Biomaterial and cellular implants: foreign surfaces where immunity and coagulation meet.

Exposure of blood to a foreign surface in the form of a diagnostic or therapeutic biomaterial device or implanted cells or tissue elicits an immediate, evolutionarily conserved thromboinflammatory response from the host. Primarily designed to protect against invading organisms after an injury, this innate response features instantaneous activation of several blood-borne, highly interactive, well-orchestrated cascades and cellular events that limit bleeding, destroy and eliminate the foreign substance or cells, and promote healing and a return to homeostasis via delicately balanced regenerative processes. In the setting of blood-contacting synthetic or natural biomaterials and implantation of foreign cells or tissues, innate responses are robust, albeit highly context specific. Unfortunately, they tend to be less than adequately regulated by the host's natural anticoagulant or anti-inflammatory pathways, thereby jeopardizing the functional integrity of the device, as well as the health of the host. Strategies to achieve biocompatibility with a sustained return to homeostasis, particularly while the device remains in situ and functional, continue to elude scientists and clinicians. In this review, some of the complex mechanisms by which biomaterials and cellular transplants provide a "hub" for activation and amplification of coagulation and immunity, thromboinflammation, are discussed, with a view toward the development of innovative means of overcoming the innate challenges.

Brain Hypoxia Is Associated With Neuroglial Injury in Humans Post-Cardiac Arrest.

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Chondrocyte Thrombomodulin Protects against Osteoarthritis.

Osteoarthritis (OA) is a prevalent form of arthritis that affects over 32.5 million adults worldwide, causing significant cartilage damage and disability. Unfortunately, there are currently no effective treatments for OA, highlighting the need for novel therapeutic approaches. Thrombomodulin (TM), a glycoprotein expressed by chondrocytes and other cell types, has an unknown role in OA. Here, we investigated the function of TM in chondrocytes and OA using various methods, including recombinant TM (rTM), transgenic mice lacking the TM lectin-like domain (TMLeD/LeD), and a microRNA (miRNA) antagomir that increased TM expression. Results showed that chondrocyte-expressed TM and soluble TM [sTM, like recombinant TM domain 1 to 3 (rTMD123)] enhanced cell growth and migration, blocked interleukin-1ß (IL-1ß)-mediated signaling and protected against knee function and bone integrity loss in an anterior cruciate ligament transection (ACLT)-induced mouse model of OA. Conversely, TMLeD/LeD mice exhibited accelerated knee function loss, while treatment with rTMD123 protected against cartilage loss even one-week post-surgery. The administration of an miRNA antagomir (miR-up-TM) also increased TM expression and protected against cartilage damage in the OA model. These findings suggested that chondrocyte TM plays a crucial role in counteracting OA, and miR-up-TM may represent a promising therapeutic approach to protect against cartilage-related disorders.

Complement contributions to COVID-19.

PURPOSE OF REVIEW: COVID-19 remains a major source of concern, particularly as new variants emerge and with recognition that patients may suffer long-term effects. Mechanisms underlying SARS-CoV-2 mediated organ damage and the associated vascular endotheliopathy remain poorly understood, hindering new drug development. Here, we highlight selected key concepts of how the complement system, a major component of innate immunity that is dysregulated in COVID-19, participates in the thromboinflammatory response and drives the vascular endotheliopathy. RECENT FINDINGS: Recent studies have revealed mechanisms by which complement is activated directly by SARS-CoV-2, and how the system interfaces with other innate thromboinflammatory cellular and proteolytic pathways involving platelets, neutrophils, neutrophil extracellular traps and the coagulation and kallikrein-kinin systems. With this new information, multiple potential sites for therapeutic intervention are being uncovered and evaluated in the clinic. SUMMARY: Infections with SARS-CoV-2 cause damage to the lung alveoli and microvascular endothelium via a process referred to as thromboinflammation. Although not alone in being dysregulated, complement is an early player, prominent in promoting the endotheliopathy and consequential organ damage, either directly and/or via the system's complex interplay with other cellular, molecular and biochemical pathways. Delineating these critical interactions is revealing novel and promising strategies for therapeutic intervention.

Absence of the lectin-like domain of thrombomodulin reduces HSV-1 lethality of mice with increased microglia responses.

