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BACKGROUND: Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a viable technique for management of non-compressible torso hemorrhage. The major limitation of the current unilobed fully-occlusive REBOA catheters is below-the-balloon ischemia-reperfusion complications. We hypothesized that partial aortic occlusion with a novel bilobed partial (p)REBOA-PRO would result in the need for less intra-aortic balloon adjustments to maintain a distal goal perfusion pressure as compared to currently available unilobed ER-REBOA. METHODS: Anesthetized (40–50 kg) swine randomized to control (no intervention), ER-REBOA or pREBOA-PRO underwent supraceliac aortic injury. REBOA groups underwent catheter placement into Zone 1 with initial balloon inflation to full occlusion for 10 minutes followed by gradual deflation to achieve and subsequently maintain half of the baseline below-the-balloon mean arterial pressure (MAP). Physiologic data and blood samples were collected at baseline and then hourly. At 4 hours, the animals were euthanized, total blood loss and urine output were recorded, and tissue samples were collected. RESULTS: Baseline physiologic data and basic labs were similar between groups. Compared to control, interventions similarly prolonged survival from a median of 18 to over 240 minutes with comparable mortality trends. Blood loss was similar between partial ER-REBOA (41%) and pREBOA-PRO (51%). Partial pREBOA-PRO required a significantly lower number of intra-aortic balloon adjustments (10 ER-REBOA vs. 3 pREBOA-PRO, p

Objective: Cardiac myosin (CM) is structurally similar to skeletal muscle myosin, which has procoagulant activity. Here, we evaluated CM’s ex vivo, in vivo, and in vitro activities related to hemostasis and thrombosis. Approach and Results: Perfusion of fresh human blood over CM-coated surfaces caused thrombus formation and fibrin deposition. Addition of CM to blood passing over collagen-coated surfaces enhanced fibrin formation. In a murine ischemia/reperfusion injury model, exogenous CM, when administered intravenously, augmented myocardial infarction and troponin I release. In hemophilia A mice, intravenously administered CM reduced tail-cut-initiated bleeding. These data provide proof of concept for CM’s in vivo procoagulant properties. In vitro studies clarified some mechanisms for CM’s procoagulant properties. Thrombin generation assays showed that CM, like skeletal muscle myosin, enhanced thrombin generation in human platelet-rich and platelet-poor plasmas and also in mixtures of purified factors Xa, Va, and prothrombin. Binding studies showed that CM, like skeletal muscle myosin, directly binds factor Xa, supporting the concept that the CM surface is a site for prothrombinase assembly. In tPA (tissue-type plasminogen activator)-induced plasma clot lysis assays, CM was antifibrinolytic due to robust CM-dependent thrombin generation that enhanced activation of TAFI (thrombin activatable fibrinolysis inhibitor). Conclusions: CM in vitro is procoagulant and prothrombotic. CM in vivo can augment myocardial damage and can be prohemostatic in the presence of bleeding. CM’s procoagulant and antifibrinolytic activities likely involve, at least in part, its ability to bind factor Xa and enhance thrombin generation. Future work is needed to clarify CM’s pathophysiology and its mechanistic influences on hemostasis or thrombosis.

Skeletal muscle myosin (SkM) has been shown to possess procoagulant activity; however, the mechanisms of this coagulation-enhancing activity involving plasma coagulation pathways and factors are incompletely understood. Here, we discovered direct interactions between immobilized SkM and coagulation factor XI (FXI) using biolayer interferometry (K

Objective Evans syndrome, the combination of immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA) or autoimmune neutropenia, is associated with a high rate of relapsed/refractory disease. There are limited data on the efficacy of splenectomy for this condition. We reviewed patient outcomes after splenectomy for Evans syndrome compared to ITP at our institution. Methods We performed a retrospective analysis of patients who underwent splenectomy for autoimmune cytopenias over a 23-year period with the intention of comparing disease relapse rates after splenectomy in patients with Evans syndrome and in those with ITP. Results During the study period, 77 patients underwent splenectomy for ITP and seven underwent splenectomy for Evans syndrome. In the Evans cohort, splenectomy led to an 85.7% initial response rate with a 42.8% rate of relapse within one year and a long-term (one-year) response rate of 42.8%. In the ITP cohort, the initial response rate was 90.9% with a long-term response rate of 70.1%. Conclusion Our data suggest that long-term remission rates after splenectomy are lower in adults with Evans syndrome compared to those with ITP, although splenectomy may still be an acceptable treatment for certain patients with Evans syndrome. Our findings underscore the need for further research and development of additional therapeutic strategies for this patient population.

Platelets engage cues of pending vascular injury through coordinated adhesion, secretion, and aggregation responses. These rapid, progressive changes in platelet form and function are orchestrated downstream of specific receptors on the platelet surface and through intracellular signaling mechanisms that remain systematically undefined. This study brings together cell physiological and phosphoproteomics methods to profile signaling mechanisms downstream of the immunotyrosine activation motif (ITAM) platelet collagen receptor GPVI. Peptide tandem mass tag (TMT) labeling, sample multiplexing, synchronous precursor selection (SPS), and triple stage tandem mass spectrometry (MS3) detected >3000 significant (false discovery rate < 0.05) phosphorylation events on >1300 proteins over conditions initiating and progressing GPVI-mediated platelet activation. With literature-guided causal inference tools, >300 site-specific signaling relations were mapped from phosphoproteomics data among key and emerging GPVI effectors (ie, FcRγ, Syk, PLCγ2, PKCδ, DAPP1). Through signaling validation studies and functional screening, other less-characterized targets were also considered within the context of GPVI/ITAM pathways, including Ras/MAPK axis proteins (ie, KSR1, SOS1, STAT1, Hsp27). Highly regulated GPVI/ITAM targets out of context of curated knowledge were also illuminated, including a system of >40 Rab GTPases and associated regulatory proteins, where GPVI-mediated Rab7 S72 phosphorylation and endolysosomal maturation were blocked by TAK1 inhibition. In addition to serving as a model for generating and testing hypotheses from omics datasets, this study puts forth a means to identify hemostatic effectors, biomarkers, and therapeutic targets relevant to thrombosis, vascular inflammation, and other platelet-associated disease states.

