Activated platelets retain and protect most of their factor XIII-A cargo from proteolytic activation and degradation

The authors thank László Muszbek for the polyclonal anti-FXIII-Aantibody,Abigail R. Ballardfor technical support, Pablo Ariel for help with the immunofluorescence imaging and analysis, Marina Sokolsky for help with nanoparticle tracking analysis,and Nigel Mackman and Kadri Kangro for reading the manuscript.The Microscopy Services Laboratory, Department of Pathology and Laboratory Medicine, is supported in part by a Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center(P30 CA016086).Peer reviewe

Activated platelets retain and protect most of their factor XIII-A cargo from proteolytic activation and degradation.

Platelet factor (F)XIII-A is a major cytoplasmic protein (~3% of total) representing ~50% of total circulating FXIII. However, mobilization of FXIII-A during platelet activation is not well defined. To determine mechanisms mediating the retention versus release of platelet FXIII-A, platelets from healthy humans and mice (F13a1-/-, Fga-/-, Plg-/-, Stim1fl/fl, Pf4-Cre and respective controls) were stimulated with thrombin, convulxin+thrombin, or calcium ionophore (A23187), in the absence or presence of inhibitors of transglutaminase activity, mRNA translation, microtubule rearrangement, calpain, and Rho GTPase. Platelet releasates and pellets were separated by (ultra)centrifugation. FXIII-A was detected by immunoblotting and immunofluorescence microscopy. Even following strong dual agonist (convulxin+thrombin) stimulation of human platelets, >80% platelet FXIII-A remained associated with the platelet pellet. In contrast, essentially all tissue factor pathway inhibitor, another cytoplasmic protein in platelets, was released to the supernatant. Pellet-associated FXIII-A was not due to de novo synthesis via platelet F13A1 mRNA. The proportion of platelet FXIII-A retained by, versus released from, activated platelets was partly dependent on STIM1 signaling, microtubule rearrangement, calpain, and RhoA activation, but did not depend on the presence of fibrinogen or plasminogen. Immunofluorescence microscopy confirmed the presence of considerable FXIII-A within the activated platelets. Whereas released FXIII-A was cleaved to FXIII-A* and could be degraded by plasmin, platelet-associated FXIII-A remained uncleaved. Retention of substantial platelet-derived FXIII-A by activated platelets, and its reduced susceptibility to thrombin- and plasmin-mediated proteolysis, suggests platelet FXIII-A is a protected pool with biological role(s) that differs from plasma FXIII

Single cell analysis of PANoptosome cell death complexes through an expansion microscopy method

In response to infection or sterile insults, inflammatory programmed cell death is an essential component of the innate immune response to remove infected or damaged cells. PANoptosis is a unique innate immune inflammatory cell death pathway regulated by multifaceted macromolecular complexes called PANoptosomes, which integrate components from other cell death pathways. Growing evidence shows that PANoptosis can be triggered in many physiological conditions, including viral and bacterial infections, cytokine storms, and cancers. However, PANoptosomes at the single cell level have not yet been fully characterized. Initial investigations have suggested that key pyroptotic, apoptotic, and necroptotic molecules including the inflammasome adaptor protein ASC, apoptotic caspase-8 (CASP8), and necroptotic RIPK3 are conserved components of PANoptosomes. Here, we optimized an immunofluorescence procedure to probe the highly dynamic multiprotein PANoptosome complexes across various innate immune cell death-inducing conditions. We first identified and validated antibodies to stain endogenous mouse ASC, CASP8, and RIPK3, without residual staining in the respective knockout cells. We then assessed the formation of PANoptosomes across innate immune cell death-inducing conditions by monitoring the colocalization of ASC with CASP8 and/or RIPK3. Finally, we established an expansion microscopy procedure using these validated antibodies to image the organization of ASC, CASP8, and RIPK3 within the PANoptosome. This optimized protocol, which can be easily adapted to study other multiprotein complexes and other cell death triggers, provides confirmation of PANoptosome assembly in individual cells and forms the foundation for a deeper molecular understanding of the PANoptosome complex and PANoptosis to facilitate therapeutic targeting

Cutting Edge: Caspase-8 Is a Linchpin in Caspase-3 and Gasdermin D Activation to Control Cell Death, Cytokine Release, and Host Defense during Influenza A Virus Infection

Programmed cell death (PCD) is essential for the innate immune response, which serves as the first line of defense against pathogens. Caspases regulate PCD, immune responses, and homeostasis. Caspase-8 specifically plays multifaceted roles in PCD pathways including pyroptosis, apoptosis, and necroptosis. However, because caspase-8-deficient mice are embryonically lethal, little is known about how caspase-8 coordinates different PCD pathways under physiological conditions. Here, we report an anti-inflammatory role of caspase-8 during influenza A virus infection. We generated viable mice carrying an uncleavable version of caspase-8 (Casp8DA/DA). We demonstrated that caspase-8 autoprocessing was responsible for activating caspase-3, thereby suppressing gasdermin D-mediated pyroptosis and inflammatory cytokine release. We also found that apoptotic and pyroptotic pathways were activated at the same time during influenza A virus infection, which enabled the cell-intrinsic anti-inflammatory function of the caspase-8-caspase-3 axis. Our findings provide new insight into the immunological consequences of caspase-8-coordinated PCD crosstalk under physiological conditions

Cutting Edge: Caspase-8 Is a Linchpin in Caspase-3 and Gasdermin D Activation to Control Cell Death, Cytokine Release, and Host Defense during Influenza A Virus Infection

