Dataset Information

Fast clearance of platelets in a commonly used mouse model for GPIbα is impeded by an anti-GPIbβ antibody derivative.

ABSTRACT:

Background

Glycoprotein (GP)Ibα plays a critical role in regulating platelet clearance. Recently, we identified the mechanosensory domain (MSD) in GPIbα and reported evidence to suggest that unfolding of the GPIbα MSD induces exposure of the Trigger sequence therein and subsequent GPIb-IX signaling that accelerates platelet clearance. In a commonly used transgenic mouse model, IL4R-IbαTg, where the Trigger sequence is constitutively exposed, constitutive GPIb-IX-mediated cellular signals are present. Clearance of their platelets is also significantly faster than that of wild-type mice. Previously, an anti-GPIbβ antibody RAM.1 was developed. RAM.1 inhibits GPIbα-dependent platelet signaling and activation. Further, RAM.1 also inhibits anti-GPIbα antibody-mediated filopodia formation.

Objective

To investigate whether RAM.1 can ameliorate trigger sequence exposure-mediated platelet clearance.

Methods

Spontaneous filopodia were measured by confocal microscopy. Other platelet signaling events were measured by flow cytometry. Endogenous platelet life span was tracked by Alexa 488-labeled anti-mouse GPIX antibody.

Result

Transfected Chinese hamster ovary cells stably expressing the same chimeric IL4R-Ibα protein complex as in IL4R-IbαTg mice also constitutively exhibit filopodia, and that such filopodia could be abolished by treatment of RAM.1. Further, transfusion of a recombinant RAM.1 derivative that is devoid of its Fc portion significantly extends the endogenous life span of IL4R-IbαTg platelets.

Conclusion

These results provide the key evidence supporting the causative link of Trigger sequence exposure to accelerated platelet clearance, and suggest that a RAM.1 derivative may be therapeutically developed to treat GPIb-IX-mediated thrombocytopenia.

SUBMITTER: Chen W

PROVIDER: S-EPMC9133214 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Publications

Fast clearance of platelets in a commonly used mouse model for GPIbα is impeded by an anti-GPIbβ antibody derivative.

Chen Wenchun W Wilson Moriah S MS Wang Yingchun Y Bergmeier Wolfgang W Lanza Francois F Li Renhao R

Journal of thrombosis and haemostasis : JTH 20220329 6

<h4>Background</h4>Glycoprotein (GP)Ibα plays a critical role in regulating platelet clearance. Recently, we identified the mechanosensory domain (MSD) in GPIbα and reported evidence to suggest that unfolding of the GPIbα MSD induces exposure of the Trigger sequence therein and subsequent GPIb-IX signaling that accelerates platelet clearance. In a commonly used transgenic mouse model, IL4R-IbαTg, where the Trigger sequence is constitutively exposed, constitutive GPIb-IX-mediated cellular signals ...[more]