IMPORTANCE: Mechanical heart valves (MHVs) pose significant thrombogenic risks to pregnant women and their fetuses, yet the choice of anticoagulation in this clinical setting remains unclear. Various therapeutic strategies carry distinct risk profiles that must be considered when making the decision about optimal anticoagulation. OBJECTIVE: We sought to review existing data and offer recommendations for the anticoagulation of pregnant women with MHVs, as well as management of anticoagulation in the peripartum period. EVIDENCE ACQUISITION: We performed a literature review of studies examining outcomes in pregnant women receiving systemic anticoagulation for mechanical valves, and also reviewed data on the safety profiles of various anticoagulant strategies in the setting of pregnancy. RESULTS: Warfarin has been shown to increase rates of embryopathy and fetal demise, although it has traditionally been the favored anticoagulant in this setting. Low-molecular-weight heparin, when dosed appropriately with close therapeutic monitoring, has been shown to be safe for both mother and fetus. CONCLUSIONS AND RELEVANCE: We favor the use of low-molecular-weight heparin with appropriate dosing and monitoring for the anticoagulation of pregnant women with MHVs. Data suggest that this approach minimizes the thrombotic risk associated with the valve while also providing safe and effective anticoagulation that can be easily managed in the peripartum period. TARGET AUDIENCE: Obstetricians and gynecologists, family physicians. LEARNING OBJECTIVES: After completing this activity, the learner should be better able to: describe the clinical considerations in choosing an anticoagulation strategy for a pregnant patient with an MHV; evaluate the existing data about the safety profile of various anticoagulation strategies and the potential benefits and risks of each approach to the mother and fetus; and discuss one recommended approach to management of mechanical valves in the pregnant patient and assess the clinical nuances associated with each individual patient’s decision.

PURPOSE—: To develop a small volume whole blood analyzer capable of measuring the hematocrit and coagulation kinetics of whole blood. METHODS AND RESULTS—: A co-planar microfluidic chamber designed to facilitate self-driven capillary action across an internal electrical chip was developed and used to measure the electric parameters of whole human blood that had been anticoagulated or allowed to clot. To promote blood clotting, select chip surfaces were coated with a prothrombin time (PT) reagent containing lipidated tissue factor (TF), which activates the extrinsic pathway of coagulation to promote thrombin generation and fibrin formation. Whole human blood was added to the microfluidic device, and voltage changes within the platform were measured and interpreted using basic resistor-capacitor (RC) circuit and fluid dynamics theory. Upon wetting of the sensing zone, a circuit between two co-planar electrodes within the sensing zone was closed to generate a rapid voltage drop from baseline. The voltage then rose due to sedimentation of red blood cells (RBC) in the sensing zone. For anticoagulated blood samples, the time for the voltage to return to baseline was dependent on hematocrit. In the presence of coagulation, the initiation of fibrin formation in the presence of the PT reagent prevented the return of voltage to baseline due to the reduced packing of RBCs in the sensing zone. CONCLUSIONS—: The technology presented in this study has potential for monitoring the hematocrit and coagulation parameters of patient samples using a small volume of whole blood, suggesting it may hold clinical utility as a point-of-care test.

Blood coagulation mechanisms play key roles in health and disease. Pilot studies using selected human plasmas showed the potential associations of plasma skeletal muscle myosin (SkM) isoforms and phenotypes with pulmonary embolism and thrombin generation, suggesting SkM may contribute to blood coagulation reactions in plasma. Here we report that ex vivo studies of the coagulability of fresh flowing human blood over SkM-coated surfaces showed that an anti-factor XI (FXI) mAb, but not an anti-tissue factor mAb, inhibited clot formation, indicating that FXI is an essential contributor for the normal observed procoagulant response of blood during its exposure to immobilized SkM. This raised the question of whether procoagulant SkM's requirement for FXI involves direct or indirect effects on FXI. To assess direct interactions between SkM and FXI, Bio-Layer Interferometry (BLI) (Octet Red system) was used to record kinetics for binding of soluble FXI to immobilized SkM. BLI data showed that FXI bound to SkM with a Kd of 0.2 nM (k on= 2.92x10 6 M -1s -1 and k off=9.25x10 -3 s -1) (Fig. 1A). In contrast, prekallikrein (PK) did not bind to the SkM (Fig.1A), indicating the specificity of SkM for binding FXI. The anti-FXI mAb1A6, which recognizes the Apple (A)3 domain of FXI, potently inhibited binding of FXI to immobilized SkM, implying SkM binds the FXI A3 domain. Studies using purified clotting factors were made to identify which FXI-related activities might be affected by SkM. When FXI activation by thrombin was evaluated under conditions where polyphosphate (PolyP) 100-mer and 700-mer enhance FXI activation, SkM concentration-dependently enhanced FXI activation by thrombin (Fig. 1B). Whereas alkaline phosphatase destroyed PolyP's ability to stimulate FXI activation by thrombin, it did not cause a reduction of SkM's ability to enhance FXI activation, indicating SkM's activity is independent of PolyP-like sequences in SkM. Small unilamellar phospholipid vesicles (20% phosphatidylserine (PS) / 80% phosphatidylcholine) did not affect FXI activation by thrombin; furthermore, reagents that neutralize procoagulant PS, i.e., lactadherin, annexin V, and phospholipase A2, did not affect SkM's enhancement of FXI activation by thrombin, indicating that this activity is not due to anionic phospholipids linked to SkM. The effects of SkM on FXI autoactivation and FXI activation by FXIIa were evaluated. As is well known, PolyP and some other anionic reagents, e.g., nucleic acid polymers, enhance not only FXI activation by thrombin but also FXI autoactivation and FXI activation by FXIIa. However, SkM did not significantly affect FXI autoactivation or FXI activation by factor XIIa, further emphasizing that SkM's enhancement of FXI activation by thrombin is not due to any PolyP-like compounds and that it is a unique property of procoagulant SkM. This also suggests that SkM has a unique mechanism for its procoagulant activity on FXI activation which is limited to the thrombin positive feedback loop. To evaluate further the basis for interactions between FXI and SkM, we employed FXI- PK chimeras because BLI binding studies showed that, in contrast to FXI, PK did not bind to SkM. Recombinant FXI proteins in which each of the four A domains of the heavy chain (designated A1 through A4) were individually replaced with the corresponding A domain from PK and were used to identify the site of factor XI to interact with SkM for FXI activation by thrombin. The FXI chimera with the substitution of the PKA1 domain was not activated by thrombin, which is consistent with the fact that the FXI A1 domain is an interactive site for thrombin. Thrombin activation of the two FXI chimeras (FXI/PKA3 and FXI/PKA4) with substitutions of either the A3 or A4 domains was not enhanced by SkM, whereas substitution of the A2 domain (FXI/PKA2) did not reduce the enhancement of activation by thrombin compared to wild type FXI. Furthermore, mAb1A6, which recognizes the A3 domain and which inhibited the prothrombotic activity of fresh blood flowing over a SkM-coated surface, potently inhibited FXI binding to SkM in BLI studies. These data strongly suggest that functional interaction sites on FXI for SkM involve the A3 and A4 domains of FXI. In summary, we found that SkM's ex vivo procoagulant activity requires FXI, that SkM enhances FXI activation by thrombin and this requires FXI's A3 and A4 domains, and that SkM's high affinity binding of FXI requires the FXI A3 domain (Fig. 1C). Figure 1 Figure 1. Disclosures Gruber: Aronora Inc.: Current Employment, Current equity holder in publicly-traded company; Oregon Health and Science University: Current Employment. Gailani: Anthos Therapeutics: Consultancy; Aronora: Membership on an entity's Board of Directors or advisory committees; Bayer Pharma: Consultancy; Bristol Myer Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ionis: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees.

