterize a specific small-molecule tracer for PET imaging that binds with high affinity to GPIIb/IIIa receptors and has suitable pharmacokinetic properties to overcome limitations of previous approaches. Methods: Binding of 18 F-GP1 to GPIIb/IIIa receptors was investigated in competition binding assays and autoradiography using a fresh cardiac thrombus from an explanted human heart. The clot-to-blood ratio for 18 F-GP1 was investigated by an in vitro blood flow model. Biodistribution and thrombus detection was investigated in cynomolgus monkeys after insertion of a roughened catheter into either the vena cava or the aorta. Results: 18 F-GP1 is an 18 F-labeled small molecule for PET imaging of thrombi. The half maximal inhibitory concentration of 18 F-GP1 to GPIIb/IIIa was 20 nM. 18 F-GP1 bound to thrombi with a mean clot-to-blood ratio of 95. Binding was specific and can be displaced by excess nonradioactive derivative. Binding was not affected by anticoagulants such as aspirin or heparin. 18 F-GP1 showed rapid blood clearance and a low background after intravenous injection in cynomolgus monkeys. Small arterial, venous thrombi, thrombotic depositions on damaged endothelial surface, and small cerebral emboli were detected in vivo by PET imaging. Conclusions: 18 F-GP1 binds specifically with high affinity to the GPIIb/IIIa receptor involved in platelet aggregation. Because of its favorable preclinical characteristics, 18 F-GP1 is currently being investigated in a human clinical study.

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Section: Discussionmentioning

confidence: 99%

¹⁸F-GP1, a Novel PET Tracer Designed for High-Sensitivity, Low-Background Detection of Thrombi

et al. 2017

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“…The genotypes of mice containing the h␣IIb transgene and homozygous for the targeted disruption of m␣IIb were confirmed by polymerase chain reaction (PCR), and direct assessment of surface expression of the receptors was performed on washed platelets prepared from platelet-rich plasma (PRP) as previously described. 9,10 Fluorescein isothiocyanate (FITC)-conjugated anti-human CD41 (HIP8; eBioscience) antibody was used to detect h␣IIb, and phycoerythrin-conjugated anti-mouse CD41 (MWReg30; BD Biosciences, San Jose, CA) antibody was used to detect m␣IIb.…”

Section: Generation and Characterization Of Murine Platelets Expressimentioning

confidence: 99%

Structural and therapeutic insights from the species specificity and in vivo antithrombotic activity of a novel αIIb-specific αIIbβ3 antagonist

Blue

Kowalska

Hirsch

et al. 2009

Blood

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We previously reported on a novel compound (Compound 1; RUC-1) identified by high-throughput screening that inhibits human ␣IIb␤3. RUC-1 did not inhibit ␣V␤3, suggesting that it interacts with ␣IIb, and flexible ligand/rigid protein molecular docking studies supported this speculation. We have now studied RUC-1's effects on murine and rat platelets, which are less sensitive than human to inhibition by Arg-Gly-Asp (RGD) peptides due to differences in the ␣IIb sequences contributing to the binding pocket. We found that RUC-1 was much less potent in inhibiting aggregation of murine and rat platelets. Moreover, RUC-1 potently inhibited fibrinogen binding to murine platelets expressing a hybrid ␣IIb␤3 receptor composed of human ␣IIb and murine ␤3, but not a hybrid receptor composed of murine ␣IIb and human ␤3. Molecular docking studies of RUC-1 were consistent with the functional data. In vivo studies of RUC-1 administered intraperitoneally at a dose of 26.5 mg/kg demonstrated antithrombotic effects in both ferric chloride carotid artery and laser-induced microvascular injury models in mice with hybrid h␣IIb/m␤3 receptors. Collectively, these data support RUC-1's specificity for ␣IIb, provide new insights into the ␣IIb binding pocket, and establish RUC-1's antithrombotic effects in vivo. (Blood. 2009;114:195-201) IntroductionWe previously published data on the identification of a novel inhibitor of ␣IIb␤3 (Compound 1; now referred to as RUC-1). 1 We speculated that it interacted exclusively with the ␣IIb portion of the Arg-Gly-Asp (RGD) binding site based on its specificity for ␣IIb␤3 compared with ␣V␤3 and molecular docking studies into the human ␣IIb␤3 headpiece suggesting that the positively charged piperazinyl nitrogen of RUC-1 interacts with the carboxyl group of D224 in ␣IIb and that the heterocyclic fused ring of RUC-1 interacts with one or more of the 3 aromatic residues that line the ␣IIb pocket. RUC-1 also is too short to span between D224 of ␣IIb and the ␤3 metal ion-dependent adhesion site (MIDAS) and lacks a carboxyl group to coordinate the MIDAS metal ion, which is an invariant feature of all other small molecule ␣IIb␤3 antagonists. [2][3][4] In the present study, we further tested whether RUC-1 demonstrates specificity for ␣IIb by taking advantage of known differences in the abilities of ␣IIb␤3 antagonists to inhibit ␣IIb␤3-mediated platelet aggregation in different species. Consistent with these data, we also found that RUC-1 could inhibit thrombus formation in vivo in transgenic mice expressing human (h) ␣IIb in complex with murine (m) ␤3, but not wild-type (WT) mice. Estimates of electrostatic and van der Waals interaction energies of RUC-1 docked into the crystal structure of human ␣IIb␤3 or molecular models of rat ␣IIb␤3, mouse ␣IIb␤3, or hybrid human ␣IIb/mouse␤3 were consistent with the functional data. In aggregate, these data have important implications for understanding the structure of the ␣IIb binding pocket and the potential antiplatelet effects of ␣IIb-specific ␣IIb␤3 antagonists. Me...