BACKGROUND: Herpes simplex virus 1 (HSV-1) can induce fatal encephalitis. Cellular factors regulate the host immunity to affect the severity of HSV-1 encephalitis. Recent reports focus on the significance of thrombomodulin (TM), especially the domain 1, lectin-like domain (TM-LeD), which modulates the immune responses to bacterial infections and toxins and various diseases in murine models. Few studies have investigated the importance of TM-LeD in viral infections, which are also regulated by the host immunity. METHODS: In vivo studies comparing wild-type and TM-LeD knockout mice were performed to determine the role of TM-LeD on HSV-1 lethality. In vitro studies using brain microglia cultured from mice or a human microglia cell line to investigate whether and how TM-LeD affects microglia to reduce HSV-1 replication in brain neurons cultured from mice or in a human neuronal cell line. RESULTS: Absence of TM-LeD decreased the mortality, tissue viral loads, and brain neuron apoptosis of HSV-1-infected mice with increases in the number, proliferation, and phagocytic activity of brain microglia. Moreover, TM-LeD deficiency enhanced the phagocytic activity of brain microglia cultured from mice or of a human microglia cell line. Co-culture of mouse primary brain microglia and neurons or human microglia and neuronal cell lines revealed that TM-LeD deficiency augmented the capacity of microglia to reduce HSV-1 replication in neurons. CONCLUSIONS: Overall, TM-LeD suppresses microglia responses to enhance HSV-1 infection.

Sustained depletion of FXIII-A by inducing acquired FXIII-B deficiency.

The activated form of coagulation factor XIII (FXIII-A2B2), FXIII-A*, is a hemostatic enzyme essential for inhibiting fibrinolysis by irreversibly crosslinking fibrin and antifibrinolytic proteins. Despite its importance, there are no modulatory therapeutics. Guided by the observation that humans deficient in FXIII-B have reduced FXIII-A without severe bleeding, we hypothesized that a suitable small interfering RNA (siRNA) targeting hepatic FXIII-B could safely decrease FXIII-A. Here we show that knockdown of FXIII-B with siRNA in mice and rabbits using lipid nanoparticles resulted in a sustained and controlled decrease in FXIII-A. The concentration of FXIII-A in plasma was reduced by 90% for weeks after a single injection and for more than 5 months with repeated injections, whereas the concentration of FXIII-A in platelets was unchanged. Ex vivo, crosslinking of α2-antiplasmin and fibrin was impaired and fibrinolysis was enhanced. In vivo, reperfusion of carotid artery thrombotic occlusion was also enhanced. Re-bleeding events were increased after challenge, but blood loss was not significantly increased. This approach, which mimics congenital FXIII-B deficiency, provides a potential pharmacologic and experimental tool to modulate FXIII-A2B2 activity.

An improved in vitro model for studying the structural and functional properties of the endothelial glycocalyx in arteries, capillaries and veins.

The endothelial glycocalyx is a dynamic structure integral to blood vessel hemodynamics and capable of tightly regulating a range of biological processes (ie, innate immunity, inflammation, and coagulation) through dynamic changes in its composition of the brush structure. Evaluating the specific roles of the endothelial glycocalyx under a range of pathophysiologic conditions has been a challenge in vitro as it is difficult to generate functional glycocalyces using commonly employed 2D cell culture models. We present a new multi-height microfluidic platform that promotes the growth of functional glycocalyces by eliciting unique shear stress forces over a continuous human umbilical vein endothelial cell monolayer at magnitudes that recapitulate the physical environment in arterial, capillary and venous regions of the vasculature. Following 72 hours of shear stress, unique glycocalyx structures formed within each region that were distinct from that observed in short (3 days) and long-term (21 days) static cell culture. The model demonstrated glycocalyx-specific properties that match the characteristics of the endothelium in arteries, capillaries and veins, with respect to surface protein expression, platelet adhesion, lymphocyte binding and nanoparticle uptake. With artery-to-capillary-to-vein transition on a continuous endothelial monolayer, this in vitro platform is an improved system over static cell culture for more effectively studying the role of the glycocalyx in endothelial biology and disease.

Persistently elevated complement alternative pathway biomarkers in COVID-19 correlate with hypoxemia and predict in-hospital mortality.