INTRODUCTION: Trauma-induced coagulopathy (TIC) and the tissue injury-provoked procoagulant profile are prevalent in severely injured patients, but their mechanisms remain unclear. Myosin, exposed by or released from tissue injury, may play a role in promoting thrombin generation and attenuating fibrinolysis. The objective of the study is to examine the effects of cardiac and skeletal muscle myosins on coagulation in whole blood using thrombelastography (TEG). MATERIALS AND METHODS: Whole blood was collected from healthy adult volunteers (n=8) and native TEGs were performed to evaluate the global coagulation response in the presence of cardiac or skeletal muscle myosin by measuring reaction (R) time (minutes), clot angle (°), and maximum amplitude (MA, mm). TEG measurements were compared using paired t-tests. RESULTS: Cardiac and skeletal muscle myosins decreased R, from 10.8 min to 8.0 min (p

To test the hypothesis that skeletal muscle myosins can directly influence blood coagulation and thrombosis, ex vivo studies of the effects of myosin on thrombogenesis in fresh human blood were conducted. Addition of myosin to blood augmented the thrombotic responses of human blood flowing over collagen-coated surfaces (300 s-1 shear rate). Perfusion of human blood over myosin-coated surfaces also caused fibrin and platelet deposition, evidencing myosin's thrombogenicity. Myosin markedly enhanced thrombin generation in both platelet-rich plasma and platelet-poor plasma, indicating that myosin promoted thrombin generation in plasma primarily independent of platelets. In purified reaction mixtures composed only of factor Xa, factor Va, prothrombin, and calcium ions, myosin greatly enhanced prothrombinase activity. The Gla domain of factor Xa was not required for myosin's prothrombinase enhancement. When binding of purified clotting factors to immobilized myosin was monitored using biolayer interferometry, factors Xa and Va each showed favorable binding interactions. Factor Va reduced by 100-fold the apparent Kd of myosin for factor Xa (Kd ∼0.48 nM), primarily by reducing koff, indicating formation of a stable ternary complex of myosin:Xa:Va. In studies to assess possible clinical relevance for this discovery, we found that antimyosin antibodies inhibited thrombin generation in acute trauma patient plasmas more than in control plasmas (P = .0004), implying myosin might contribute to acute trauma coagulopathy. We posit that myosin enhancement of thrombin generation could contribute either to promote hemostasis or to augment thrombosis risk with consequent implications for myosin's possible contributions to pathophysiology in the setting of acute injuries.

Thrombus growth at the site of vascular injury is mediated by the sequential events of platelet recruitment, activation and aggregation concomitant with the initiation of the coagulation cascade, resulting in local thrombin generation and fibrin formation. While the biorheology of a localized thrombus formation has been well studied, it is unclear whether local sites of thrombin generation propagate platelet activation within the bloodstream. In order to study the physical biology of platelet activation downstream of sites of thrombus formation, we developed a platform to measure platelet activation and microaggregate formation in the bloodstream. Our results show that thrombi formed on collagen and tissue factor promote activation and aggregation of platelets in the bloodstream in a convection-dependent manner. Pharmacological inhibition of the coagulation factors (F) X, XI or thrombin dramatically reduced the degree of distal platelet activation and microaggregate formation in the bloodstream without affecting the degree of local platelet deposition and aggregation on a surface of immobilized collagen. Herein we describe the development and an example of the utility of a platform to study platelet activation and microaggregate formation in the bloodstream (convection-limited regime) relative to the local site of thrombus formation.

Cancer-associated thrombosis is a common first presenting sign of malignancy and is currently the second leading cause of death in cancer patients after their malignancy. However, the molecular mechanisms underlying cancer-associated thrombosis remain undefined. In this study, we aimed to develop a better understanding of how cancer cells affect the coagulation cascade and platelet activation to induce a prothrombotic phenotype. Our results show that colon cancer cells trigger platelet activation in a manner dependent on cancer cell tissue factor (TF) expression, thrombin generation, activation of the protease-activated receptor 4 (PAR4) on platelets and consequent release of ADP and thromboxane A2. Platelet-colon cancer cell interactions potentiated the release of platelet-derived extracellular vesicles (EVs) rather than cancer cell-derived EVs. Our data show that single colon cancer cells were capable of recruiting and activating platelets and generating fibrin in plasma under shear flow. Finally, in a retrospective analysis of colon cancer patients, we found that the number of venous thromboembolism events was 4.5 times higher in colon cancer patients than in a control population. In conclusion, our data suggest that platelet-cancer cell interactions and perhaps platelet procoagulant EVs may contribute to the prothrombotic phenotype of colon cancer patients. Our work may provide rationale for targeting platelet-cancer cell interactions with PAR4 antagonists together with aspirin and/or ADP receptor antagonists as a potential intervention to limit cancer-associated thrombosis, balancing safety with efficacy.