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“…on April 8, 2019. by guest www.bloodjournal.org From human platelet incorporation, 100 mL of 250 mM tirofiban (Ben Venue Laboratories), a species-specific inhibitor of human CD41, 31 was injected after control injuries had been recorded. After tirofiban infusion, additional injuries were made.…”

Section: Cremaster Laser Injury Functional Studiesmentioning

confidence: 99%

Comparative analysis of human ex vivo–generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale

Wang

Hayes

Jarocha

et al. 2015

Blood

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• Infused human megakaryocytes release young platelets in the lungs with characteristics similar to donor platelets.• Platelets released from ex vivo-derived megakaryocytes are preactivated and compare poorly to donor platelets.Thrombopoiesis is the process by which megakaryocytes release platelets that circulate as uniform small, disc-shaped anucleate cytoplasmic fragments with critical roles in hemostasis and related biology. The exact mechanism of thrombopoiesis and the maturation pathways of platelets released into the circulation remain incompletely understood. We showed that ex vivo-generated murine megakaryocytes infused into mice release platelets within the pulmonary vasculature. Here we now show that infused human megakaryocytes also release platelets within the lungs of recipient mice. In addition, we observed a population of platelet-like particles (PLPs) in the infusate, which include platelets released during ex vivo growth conditions. By comparing these 2 platelet populations to human donor platelets, we found marked differences: platelets derived from infused megakaryocytes closely resembled infused donor platelets in morphology, size, and function. On the other hand, the PLP was a mixture of nonplatelet cellular fragments and nonuniform-sized, preactivated platelets mostly lacking surface CD42b that were rapidly cleared by macrophages. These data raise a cautionary note for the clinical use of human platelets released under standard ex vivo conditions. In contrast, human platelets released by intrapulmonary-entrapped megakaryocytes appear more physiologic in nature and nearly comparable to donor platelets for clinical application. (Blood. 2015;125(23):3627-3636)

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Species differences in small molecule binding to αIIbβ3 are the result of sequence differences in 2 loops of the αIIb β propeller

Cited by 14 publications

References 47 publications

¹⁸F-GP1, a Novel PET Tracer Designed for High-Sensitivity, Low-Background Detection of Thrombi

¹⁸F-GP1, a Novel PET Tracer Designed for High-Sensitivity, Low-Background Detection of Thrombi

Structural and therapeutic insights from the species specificity and in vivo antithrombotic activity of a novel αIIb-specific αIIbβ3 antagonist

Comparative analysis of human ex vivo–generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale

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Species differences in small molecule binding to αIIbβ3 are the result of sequence differences in 2 loops of the αIIb β propeller

Cited by 14 publications

References 47 publications

18F-GP1, a Novel PET Tracer Designed for High-Sensitivity, Low-Background Detection of Thrombi

18F-GP1, a Novel PET Tracer Designed for High-Sensitivity, Low-Background Detection of Thrombi

Structural and therapeutic insights from the species specificity and in vivo antithrombotic activity of a novel αIIb-specific αIIbβ3 antagonist

Comparative analysis of human ex vivo–generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale

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Product

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¹⁸F-GP1, a Novel PET Tracer Designed for High-Sensitivity, Low-Background Detection of Thrombi

¹⁸F-GP1, a Novel PET Tracer Designed for High-Sensitivity, Low-Background Detection of Thrombi