Mechanisms underlying the SARS-CoV-2-triggered hyperacute thrombo-inflammatory response that causes multi-organ damage in coronavirus disease 2019 (COVID-19) are poorly understood. Several lines of evidence implicate overactivation of complement. To delineate the involvement of complement in COVID-19, we prospectively studied 25 ICU-hospitalized patients for up to 21 days. Complement biomarkers in patient sera and healthy controls were quantified by enzyme-linked immunosorbent assays. Correlations with respiratory function and mortality were analyzed. Activation of complement via the classical/lectin pathways was variably increased. Strikingly, all patients had increased activation of the alternative pathway (AP) with elevated levels of activation fragments, Ba and Bb. This was associated with a reduction of the AP negative regulator, factor (F) H. Correspondingly, terminal pathway biomarkers of complement activation, C5a and sC5b-9, were significantly elevated in all COVID-19 patient sera. C5a and AP constituents Ba and Bb, were significantly associated with hypoxemia. Ba and FD at the time of ICU admission were strong independent predictors of mortality in the following 30 days. Levels of all complement activation markers were sustained throughout the patients' ICU stays, contrasting with the varying serum levels of IL-6, C-reactive protein, and ferritin. Severely ill COVID-19 patients have increased and persistent activation of complement, mediated strongly via the AP. Complement activation biomarkers may be valuable measures of severity of lung disease and the risk of mortality. Large-scale studies will reveal the relevance of these findings to thrombo-inflammation in acute and post-acute COVID-19.

VEGF-Induced Endothelial Podosomes via ROCK2-Dependent Thrombomodulin Expression Initiate Sprouting Angiogenesis.

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Recombinant thrombomodulin domain 1 rescues pathological angiogenesis by inhibition of HIF-1α-VEGF pathway.

Pathological angiogenesis (PA) contributes to various ocular diseases, including age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity, which are major causes of blindness over the world. Current treatments focus on anti-vascular endothelial growth factor (VEGF) therapy, but persistent avascular retina, recurrent intravitreal neovascularization, and general adverse effects are reported. We have previously found that recombinant thrombomodulin domain 1 (rTMD1) can suppress vascular inflammation. However, the function of rTMD1 in VEGF-induced PA remains unknown. In this study, we found that rTMD1 inhibited VEGF-induced angiogenesis in vitro. In an oxygen induced retinopathy (OIR) animal model, rTMD1 treatment significantly decreased retinal neovascularization but spared normal physiological vessel growth. Furthermore, loss of TMD1 significantly promoted PA in OIR. Meanwhile, hypoxia-inducible factor-1α, the transcription factor that upregulates VEGF, was suppressed after rTMD1 treatment. The levels of interleukin-6, and intercellular adhesion molecule-1 were also significantly suppressed. In conclusion, our results indicate that rTMD1 not only has dual effects to suppress PA and inflammation in OIR, but also can be a potential HIF-1α inhibitor for clinical use. These data bring forth the possibility of rTMD1 as a novel therapeutic agent for PA.

Releasates of riboflavin/UV-treated platelets: Microvesicles suppress cytokine-mediated endothelial cell migration/proliferation.

BACKGROUND: Accelerated development of the platelet (PLT) storage lesion upon pathogen inactivation (PI) is associated with the release of proteins from granules and platelet microvesicles (PMVs). Whether PI treatments alter the interaction between PLT factors and the vessel endothelium is of interest in understanding the risk profile of these technologies. STUDY DESIGN AND METHODS: In a pool-and-split study, one platelet concentrate (PC) was treated with riboflavin/UV (RF/UV) light, while the other one was kept as an untreated control. Releasates and PMV-depleted releasates were prepared by differential centrifugation steps on days 0, 1, 5, and 7 of storage. Cytokine/chemokine release following PI treatment was analyzed by an antibody array, and results were verified by the enzyme-linked immunosorbent assay. PMVs were enumerated by CD41 labeling and flow cytometry. Wound scratch assays were performed using cultured Ea.hy926 cells exposed to the differently prepared releasates. Effects of releasates on the phosphorylation levels of kinases ERK and p38 expressed by endothelial cells were analyzed by immunoblot. RESULTS: Cytokine/chemokine assays identified a 2-fold increase in epidermal growth factor released from PCs treated with RF/UV light compared with control. PMV count increased ~100-fold following PI treatment. Unmodified releasates and PMV-depleted releasates displayed different contributions to the kinetics of endothelial cell wound closure. This observation was associated with an increased ERK versus unaltered p38 activation in the endothelial cells. CONCLUSION: This study identified an inhibitory impact of PMVs on endothelial cell migration/proliferation upon stimulation by released cytokines and PMVs from PLTs treated with RF/UV light for endothelial cell wound closure.

Exploring traditional and nontraditional roles for thrombomodulin.