Objective— Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood promotes thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo. Here, we sought to determine whether presence of long-chain polyP or bacteria in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Approach and Results— Long-chain polyP promoted platelet P-selectin expression, microaggregate formation, and platelet consumption in flowing whole blood in a contact activation pathway-dependent manner. Moreover, long-chain polyP promoted local fibrin formation on collagen under shear flow in a FXI-dependent manner. Distal to the site of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the blood flow in a FXI- and FXII-dependent manner. In a murine model, long-chain polyP promoted platelet deposition and fibrin generation in lungs in a FXII-dependent manner. In a nonhuman primate model of bacterial sepsis, pre-treatment of animals with an antibody blocking FXI activation by FXIIa reduced lethal dose 100 Staphylococcus aureus –induced platelet and fibrinogen consumption. Conclusions— This study demonstrates that bacterial-type long-chain polyP promotes platelet activation in a FXII-dependent manner in flowing blood, which may contribute to sepsis-associated thrombotic processes, consumptive coagulopathy, and thrombocytopenia.

Background: Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood can promote thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo . Aim: Determine whether presence of long-chain polyP in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Methods/Results: The addition of long-chain polyP to human whole blood promoted platelet P-selectin expression, microaggregate formation and platelet consumption in the bloodstream under shear in a FXII-dependent manner. Moreover, long-chain polyP accelerated thrombus formation on immobilized collagen surfaces under shear flow in a thrombin generation-dependent manner. Distal to the site of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the bloodstream. Inhibiting contact activation of coagulation using established and novel agents reduced fibrin formation on collagen as well as platelet consumption in the bloodstream distal to the site of thrombus formation. In vivo , FXII deficiency was protective against long-chain polyP occlusive lung thrombus formation in mice. Lastly, in a non-human primate model of sepsis, pretreatment of animals with an antibody blocking FXI activation by FXIIa (14E11) diminished LD 100 S. aureus -induced platelet and fibrinogen consumption. Conclusions: This study demonstrates that bacterial-type long-chain polyP promotes FXII-mediated thrombin generation and platelet activation in the flowing blood and could contribute to sepsis-associated thrombotic processes, consumptive coagulopathy and thrombocytopenia.

Background: Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood can promote thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo . Aim: Determine whether presence of long-chain polyP in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Methods/Results: The addition of long-chain polyP to human whole blood promoted platelet P-selectin expression, microaggregate formation and platelet consumption in the bloodstream under shear in a FXII-dependent manner. Moreover, long-chain polyP accelerated thrombus formation on immobilized collagen surfaces under shear flow in a thrombin generation-dependent manner. Distal to the site of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the bloodstream. Inhibiting contact activation of coagulation using established and novel agents reduced fibrin formation on collagen as well as platelet consumption in the bloodstream distal to the site of thrombus formation. In vivo , FXII deficiency was protective against long-chain polyP occlusive lung thrombus formation in mice. Lastly, in a non-human primate model of sepsis, pretreatment of animals with an antibody blocking FXI activation by FXIIa (14E11) diminished LD 100 S. aureus -induced platelet and fibrinogen consumption. Conclusions: This study demonstrates that bacterial-type long-chain polyP promotes FXII-mediated thrombin generation and platelet activation in the flowing blood and could contribute to sepsis-associated thrombotic processes, consumptive coagulopathy and thrombocytopenia.

Intracellular signaling systems associated with the platelet glycoprotein VI receptor (GPVI) are essential to hemostasis and also initiate atherothrombotic events in cardiovascular disease contexts. While many separable molecular entities linked to GPVI-mediated platelet activation have been characterized, a systemic understanding of GPVI signaling over the context of the entire platelet proteome remains far from complete. To test the hypothesis that specific, intact signaling systems downstream of GPVI regulate platelet function, we used a combination of tandem mass tag (TMT) labeling and high-resolution, multidimensional mass spectrometry and informatics tools to quantify and model protein phosphorylation events reproducibly measurable in platelets from healthy human subjects (n=5) in response to the GPVI specific agonist collagen-related peptide (CRP). Following CRP treatment, platelets rapidly regulated the phosphorylation state of 1004 serine, threonine and tyrosine residues on 542 proteins (fold change > 1.5; false discovery rate < 0.05). In addition to regulatory sites on kinases, phospholipases and other well-known targets, the majority of phosphorylation events following CRP stimulation occurred on proteins with uncharacterized roles in platelet function. CausalPath analysis of this data mapped 46 high confidence pathway relations, suggesting that Syk, PKCs and MAPKs ultimately drive the phosphorylation of several novel effectors specifically downstream of GPVI in platelet regulation, including CRMP-2, RAD23B and caldesmon. In addition to comprehensively mapping out known signaling relations while illuminating previously unrecognized regulatory mechanisms of platelet function around GPVI, our work provides a platform to generate novel, mechanistic and testable hypotheses from omics data sets for subsequent discovery-driven efforts to identify platelet-based therapeutic and biomarker targets relevant to cardiovascular disease risk, progression and mortality.

The hypoxia-inducible factors HIF-1α and HIF-2α are important regulators of the chondrocyte phenotype but little is known about HIF-3α in cartilage. The objective of this study was to characterize HIF-3α (HIF3A) expression during chondrocyte differentiation in vitro and in native cartilage tissues. HIF3A, COL10A1, and MMP13 were quantified in mesenchymal stem cells (MSCs) and articular chondrocytes from healthy and osteoarthritic (OA) tissue in three-dimensional cultures and in human embryonic epiphyses and adult articular cartilage. HIF3A was found to have an inverse association with hypertrophic markers COL10A1 and MMP13 in chondrogenic cells and tissues. In healthy chondrocytes, HIF3A was induced by dexamethasone and increased during redifferentiation. By comparison, HIF3A expression was extremely low in chondrogenically differentiated MSCs expressing high levels of COL10A1 and MMP13. HIF3A was also lower in redifferentiated OA chondrocytes than in healthy chondrocytes. In human embryonic epiphyseal tissue, HIF3A expression was lowest in the hypertrophic zone. Distinct splice patterns were also found in embryonic cartilage when compared with adult articular cartilage and redifferentiated chondrocytes. These in vitro and in vivo findings suggest that HIF3A levels are indicative of the hypertrophic state of chondrogenic cells and one or more splice variants may be important regulators of the chondrocyte phenotype.

