Biology subjects

Godwin, M.

Publications and source records attributed to Godwin, M..

5 recordsLinked to original sources

bioRxiv2024

Targeting an Olfactory Receptor Mitigates High Residual Platelet Reactivity and Arterial Thrombosis Through Actin Cytoskeleton Depolymerization

BackgroundDespite antiplatelet therapy, some patients remain at high risk for ischemic events due to medication non-responsiveness or High Residual Platelet Reactivity (HRPR). Our goal was to target an orphan G-Protein-Coupled Receptor (GPCR) on platelets belonging to the olfactory receptor family as a new antithrombotic strategy. MethodsUsing an engineered reporter cell line expressing Olfactory Receptor 2L13 (OR2L13) that was recently identified as an orphan GPCR to limit platelet reactivity, we performed a high-throughput screen (HST) of non-odorant bioactive compounds with counter-screen validation, followed by studies to determine changes in platelet function in healthy subjects and in patients with coronary artery disease and peripheral artery disease. Phospho-proteomics identified key signal transduction pathways, and a variety of ex vivo assays and in vivo functional studies examined the impact of an identified non-odorant compound on platelet signaling, platelet biomechanics, and thrombosis. ResultsWe identified 6 ligands specific for OR2L13 that function as receptor agonists, leading to the suppression of platelet aggregation and -granule exocytosis via P2Y12, PAR1, Thromboxane Receptor (TxR) and glycoprotein VI (GPVI) receptors, suggesting involvement of common downstream mediator. The lead OR2L13 agonist identified phosphorylates heat shock protein 27 (HSP27) and depolymerizes the platelet filamentous actin cytoskeleton which was further confirmed in a clot retraction assay (CCF0054500 clot area 70.6 vs. vehicle clot area 5.2, P<0.0001). This anti-thrombotic effect of the OR2L13 agonist was reversed by a HSP27 inhibitor (clot area 3.6, P<0.0001). In a murine cremaster arterial injury model, platelet accumulation at the injury site was reduced by 88.9% by the lead OR2L13 agonist compared to vehicle (P<0.0003) without altering fibrin generation in vivo, or interfering with the coagulation cascade, and without impairing the protective mechanism of platelet hemostasis. ConclusionWe describe and characterize the first non-olfactory tool to target an olfactory receptor for the purpose of inhibiting platelet activation and thrombosis through downstream HSP27 in a comprehensive investigation using a first-of-its kind platelet inhibitor targeting an orphan platelet GPCR.

physiology↗

bioRxiv2023

Protection of β2GPI Deficient Mice from Thrombosis Reflects a Defect in PAR3-facilitated Platelet Activation

BackgroundAntibodies to {beta}2-glycoprotein I ({beta}2GPI) cause thrombosis in antiphospholipid syndrome, however the role of {beta}2GPI itself in regulation of coagulation pathways in vivo is not well understood. MethodsWe developed {beta}2GPI-deficient mice (Apoh-/-) by deleting exon 2 and 3 of Apoh using CRISPR/Cas9 and compared the propensity of wild-type (WT) and Apoh-/-mice to develop thrombosis using rose bengal and FeCl3-induced carotid thrombosis, laser-induced cremaster arteriolar injury, and inferior vena cava (IVC) stasis models. We also compared tail bleeding times and assessed platelet activation in WT and Apoh-/- mice in the absence and presence of exogenous {beta}2GPI. ResultsCompared to WT littermates, Apoh-/- mice demonstrated a prolonged time to occlusion of the carotid artery after exposure to rose bengal or FeCl3, and reduced platelet and fibrin accumulation in cremasteric arterioles after laser injury. Similarly, significantly smaller thrombi were retrieved from the IVC of Apoh-/-mice 48 hours after IVC occlusion. The activated partial thromboplastin time (aPTT) and prothrombin time, as well as aPTT reagent- and tissue factor-induced thrombin generation times using plasma from Apoh-/- and WT mice revealed no differences. However, we observed significant prolongation of tail bleeding in Apoh-/- mice, and reduced P-selectin expression and binding of fibrinogen to the activated 2b{beta}3 integrin on platelets from these mice after stimulation with low thrombin concentrations; these changes were reversed by exogenous {beta}2GPI. An antibody to PAR3 blocked thrombin-induced activation of WT, but not Apoh-/- platelets, as well as the ability of {beta}2GPI to restore the activation response of Apoh-/- platelets to thrombin. {beta}2GPI deficiency did not affect platelet activation by a PAR4-activator peptide, or ADP. ConclusionsIn mice, {beta}2GPI may mediate procoagulant activity by enhancing the ability of PAR3 to present thrombin to PAR4, promoting platelet activation at low thrombin concentrations. Key PointsO_LI{beta}2GPI deficient mice are protected from experimental arterial, venous, and microvascular thrombosis. C_LIO_LI{beta}2GPI deficient mice display prolonged tail bleeding times and reduced PAR3-facilitated platelet activation by low concentrations of thrombin. C_LI

cell biology↗

bioRxiv2023

Dysregulated Platelet Function and Thrombosis in Patients with Post-Acute Sequelae of COVID-19