Thrombomodulin (TM) is an integral component of a multimolecular system, localized primarily to the vascular endothelium, that integrates crucial biological processes and biochemical pathways, including those related to coagulation, innate immunity, inflammation, and cell proliferation. These are designed to protect the host from injury and promote healing. The "traditional" role of TM in hemostasis was determined with its discovery in the 1980s as a ligand for thrombin and a critical cofactor for the major natural anticoagulant protein C system and subsequently for thrombin-mediated activation of the thrombin activatable fibrinolysis inhibitor (also known as procarboxypeptidase B2). Studies in the past 2 decades are redefining TM as a molecule with many properties, exhibited via its multiple domains, through its interacting partners, complex regulated expression, and synthesis by cells other than the endothelium. In this report, we review some of the recently reported diverse properties of TM and how these may impact on our understanding of the pathogenesis of several diseases.

Alteration of blood clotting and lung damage by protamine are avoided using the heparin and polyphosphate inhibitor UHRA.

Anticoagulant therapy-associated bleeding and pathological thrombosis pose serious risks to hospitalized patients. Both complications could be mitigated by developing new therapeutics that safely neutralize anticoagulant activity and inhibit activators of the intrinsic blood clotting pathway, such as polyphosphate (polyP) and extracellular nucleic acids. The latter strategy could reduce the use of anticoagulants, potentially decreasing bleeding events. However, previously described cationic inhibitors of polyP and extracellular nucleic acids exhibit both nonspecific binding and adverse effects on blood clotting that limit their use. Indeed, the polycation used to counteract heparin-associated bleeding in surgical settings, protamine, exhibits adverse effects. To address these clinical shortcomings, we developed a synthetic polycation, Universal Heparin Reversal Agent (UHRA), which is nontoxic and can neutralize the anticoagulant activity of heparins and the prothrombotic activity of polyP. Sharply contrasting protamine, we show that UHRA does not interact with fibrinogen, affect fibrin polymerization during clot formation, or abrogate plasma clotting. Using scanning electron microscopy, confocal microscopy, and clot lysis assays, we confirm that UHRA does not incorporate into clots, and that clots are stable with normal fibrin morphology. Conversely, protamine binds to the fibrin clot, which could explain how protamine instigates clot lysis and increases bleeding after surgery. Finally, studies in mice reveal that UHRA reverses heparin anticoagulant activity without the lung injury seen with protamine. The data presented here illustrate that UHRA could be safely used as an antidote during adverse therapeutic modulation of hemostasis.

The Structural Basis for Complement Inhibition by Gigastasin, a Protease Inhibitor from the Giant Amazon Leech.

Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway.

Polyphosphate is a novel cofactor for regulation of complement by a serpin, C1 inhibitor.

The complement system plays a key role in innate immunity, inflammation, and coagulation. The system is delicately balanced by negative regulatory mechanisms that modulate the host response to pathogen invasion and injury. The serpin, C1-esterase inhibitor (C1-INH), is the only known plasma inhibitor of C1s, the initiating serine protease of the classical pathway of complement. Like other serpin-protease partners, C1-INH interaction with C1s is accelerated by polyanions such as heparin. Polyphosphate (polyP) is a naturally occurring polyanion with effects on coagulation and complement. We recently found that polyP binds to C1-INH, prompting us to consider whether polyP acts as a cofactor for C1-INH interactions with its target proteases. We show that polyP dampens C1s-mediated activation of the classical pathway in a polymer length- and concentration-dependent manner by accelerating C1-INH neutralization of C1s cleavage of C4 and C2. PolyP significantly increases the rate of interaction between C1s and C1-INH, to an extent comparable to heparin, with an exosite on the serine protease domain of the enzyme playing a major role in this interaction. In a serum-based cell culture system, polyP significantly suppressed C4d deposition on endothelial cells, generated via the classical and lectin pathways. Moreover, polyP and C1-INH colocalize in activated platelets, suggesting that their interactions are physiologically relevant. In summary, like heparin, polyP is a naturally occurring cofactor for the C1s:C1-INH interaction and thus an important regulator of complement activation. The findings may provide novel insights into mechanisms underlying inflammatory diseases and the development of new therapies.

Cross Talk Pathways Between Coagulation and Inflammation.

Anatomic pathology studies performed over 150 years ago revealed that excessive activation of coagulation occurs in the setting of inflammation. However, it has taken over a century since these seminal observations were made to delineate the molecular mechanisms by which these systems interact and the extent to which they participate in the pathogenesis of multiple diseases. There is, in fact, extensive cross talk between coagulation and inflammation, whereby activation of one system may amplify activation of the other, a situation that, if unopposed, may result in tissue damage or even multiorgan failure. Characterizing the common triggers and pathways are key for the strategic design of effective therapeutic interventions. In this review, we highlight some of the key molecular interactions, some of which are already showing promise as therapeutic targets for inflammatory and thrombotic disorders.