PURPOSE: Platelet apheresis units are transfused into patients to mitigate or prevent bleeding. In a hospital, platelet apheresis units are transported from the transfusion service to the healthcare teams via two methods: a pneumatic tubing system (PTS) or ambulatory transport. Whether PTS transport affects the activity and utility of platelet apheresis units is unclear. METHODS: We quantified the gravitational forces and transport time associated with PTS and ambulatory transport within our hospital. Washed platelets and supernatants were prepared from platelet apheresis units prior to transport as well as following ambulatory or PTS transport. For each group, we compared resting and agonist-induced platelet activity and platelet aggregate formation on collagen or von Willebrand factor (VWF) under shear, platelet VWF-receptor expression and VWF multimer levels. RESULTS: Subjection of platelet apheresis units to rapid acceleration/deceleration forces during PTS transport did not pre-activate platelets or their ability to activate in response to platelet agonists as compared to ambulatory transport. Platelets within platelet apheresis units transported via PTS retained their ability to adhere to surfaces of VWF and collagen under shear, although platelet aggregation on collagen and VWF was diminished as compared to ambulatory transport. VWF multimer levels and platelet GPIb receptor expression was unaffected by PTS transport as compared to ambulatory transport. CONCLUSIONS: Subjection of platelet apheresis units to PTS transport did not significantly affect the baseline or agonist-induced levels of platelet activation as compared to ambulatory transport. Our case study suggests that PTS transport may not significantly affect the hemostatic potential of platelets within platelet apheresis units.

Human platelets express two protease-activated receptors (PARs), PAR1 (F2R) and PAR4 (F2RL3), which are activated by a number of serine proteases that are generated during pathological events and cause platelet activation. Recent interest has focused on PAR4 as a therapeutic target, given PAR4 seems to promote experimental thrombosis and procoagulant microparticle formation, without a broadly apparent role in hemostasis. However, it is not yet known whether PAR4 activity plays a role in platelet-leukocyte interactions, which are thought to contribute to both thrombosis and acute or chronic thrombo-inflammatory processes. We sought to determine whether PAR4 activity contributes to granule secretion from activated platelets and platelet-leukocyte interactions. We performed in vitro and ex vivo studies of platelet granule release and platelet-leukocyte interactions in the presence of PAR4 agonists including PAR4 activating peptide, thrombin, cathepsin G, and plasmin in combination with small-molecule PAR4 antagonists. Activation of human platelets with thrombin, cathepsin G, or plasmin potentiated platelet dense granule secretion that was specifically impaired by PAR4 inhibitors. Platelet-leukocyte interactions and platelet P-selectin exposure the following stimulation with PAR4 agonists were also impaired by activated PAR4 inhibition in either a purified system or in whole blood. These results indicate PAR4-specific promotion of platelet granule release and platelet-leukocyte aggregate formation and suggest that pharmacological control of PAR4 activity could potentially attenuate platelet granule release or platelet-leukocyte interaction-mediated pathological processes.

Background: Under normal conditions, endothelial cells (ECs) govern blood flow dynamics including providing a barrier between blood and tissue and regulating platelet aggregation and thrombin generation in the bloodstream. In turn, blood components, primarily platelets and coagulation factors such as thrombin, regulate EC barrier integrity. The breakdown of EC barrier function is a hallmark of a variety of vascular diseases. In sepsis, for example, the dysfunction of vascular ECs has been correlated with poorer outcomes due to hemorrhage and multi-organ failure associated with consumption of platelets and coagulation factors into clots within the microcirculation, a condition termed disseminated intravascular coagulation (DIC). Aim: Develop an endothelialized flow chamber to study the platelet-endothelium interface. Methods and Results: We developed a 3D-chamber with a perfuseable cylindrical microvessel embedded in an extracellular matrix (ECM) material. This model allows for the study of the role of thrombin generation and platelet aggregation in endothelial barrier leak development and repair in healthy as well as inflamed microvessels. Incorporation of subendothelial matrix proteins in these 3D-microvessel devices expands the capacity of the microfluidic studies to investigate blood cell extravasation and enables the control of physical parameters such as transmural pressure and interstitial flow through the ECM. Conclusion: This model may provide insight into the pathophysiology of different disease states and serve as an expedient platform for therapy design and testing. The platelet-endothelium interface under shear flow. Diagram ( A ) and an experimental prototype ( B ) of a 3D-perfuseable device. Microvessel phenotype (following treatment with vehicle or 10 ng/mL TNFα) pre- and post- perfusion with recalcified whole blood for 33 min as visualized by differential interference contrast, DIC, ( C ) and fluorescence microscopy ( D ).

Background: Terminal complications of bacterial sepsis include development of consumptive coagulopathy referred to as disseminated intravascular coagulation. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway is capable of promoting thrombin generation and platelet activation and consumption in whole blood distal to thrombus formation under shear ex vivo and in vivo . Aim: Test the hypothesis that the presence of long-chain polyP in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Methods and Results: Presence of long-chain polyP in whole blood promoted platelet aggregation on immobilized collagen surfaces under shear flow. Long-chain polyP enhanced fibrin formation and shortened clotting times of plasma and whole blood. The addition of long-chain polyP promoted platelet P-selectin expression, microaggregate formation and platelet consumption in the bloodstream under shear in a FXII-dependent manner. Moreover, long-chain polyP accelerated thrombus formation on immobilized collagen surfaces under shear flow. Distal to the sites of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the bloodstream. Inhibiting contact activation of the coagulation pathway reduced fibrin formation on collagen as well as platelet consumption in the bloodstream distal to the site of thrombus formation. Conclusions: This study demonstrates that bacterial-type long-chain polyP promotes FXII-mediated thrombin generation and platelet activation in the flowing blood and could exaggerate sepsis-associated thrombotic processes, consumptive coagulopathy and thrombocytopenia.