BackgroundPost-acute sequelae of COVID-19 (PASC), also referred as Long-COVID, sometimes follows COVID-19, a disease caused by SARS-CoV-2. While SARS-CoV-2 is well-known to promote a prothrombotic state, less is known about the thrombosis risk in PASC. AimOur objective was to evaluate the platelet function and thrombotic potential in patients following recovery from SARS-CoV-2 with clear symptoms of PASC. MethodsPASC patients and matched healthy controls were enrolled in the study on average 15 months after documented SARS-CoV-2 infection. Platelet activation was evaluated by Light Transmission Aggregometry (LTA) and flow cytometry in response to platelet surface receptor agonists. Thrombosis in platelet-deplete plasma was evaluated by Factor Xa activity. A microfluidics system assessed thrombosis in whole blood under shear stress conditions. ResultsA mild increase in platelet aggregation in PASC patients through the thromboxane receptor was observed and platelet activation through the glycoprotein VI (GPVI) receptor was decreased in PASC patients compared to age- and sex-matched healthy controls. Thrombosis under shear conditions as well as Factor Xa activity were reduced in PASC patients. Plasma from PASC patients was an extremely potent activator of washed, healthy platelets - a phenomenon not observed when stimulating healthy platelets after incubation with plasma from healthy individuals. ConclusionsPASC patients show dysregulated responses in platelets and coagulation in plasma, likely caused by a circulating molecule that promotes thrombosis. A hitherto undescribed protective response appears to exists in PASC patients to counterbalance ongoing thrombosis that is common to SARS-CoV-2 infection.

pharmacology and toxicology↗

bioRxiv2022

Cellular, Molecular, and Enzymatic Signatures ofThrombi are Vascular Bed-Dependent

BackgroundThe contribution of arterial and venous thrombi to vascular remodeling is unclear. While catheter-extraction of thrombus in cerebrovascular accident (CVA) is time-sensitive, similar urgency is rare in managing venous thromboembolism (VTE). ObjectivesOur goal was to determine molecular cellular signatures of thrombus extracted by catheter from various vascular beds to gain insight into vascular remodeling. MethodsTwenty-five patients underwent catheter-directed thrombectomy (CDT), 13 for acute CVA, 8 for pulmonary embolism (PE), and 4 for deep vein thrombosis (DVT). Protein and RNA extracted from thrombus was evaluated by immunoblotting and sequencing, respectively. Thrombus-derived enzymes for which substrate is present in the blood vessel wall were examined for enzymatic activity. ResultsTime from symptom onset to thrombus extraction was 7.7 {+/-} 1.9 hours for CVA and 109 {+/-} 55 hours for VTE. Protein concentration, white blood cell and red blood cell content were all greater in venous compared with arterial thrombus while platelet content was similar. Both venous and arterial thrombus contained multiple Matrix Metalloproteinase (MMP) isoforms. MMP9 specific activity was greater in venous than in arterial thrombus (57 {+/-} 6 ng/mL.g protein-1 vs. 24 {+/-} 8 ng/mL.g protein-1, P=0.0051). ConclusionsArterial and venous thrombus have dissimilar phenotypes, each with biologically-active enzymes known to remodel blood vessels, and enzymatic activity proportional to the white blood cell content which increases with thrombus age. These data suggest a mechanistically-important role for early CDT to avoid the consequences of irreversible vascular remodeling. Condensed AbstractEmergent extraction of acute thrombus from arterial vascular beds restores limb and end-organ perfusion and is widely-accepted to be the standard of care. Extraction of thrombus from venous vascular beds, however, is rarely considered urgent, even though many patients subsequently develop debilitating symptoms. By capitalizing on privileged thrombus extracted from multiple vascular beds, we gained mechanistic insight regarding the cellular composition and cell-derived enzymes secreted from thrombus that may remodel the vessel wall. This study shows thrombi are biologically-active entities, continuously recruiting circulating cells that secrete enzymes both proportional to thrombus age and the time of patient presentation.

biochemistry↗

bioRxiv2018

DAF-16/Foxo suppresses the transgenerational sterility of prg-1 piRNA mutants via a systemic small RNA pathway

Mutation of the daf-2 insulin/IGF-1 receptor activates the DAF-16/Foxo transcription factor to suppress the transgenerational sterility phenotype of prg-1/piRNA mutants that are deficient for piRNA-mediated genome silencing. As with PRG-1/piRNAs, mutations in the nuclear RNA interference gene nrde-1 compromised germ cell immortality, but deficiency for daf-2 did not suppress the transgenerational sterility of nrde-1 or nrde-4 single mutants or of prg-1; nrde-4 or prg-1; hrde-1 double mutants. NRDE-1 and NRDE-4 promote transcriptional silencing in somatic cells via the nuclear Argonaute protein NRDE-3, which was dispensable for germ cell immortality. However, daf-2 deficiency failed to promote germ cell immortality in prg-1; nrde-3 mutants. Consistently, we found that DAF-16 activity in somatic cells suppressed the transgenerational sterility of prg-1 mutants via the SID-1 dsRNA transmembrane channel that promotes systemic RNAi as well as Dicer, the dsRNA binding protein RDE-4 and the RDRP RRF-3. We conclude that DAF-16 activates a cell-non-autonomous systemic RNAi pathway that promotes small RNA-mediated genome silencing in germ cells to suppress loss of the genomic immune surveillance factor Piwi/PRG-1. Author SummarySmall RNAs can promote genome silencing. The Argonaute protein Piwi interacts with thousands of small RNAs termed piRNAs in germ cells to suppress expression of transposons and foreign genetic elements. However, the Piwi silencing system may be commonly targeted by viral or transposon genomic parasites that seek to suppress the endogenous defences against their expression and replication. Activation of the DAF-16 stress response pathway promotes adult longevity and can also abolish the transgenerational sterility of C. elegans Piwi mutants. We found that DAF-16 accomplishes this by activating a somatic small RNA pathway where small RNAs are initially produced in the soma and are then transported into the germline to suppress expression of a toxic genetic locus in Piwi mutants. Thus, the DAF-16 stress response pathway activates a systemic small RNA cascade to suppress defects in the Piwi/piRNA genome silencing system.

genetics↗