Essential Role for Survivin in the Proliferative Expansion of Progenitor and Mature B Cells.

Survivin is a member of the inhibitor of apoptosis family of proteins and a biomarker of poor prognosis in aggressive B cell non-Hodgkin's lymphoma. In addition to its role in inhibition of apoptosis, survivin also regulates mitosis. In this article, we show that deletion of survivin during early B cell development results in a complete block at the cycling pre-B stage. In the periphery, B cell homeostasis is not affected, but survivin-deficient B cells are unable to mount humoral responses. Correspondingly, we show that survivin is required for cell division in response to mitogenic stimulation. Thus, survivin is essential for proliferation of B cell progenitors and activated mature B cells, but is dispensable for B cell survival. Moreover, a small-molecule inhibitor of survivin strongly impaired the growth of representative B lymphoma lines in vitro, supporting the validity of survivin as an attractive therapeutic target for high-grade B cell non-Hodgkin's lymphoma.

Hepatocellular carcinoma repression by TNFα-mediated synergistic lethal effect of mitosis defect-induced senescence and cell death sensitization.

UNLABELLED: Hepatocellular carcinoma (HCC) is a cancer lacking effective therapies. Several measures have been proposed to treat HCCs, such as senescence induction, mitotic inhibition, and cell death promotion. However, data from other cancers suggest that single use of these approaches may not be effective. Here, by genetic targeting of Survivin, an inhibitor of apoptosis protein (IAP) that plays dual roles in mitosis and cell survival, we identified a tumor necrosis factor alpha (TNFα)-mediated synergistic lethal effect between senescence and apoptosis sensitization in malignant HCCs. Survivin deficiency results in mitosis defect-associated senescence in HCC cells, which triggers local inflammation and increased TNFα. Survivin inactivation also sensitizes HCC cells to TNFα-triggered cell death, which leads to marked HCC regression. Based on these findings, we designed a combination treatment using mitosis inhibitor and proapoptosis compounds. This treatment recapitulates the therapeutic effect of Survivin deletion and effectively eliminates HCCs, thus representing a potential strategy for HCC therapy. CONCLUSION: Survivin ablation dramatically suppresses human and mouse HCCs by triggering senescence-associated TNFα and sensitizing HCC cells to TNFα-induced cell death. Combined use of mitotic inhibitor and second mitochondrial-derived activator of caspases mimetic can induce senescence-associated TNFα and enhance TNFα-induced cell death and synergistically eliminate HCC. (Hepatology 2016;64:1105-1120).

HUS and the case for complement.

Hemolytic-uremic syndrome (HUS) is a thrombotic microangiopathy that is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. Excess complement activation underlies atypical HUS and is evident in Shiga toxin-induced HUS (STEC-HUS). This Spotlight focuses on new knowledge of the role of Escherichia coli-derived toxins and polyphosphate in modulating complement and coagulation, and how they affect disease progression and response to treatment. Such new insights may impact on current and future choices of therapies for STEC-HUS.

Polyphosphate suppresses complement via the terminal pathway.

Polyphosphate, synthesized by all cells, is a linear polymer of inorganic phosphate. When released into the circulation, it exerts prothrombotic and proinflammatory activities by modulating steps in the coagulation cascade. We examined the role of polyphosphate in regulating the evolutionarily related proteolytic cascade complement. In erythrocyte lysis assays, polyphosphate comprising more than 1000 phosphate units suppressed total hemolytic activity with a concentration to reduce maximal lysis to 50% that was 10-fold lower than with monophosphate. In the ion- and enzyme-independent terminal pathway complement assay, polyphosphate suppressed complement in a concentration- and size-dependent manner. Phosphatase-treated polyphosphate lost its ability to suppress complement, confirming that polymer integrity is required. Sequential addition of polyphosphate to the terminal pathway assay showed that polyphosphate interferes with complement only when added before formation of the C5b-7 complex. Physicochemical analyses using native gels, gel filtration, and differential scanning fluorimetry revealed that polyphosphate binds to and destabilizes C5b,6, thereby reducing the capacity of the membrane attack complex to bind to and lyse the target cell. In summary, we have added another function to polyphosphate in blood, demonstrating that it dampens the innate immune response by suppressing complement. These findings further establish the complex relationship between coagulation and innate immunity.