Background: ADAMTS13 cleaves and inactivates von Willebrand factor (VWF), which binds collagen, facilitating platelet adhesion under vascular injury. But is still uncertain how ADAMTS13 activity is regulated. Thrombin and plasmin have been shown to cleave ADAMTS13. Based on the fact that elevated levels of FXI is an independent risk factor for deep vein thrombosis and ischemic stroke, we hypothesize that FXIa inactivates ADAMTS13 leading to platelet aggregation and thrombus formation. Aim: To determine the functional role of inactivation of ADAMTS13 by FXIa. Methods and Results: Recombinant ADAMTS13 (250 nM) was incubated with FXIa (50 nM) for increasing times (0-3 hours) at 37 o C before being analyzed by western blot using an anti-ADAMTS13 antibody against the two CUB domains (C-terminal) or against the metalloproteinase (MET) domain (N-terminal). Our results show that FXIa caused the disappearance of the ADAMTS13 band (~200 kDa) and the appearance of a band at ~150 kDa when the samples were analyzed with the anti-MET antibody and a ~50 kDa band when the samples were analyzed with the anti-CUB antibody. The presence of aprotinin, which inhibits FXIa activity, blocked the degradation of ADAMTS13. Kallikrein or FXIIa were unable to cleave ADAMTS13. Using a cell surface immunoassay we observed that after incubation with FXIa, the detection of the CUB domain from ADAMTS13 was lost from endothelial cells surface. The incubation of ADAMTS13 with FXIa caused an increase in ADAMTS13 activity as measured by a fluorogenic substrate (FRETS). Conclusion: ADAMTS13 circulates in a closed conformation, which is maintained by a CUB-spacer domain binding interaction. ADAMTS13 becomes conformationally activated through interaction of its CUBs domains with VWF. Here we show that FXIa-mediated deletion of ADAMTS13-CUB domains enhances its capacity to cleave FRETS and blocks the interaction with VWF. Our results suggest that FXIa may limit ADAMTS13-mediated VWF inactivation.

The integration of biomaterials and understanding of vascular biology has led to the development of perfusable endothelialized flow models, which have been used as valuable tools to study the platelet-endothelium interface under shear. In these models, the parameters of geometry, compliance, biorheology, and cellular complexity are varied to recapitulate the physical biology of platelet recruitment and activation under physiologically relevant conditions of blood flow. In this review, we summarize the mechanistic insights learned from perfusable microvessel models and discuss the potential utility as well as challenges of endothelialized microfluidic devices to study platelet function in the bloodstream in vitro.

Thrombin generation and fibrin formation can cause occlusive thrombosis and myocardial infarction is caused by occlusive thrombi. Exposure and release of cardiac myosin (CM) are linked to myocardial infarction, but CM has not been accorded any thrombotic functional significance. Skeletal muscle myosin (SkM), which is structurally similar to CM, was previously shown to exert procoagulant activities (Deguchi H et al, Blood. 2016;128:1870), leading us to undertake new studies of the in vitro and in vivo procoagulant activities of CM. First, the setting of hemophilia A with its remarkable bleeding risk was used to evaluate the procoagulant properties of CM. In studies of human hemophilia plasma and of murine acquired hemophilia A plasma, CM was added to these plasmas and tissue factor (TF)-induced thrombin generation assays were performed. Plasmas included human hemophilia A plasma and C57BL/6J mouse plasma with anti-FVIII antibody (GMA-8015; 5 microgram/mL final). CM showed strong procoagulant effects in human hemophilia A plasma, which is naturally deficient in factor VIII ( Second, studies of the effects of CM on thrombogenesis ex vivo using fresh human flowing blood showed that perfusion of blood over CM-coated surfaces at 300 s-1 shear rate caused extensive fibrin deposition. Addition of CM to blood also promoted the thrombotic responses of human blood flowing over collagen-coated surfaces, evidence of CM's thrombogenicity. Further studies showed that CM enhanced thrombin generation in platelet rich plasma and platelet poor plasma, indicating that CM promotes thrombin generation in plasma primarily independently of platelets. To address the mechanistic insights for CM's procoagulant activity, purified coagulation factors were employed. In a purified system composed of factor Xa, factor Va, prothrombin and calcium ions, CM greatly enhanced prothrombinase activity. Experiments using Gla-domainless factor Xa showed that the Gla domain of factor Xa was not required for CM's prothrombinase enhancement in contrast to phospholipid-enhanced prothrombinase activity which requires that Gla domain. Binding studies showed that CM directly binds factor Xa. In summary, here we show that CM is procoagulant due to its ability to bind factor Xa and strongly promote thrombin generation. In summary, CM acts as procoagulant by its ability to bind factor Xa and strongly promote thrombin generation both in vivo an in vitro. These provocative findings raise many questions about whether and how the protective pro-hemostatic properties or the pathogenic prothrombotic properties of CM contribute to pathophysiology in the coronary circulation. This discovery raises many questions about CM and coronary pathophysiology, and future CM research may enable novel translations of new knowledge regarding CM's procoagulant activities for coronary health and disease. Figure Disclosures Mosnier: The Scripps Research Institute: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Ruggeri:MERU-VasImmune Inc.: Equity Ownership, Other: CEO and Founder.

In the contact activation pathway of the coagulation, zymogen factor XII (FXII) is converted to FXIIa, which triggers activation of FXI leading to the activation of FIX and subsequent thrombin generation and fibrin formation. Feedback activation of FXI by thrombin has been shown to promote thrombin generation in a FXII-independent manner and FXIIa can bypass FXI to directly activate FX and prothrombin in the presence of highly negatively charged molecules, such as long-chain polyphosphates (LC polyP). We sought to determine whether activation of FXII or FXI differentially regulate the physical biology of fibrin formation. Fibrin formation was initiated with tissue factor, ellagic acid (EA), or LC polyP in the presence of inhibitors of FXI and FXII. Our data demonstrated that inhibition of FXI decreased the rate of fibrin formation and fiber network density, and increased the fibrin network strength and rate of fibrinolysis when gelation was initiated via the contact activation pathway with EA. FXII inhibition decreased the fibrin formation and fibrin density, and increased the fibrinolysis rate only when fibrin formation was initiated via the contact activation pathway with LC polyP. Overall, we demonstrate that inhibition of FXI and FXII distinctly alter the biophysical properties of fibrin.