Programmed cell death (PCD) is essential for the innate immune response, which serves as the first line of defense against pathogens. Caspases regulate PCD, immune responses, and homeostasis. Caspase-8 specifically plays multifaceted roles in PCD pathways including pyroptosis, apoptosis, and necroptosis. However, because caspase-8-deficient mice are embryonically lethal, little is known about how caspase-8 coordinates different PCD pathways under physiological conditions. Here, we report an anti-inflammatory role of caspase-8 during influenza A virus infection. We generated viable mice carrying an uncleavable version of caspase-8 (Casp8DA/DA). We demonstrated that caspase-8 autoprocessing was responsible for activating caspase-3, thereby suppressing gasdermin D-mediated pyroptosis and inflammatory cytokine release. We also found that apoptotic and pyroptotic pathways were activated at the same time during influenza A virus infection, which enabled the cell-intrinsic anti-inflammatory function of the caspase-8-caspase-3 axis. Our findings provide new insight into the immunological consequences of caspase-8-coordinated PCD crosstalk under physiological conditions.Y

High risk oral contraceptive hormones do not directly enhance endothelial cell procoagulant activity in vitro

Background Oral contraceptive (OC) use increases venous thromboembolism risk 2-5-fold. Procoagulant changes can be detected in plasma from OC users even without thrombosis, but cellular mechanisms that provoke thrombosis have not been identified. Endothelial cell (EC) dysfunction is thought to initiate venous thromboembolism. It is unknown whether OC hormones provoke aberrant procoagulant activity in ECs. Objective Characterize the effect of high-risk OC hormones (ethinyl estradiol [EE] and drospirenone) on EC procoagulant activity and the potential interplay with nuclear estrogen receptors ERα and ERβ and inflammatory processes. Methods Human umbilical vein and dermal microvascular ECs (HUVEC and HDMVEC, respectively) were treated with EE and/or drospirenone. Genes encoding the estrogen receptors ERα and ERβ (ESR1 and ESR2, respectively) were overexpressed in HUVEC and HDMVEC via lentiviral vectors. EC gene expression was assessed by RT-qPCR. The ability of ECs to support thrombin generation and fibrin formation was measured by calibrated automated thrombography and spectrophotometry, respectively. Results Neither EE nor drospirenone, alone or together, changed expression of genes encoding anti- or procoagulant proteins (TFPI, THBD, F3), integrins (ITGAV, ITGB3), or fibrinolytic mediators (SERPINE1, PLAT). EE and/or drospirenone did not increase EC-supported thrombin generation or fibrin formation, either. Our analyses indicated a subset of individuals express ESR1 and ESR2 transcripts in human aortic ECs. However, overexpression of ESR1 and/or ESR2 in HUVEC and HDMVEC did not facilitate the ability of OC-treated ECs to support procoagulant activity, even in the presence of a pro-inflammatory stimulus. Conclusions The OC hormones EE and drospirenone do not directly enhance thrombin generation potential of primary ECs in vitro

High risk oral contraceptive hormones do not directly enhance endothelial cell procoagulant activity in vitro.

BackgroundOral contraceptive (OC) use increases venous thromboembolism risk 2-5-fold. Procoagulant changes can be detected in plasma from OC users even without thrombosis, but cellular mechanisms that provoke thrombosis have not been identified. Endothelial cell (EC) dysfunction is thought to initiate venous thromboembolism. It is unknown whether OC hormones provoke aberrant procoagulant activity in ECs.ObjectiveCharacterize the effect of high-risk OC hormones (ethinyl estradiol [EE] and drospirenone) on EC procoagulant activity and the potential interplay with nuclear estrogen receptors ERα and ERβ and inflammatory processes.MethodsHuman umbilical vein and dermal microvascular ECs (HUVEC and HDMVEC, respectively) were treated with EE and/or drospirenone. Genes encoding the estrogen receptors ERα and ERβ (ESR1 and ESR2, respectively) were overexpressed in HUVEC and HDMVEC via lentiviral vectors. EC gene expression was assessed by RT-qPCR. The ability of ECs to support thrombin generation and fibrin formation was measured by calibrated automated thrombography and spectrophotometry, respectively.ResultsNeither EE nor drospirenone, alone or together, changed expression of genes encoding anti- or procoagulant proteins (TFPI, THBD, F3), integrins (ITGAV, ITGB3), or fibrinolytic mediators (SERPINE1, PLAT). EE and/or drospirenone did not increase EC-supported thrombin generation or fibrin formation, either. Our analyses indicated a subset of individuals express ESR1 and ESR2 transcripts in human aortic ECs. However, overexpression of ESR1 and/or ESR2 in HUVEC and HDMVEC did not facilitate the ability of OC-treated ECs to support procoagulant activity, even in the presence of a pro-inflammatory stimulus.ConclusionsThe OC hormones EE and drospirenone do not directly enhance thrombin generation potential of primary ECs in vitro

An Microsoft excel document containing quantitative parameters from thrombin generation and fibrin formation experiments.

An Microsoft excel document containing quantitative parameters from thrombin generation and fibrin formation experiments.</p

OC hormones do not enhance EC-supported fibrin formation.

HUVEC and HDMVEC were treated with vehicle (0.7% ethanol [veh]), 10 ng/mL TNF⍺, 1 nM EE, 100 nM drospirenone (DRS), or EE and drospirenone (E+D) for 24 hours before RNA was extracted. Transcripts encoding (A) integrin ɑv (ITGAV), (B) integrin β3 (ITGB3), (C) plasminogen activator inhibitor type I (SERPINE1), and (D) tissue plasminogen activator (PLAT) were measured by RT-qPCR and normalized to the untreated control (N = 3–12; Bars = mean + SEM). TNFɑ was compared to untreated cells, whereas EE and drospirenone treatments were compared to vehicle (*p(E) HUVEC and (F) HDMVEC was measured by turbidity in recalcified NPP (curves representative of N = 4–5). Fibrin formation parameters and statistical analyses are provided in S2 File.</p