PMID: 35305057

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Project description:BackgroundThe glycoprotein (GP) Ib-IX-V complex is a unique platelet plasma membrane receptor, which is essential for platelet adhesion and thrombus formation. GPIbα, part of the GPIb-IX-V complex, has several physiological ligands such as von Willebrand factor (vWF), thrombospondin and distinct coagulation factors, which trigger platelet activation. Despite having an important role, intracellular GPIb-IX-V signaling and its regulation by other pathways are not well defined. Our aim was to establish the intracellular signaling response of selective GPIbα activation in human platelets, in particular the role of the tyrosine kinase Syk and its regulation by cAMP/PKA and cGMP/PKG pathways, respectively. We addressed this using echicetin beads (EB), which selectively bind to GPIbα and induce platelet aggregation.MethodsPurified echicetin from snake Echis carinatus venom was validated by mass spectrometry. Washed human platelets were incubated with EB, in the presence or absence of echicetin monomers (EM), Src family kinase (SFK) inhibitors, Syk inhibitors and the cAMP- and cGMP-elevating agents iloprost and riociguat, respectively. Platelet aggregation was analyzed by light transmission aggregometry, protein phosphorylation by immunoblotting. Intracellular messengers inositolmonophosphate (InsP1) and Ca2+i were measured by ELISA and Fluo-3 AM/FACS, respectively.ResultsEB-induced platelet aggregation was dependent on integrin αIIbβ3 and secondary mediators ADP and TxA2, and was antagonized by EM. EB stimulated Syk tyrosine phosphorylation at Y352, which was SFK-dependent and Syk-independent, whereas Y525/526 phosphorylation was SFK-dependent and partially Syk-dependent. Furthermore, phosphorylation of both Syk Y352 and Y525/526 was completely integrin αIIbβ3-independent but, in the case of Y525/526, was partially ADP/TxA2-dependent. Syk activation, observed as Y352/ Y525/Y526 phosphorylation, led to the phosphorylation of direct substrates (LAT Y191, PLCγ2 Y759) and additional targets (Akt S473). PKA/PKG pathways inhibited EB-induced platelet aggregation and Akt phosphorylation but, surprisingly, enhanced Syk and LAT/PLCγ2 tyrosine phosphorylation. A similar PKA/PKG effect was confirmed with convulxin-/GPVI-stimulated platelets. EB-induced InsP1 accumulation/InsP3 production and Ca2+-release were Syk-dependent, but only partially inhibited by PKA/PKG pathways.ConclusionEB and EM are specific agonists and antagonists, respectively, of GPIbα-mediated Syk activation leading to platelet aggregation. The cAMP/PKA and cGMP/PKG pathways do not inhibit but enhance GPIbα-/GPVI-initiated, SFK-dependent Syk activation, but strongly inhibit further downstream responses including aggregation. These data establish an important intracellular regulatory network induced by GPIbα.

Project description:BackgroundSeveral validated scales have been developed to measure frailty, yet the direct relationship between these measures and their scores remains unknown. To bridge this gap, we created a crosswalk of the most commonly used frailty scales.MethodsWe used data from 7070 community-dwelling older adults who participated in National Health and Aging Trends Study (NHATS) Round 5 to construct a crosswalk among frailty scales. We operationalized the Study of Osteoporotic Fracture Index (SOF), FRAIL Scale, Frailty Phenotype, Clinical Frailty Scale (CFS), Vulnerable Elder Survey-13 (VES-13), Tilburg Frailty Indictor (TFI), Groningen Frailty Indicator (GFI), Edmonton Frailty Scale (EFS), and 40-item Frailty Index (FI). A crosswalk between FI and the frailty scales was created using the equipercentile linking method, a statistical procedure that produces equivalent scoring between scales according to percentile distributions. To demonstrate its validity, we determined the 4-year mortality risk across all scales for low-risk (equivalent to FI <0.20), moderate-risk (FI 0.20 to <0.40), and high-risk (FI ≥0.40) categories.ResultsUsing NHATS, the feasibility of calculating frailty scores was at least 90% for all nine scales, with the FI having the highest number of calculable scores. Participants considered frail on FI (cutpoint of 0.25) corresponded to the following scores on each frailty measure: SOF 1.3, FRAIL 1.7, Phenotype 1.7, CFS 5.3, VES-13 5.5, TFI 4.4, GFI 4.8, and EFS 5.8. Conversely, individuals considered frail according to the cutpoint of each frailty measure corresponded to the following FI scores: 0.37 for SOF, 0.40 for FRAIL, 0.42 for Phenotype, 0.21 for CFS, 0.16 for VES-13, 0.28 for TFI, 0.21 for GFI, and 0.37 for EFS. Across frailty scales, the 4-year mortality risks between the same categories were similar in magnitude.ConclusionOur results provide clinicians and researchers with a useful tool to directly compare and interpret frailty scores across scales.

Dataset Information

Fast clearance of platelets in a commonly used mouse model for GPIbα is impeded by an anti-GPIbβ antibody derivative.

Background

Objective

Methods

Result

Conclusion

Publications

Fast clearance of platelets in a commonly used mouse model for GPIbα is impeded by an anti-GPIbβ antibody derivative.

Similar Datasets

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