The reaction dynamics of a complex mixture of cells and proteins, such as blood, in branched circulatory networks within the human microvasculature or extravascular therapeutic devices such as extracorporeal oxygenation machine (ECMO) remains ill-defined. In this report we utilize a multi-bypass microfluidics ladder network design with dimensions mimicking venules to study patterns of blood platelet aggregation and fibrin formation under complex shear. Complex blood fluid dynamics within multi-bypass networks under flow were modeled using COMSOL. Red blood cells and platelets were assumed to be non-interacting spherical particles transported by the bulk fluid flow, and convection of the activated coagulation factor II, thrombin, was assumed to be governed by mass transfer. This model served as the basis for predicting formation of local shear rate gradients, stagnation points and recirculation zones as dictated by the bypass geometry. Based on the insights from these models, we were able to predict the patterns of blood clot formation at specific locations in the device. Our experimental data was then used to adjust the model to account for the dynamical presence of thrombus formation in the biorheology of blood flow. The model predictions were then compared to results from experiments using recalcified whole human blood. Microfluidic devices were coated with the extracellular matrix protein, fibrillar collagen, and the initiator of the extrinsic pathway of coagulation, tissue factor. Blood was perfused through the devices at a flow rate of 2 µL/min, translating to physiologically relevant initial shear rates of 300 and 700 s-1 for main channels and bypasses, respectively. Using fluorescent and light microscopy, we observed distinct flow and thrombus formation patterns near channel intersections at bypass points, within recirculation zones and at stagnation points. Findings from this proof-of-principle ladder network model suggest a specific correlation between microvascular geometry and thrombus formation dynamics under shear. This model holds potential for use as an integrative approach to identify regions susceptible to intravascular thrombus formation within the microvasculature as well as extravascular devices such as ECMO.

Objective— Coagulation factor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor–coated surfaces in vitro and in vivo by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was aimed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream. Approach and Results— Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization, and fibrin formation on immobilized collagen and tissue factor under shear flow, ex vivo. Downstream of the thrombus formed on immobilized collagen or collagen and 10 pmol/L tissue factor, platelet CD62P expression, microaggregate formation, and progressive platelet consumption were significantly reduced in the presence of FXI function-blocking antibodies or a thrombin inhibitor in a shear rate– and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation. Conclusions— This study demonstrates that the FXI–thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlight FXI as a novel therapeutic target for inhibiting distal platelet consumption without affecting proximal platelet adhesion.

Receptor-induced NF-κB activation is controlled by NEMO, the NF-κB essential modulator. Hypomorphic NEMO mutations result in X-linked ectodermal dysplasia with anhidrosis and immunodeficiency, also referred to as NEMO syndrome. Here we describe a distinct group of patients with NEMO C-terminal deletion (ΔCT-NEMO) mutations. Individuals harboring these mutations develop inflammatory skin and intestinal disease in addition to ectodermal dysplasia with anhidrosis and immunodeficiency. Both primary cells from these patients, as well as reconstituted cell lines with this deletion, exhibited increased IκB kinase (IKK) activity and production of proinflammatory cytokines. Unlike previously described loss-of-function mutations, ΔCT-NEMO mutants promoted increased NF-κB activation in response to TNF and Toll-like receptor stimulation. Investigation of the underlying mechanisms revealed impaired interactions with A20, a negative regulator of NF-κB activation, leading to prolonged accumulation of K63-ubiquitinated RIP within the TNFR1 signaling complex. Recruitment of A20 to the C-terminal domain of NEMO represents a novel mechanism limiting NF-κB activation by NEMO, and its absence results in autoinflammatory disease.

Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer worldwide. Although there are numerous treatment options for HNSCC, such as surgery, cytotoxic chemotherapy, molecularly targeted systemic therapeutics, and radiotherapy, overall survival has not significantly improved in the last 50 years. This suggests a need for a better understanding of how these cancer cells respond to current treatments in order to improve treatment paradigms. Ionizing radiation (IR) promotes cancer cell death through the creation of cytotoxic DNA lesions, including single strand breaks, base damage, crosslinks, and double strand breaks (DSBs). As unrepaired DSBs are the most cytotoxic DNA lesion, defining the downstream cellular responses to DSBs are critical for understanding the mechanisms of tumor cell responses to IR. The effects of experimental IR on HNSCC cells beyond DNA damage in vitro are ill-defined. Here we combined label-free, quantitative phase and fluorescent microscopy to define the effects of IR on the dry mass and volume of the HNSCC cell line, UM-SCC-22A. We quantified nuclear and cytoplasmic subcellular density alterations resulting from 8 Gy X-ray IR and correlated these signatures with DNA and γ-H2AX expression patterns. This study utilizes a synergistic imaging approach to study both biophysical and biochemical alterations in cells following radiation damage and will aid in future understanding of cellular responses to radiation therapy.

Activation of the NF-κB family of transcription factors is related to diverse biological processes, such as development, immune responses, and inflammation. As a result, mutations affecting the NF-κB signaling pathway cause syndromes characterized by immunodeficiency and autoinflammaotory disease. Recently, we identified the C-terminus of NEMO to be a regulator of NF-κB signaling, as a class of NEMO mutations lacking the C-terminus (ΔCT-NEMO) confer gain-of-function properties to the IKK complex due to impaired recruitment of A20 to the TNFR. Here, we investigated details by which A20 recruitment to the TNFR suppresses NF-κB signaling. The C-terminus of NEMO has been previously identified to preferentially recognize K63-linked polyubiquitin that in its unanchored form can mediate NEMO/A20 interaction. We found that the IKK complex containing ΔCT-NEMO forms but not other mutant forms of NEMO exhibit impaired K63 polyubiquitin binding ability. In addition to unanchored K63-lilnked polyubiquitin, we hypothesized that direct polyubiquitination of A20 could enable a functional interaction between A20 and NEMO. Using mass spectroscopy, we identified 4 lysine residues on A20 to be ubiquitination sites. Mutation of these lysines to arginine impaired A20’s ability to suppress TNF induced NF-κB activation. We noted stabilization of NEMO in A20 deficient cells following TNF stimulation. Biochemical studies in Jurkat and 293 cells revealed A20 dependent K48 linked polyubiquitination of NEMO at the TNFR following receptor stimulation. Our data suggest a mechanism of NF-κB regulation in which A20 is directly K63 polyubiquitinated to functionally interacts with NEMO which it targets by K48 linkages proteasomal degradation.

Introduction Regulated NF- κ B activation during development and the immune response is controlled by NEMO, the NF- κ B essential modulator. Mutations in the IKBG gene encoding NEMO that result in loss of function in NEMO result in the syndrome of Ectodermal Dysplasia with Anhidrosis and Immunodeficiency (EDA-ID), also referred to as NEMO syndrome. Methods By detailed functional analysis of primary patient cells and a NEMO deficient T cell line reconstituted with mutant forms of NEMO we describe a distinct group of NEMO mutations with C-terminal deletion (ΔCT NEMO) which result in EDA-ID combined severe skin and intestinal inflammatory disease. Results T cells reconstituted with ΔCT NEMO mutations exhibited increased constitutive NF- κ Bactivity, and increased activation following TNF or Flagellin stimulation due to upregulated intrinsic IKK complex activity, but were unresponsive to TCR stimulation. ΔCT NEMO mutations reduced NEMO/IKK complex interactions with K63- but not linear (also referred to M1) polyubiquitin chains. Skewed M1/K63 polyubiquitin binding by ΔCT NEMO led to increased association with RIP1 and stabilization of RIP1 within the NEMO signaling complex. These gain-of-function phenotypes were blocked by mutations targeting the NEMO residues in the UBAN domain essential for binding to linear but not K63-linked polyubiquitin chains. Conclusion Our findings reveal altered recognition of polyubiquitin modifications by NEMO as a receptor-specific control switch for IKK activity, and identifies the NEMO C-terminus as a negative regulator of induced NF- κ B activation by innate but not adaptive immune receptors.

Hypomorphic mutation of the NF-kB essential modulator (NEMO) causes a spectrum of inflammatory disease and immunodeficiency. NEMO is critical in immunity, regulating the activation of transcription factors NF-kB and IRF3 to control inflammation and antiviral type I interferon. In an individual with persistent inflammatory disease, we identified a synonymous mutation in the gene encoding NEMO that enhances alternative splicing and production of a mutant protein (Δ-NEMO). Co-immunoprecipitation studies in patient T cells revealed impaired association of Δ-NEMO with TANK (TRAF family member-associated NF-kB activator) and, consequently, with the IRF3 kinase TANK-binding kinase 1 (TBK1). Concomitantly, patient fibroblasts displayed impaired production of IRF3-dependent type I and III interferon in response to poly(I:C) and permitted increased virus propagation. However, cells containing Δ-NEMO exhibited enhanced IRF3 nuclear translocation and binding to the IFNB1 promoter. Our results suggest that defective interferon production in this patient lies instead in impaired NF-kB activation, as poly(I:C)- and 5’pppRNA induced translocation of NF-kB in patient fibroblasts was reduced. This impairment in NF-kB was specific, as TNF-induced NF-kB activation and gene induction was normal. These data describe a novel mechanism of autoinflammatory disease due to a type I interferon deficiency. Thus, this represents a model of inflammatory diseases responsive to type I IFN therapy.

Toll-like receptor 3 and the RIG-I-like family of receptors (RLRs) mediate antiviral signaling through NEMO-dependent activation of transcription factors NF-κB and IRF3, which are recognized for their roles in inflammation and the induction of type I interferon. We identified an individual with Ectodermal Dysplasia, a history of in utero cytomegalovirus exposure, and persistent inflammatory disease. Sanger sequencing revealed a de novo synonymous mutation in IKBKG, which encodes NEMO, the NF-κB essential modulator. The mutation results in an mRNA splicing defect which leads to the production of a mutant protein containing an in-frame deletion of 51 residues, lacking a previously identified TANK (TRAF family member-associated NFKB activator) interaction domain. Co-immunoprecipitation studies revealed that the mutant form of NEMO from patient T cells is unable to recruit the IRF3 kinase TANK-binding kinase-1 (TBK1), due to impaired association with TANK. Patient fibroblasts stimulated with the RLR ligand poly (I:C) led to impaired induction of IRF3 response genes IFNB1, CCL5, CXCL10, IP10, OASL, and DDX58. Consequently, dermal fibroblasts infected with RSV or hPIV3 demonstrated increased virus propagation relative to healthy control cells. Impaired gene expression occurred despite enhanced nuclear translocation of IRF3, suggesting that the mechanism of impaired activation is not a simple defect in nuclear locof the transcription factor. In contrast to the impaired type I IFN response, canonical IKK activation and gene induction in dermal fibroblasts appeared relatively normal following TNF treatment. These results illustrate a novel mechanism of autoinflammatory disease due to aberrant expression of an alternately spliced NEMO and deficient IRF3-mediated response.

The RIG-I-like receptors mediate antiviral signaling through NEMO-dependent activation of transcription factors NF-kB and IRF3, which are recognized for their roles in inflammation and the induction of Type I Interferon, respectively. We identified an individual with increased susceptibility to viral infection and persistent inflammatory disease. Sanger sequencing revealed a de novo synonymous mutation in the gene encoding NEMO that results in an in-frame splicing event and exclusive production of a shortened protein. To understand the role of NEMO in regulating the antiviral response, we stimulated patient and healthy control fibroblasts with RIG-I-like receptor ligand poly(I:C), which mimics viral RNA. Induction of IRF3 response genes IFNB1 and CXCL10 was reduced 35-fold and 500-fold (n=2), respectively, compared to healthy control, and patient cells were unable to control viral infection. In contrast, TNF induced the expression of proinflammatory genes TNFSF10 and IL17 7-fold and 4-fold higher in patient cells than in healthy control cells. Increased TNF induced gene expression was accompanied by increased p65 nuclear translocation indicative of increased canonical IKK activation. These results suggest that aberrant expression of an alternately spliced NEMO isoform constitutes a novel autoinflammatory disease characterized by unbalanced NF-kB and IRF3 mediated responses to viral infection.