Hot under the clot: venous thrombogenesis is an inflammatory process.

ABSTRACT: Venous thrombosis (VT) is a serious medical condition in which a blood clot forms in deep veins, often causing limb swelling and pain. Current antithrombotic therapies carry significant bleeding risks resulting from targeting essential coagulation factors. Recent advances in this field have revealed that the cross talk between the innate immune system and coagulation cascade is a key driver of VT pathogenesis, offering new opportunities for potential therapeutic interventions without inducing bleeding complications. This review summarizes and discusses recent evidence from preclinical models on the role of inflammation in VT development. We highlight the major mechanisms by which endothelial cell activation, Weibel-Palade body release, hypoxia, reactive oxygen species, inflammasome, neutrophil extracellular traps, and other immune factors cooperate to initiate and propagate VT. We also review emerging clinical data describing anti-inflammatory approaches as adjuncts to anticoagulation in VT treatment. Finally, we identify key knowledge gaps and future directions that could maximize the benefit of anti-inflammatory therapies in VT. Identifying and targeting the inflammatory factors driving VT, either at the endothelial cell level or within the clot, may pave the way for new therapeutic possibilities for improving VT treatment and reducing thromboembolic complications without increasing bleeding risk.

Complement Factor H Inhibits CD47-Mediated Resolution of Inflammation.

Variants of the CFH gene, encoding complement factor H (CFH), show strong association with age-related macular degeneration (AMD), a major cause of blindness. Here, we used murine models of AMD to examine the contribution of CFH to disease etiology. Cfh deletion protected the mice from the pathogenic subretinal accumulation of mononuclear phagocytes (MP) that characterize AMD and showed accelerated resolution of inflammation. MP persistence arose secondary to binding of CFH to CD11b, which obstructed the homeostatic elimination of MPs from the subretinal space mediated by thrombospsondin-1 (TSP-1) activation of CD47. The AMD-associated CFH(H402) variant markedly increased this inhibitory effect on microglial cells, supporting a causal link to disease etiology. This mechanism is not restricted to the eye, as similar results were observed in a model of acute sterile peritonitis. Pharmacological activation of CD47 accelerated resolution of both subretinal and peritoneal inflammation, with implications for the treatment of chronic inflammatory disease.

Efficacy of platelet-inspired hemostatic nanoparticles on bleeding in von Willebrand disease murine models.

The lack of innovation in von Willebrand disease (VWD) originates from many factors including the complexity and heterogeneity of the disease but also from a lack of recognition of the impact of the bleeding symptoms experienced by patients with VWD. Recently, a few research initiatives aiming to move past replacement therapies using plasma-derived or recombinant von Willebrand factor (VWF) concentrates have started to emerge. Here, we report an original approach using synthetic platelet (SP) nanoparticles for the treatment of VWD type 2B (VWD-2B) and severe VWD (type 3 VWD). SP are liposomal nanoparticles decorated with peptides enabling them to concomitantly bind to collagen, VWF, and activated platelets. In vitro, using various microfluidic assays, we show the efficacy of SPs to improve thrombus formation in VWF-deficient condition (with human platelets) or using blood from mice with VWD-2B and deficient VWF (VWF-KO, ie, type 3 VWD). In vivo, using a tail-clip assay, SP treatment reduced blood loss by 35% in mice with VWD-2B and 68% in mice with VWF-KO. Additional studies using nanoparticles decorated with various combinations of peptides demonstrated that the collagen-binding peptide, although not sufficient by itself, was crucial for SP efficacy in VWD-2B; whereas all 3 peptides appeared necessary for mice with VWF-KO. Clot imaging by immunofluorescence and scanning electron microscopy revealed that SP treatment of mice with VWF-KO led to a strong clot, similar to those obtained in wild-type mice. Altogether, our results show that SP could represent an attractive therapeutic alternative for VWD, especially considering their long half-life and stability.

S100A8/A9 drives the formation of procoagulant platelets through GPIbα.

S100A8/A9, also known as "calprotectin" or "MRP8/14," is an alarmin primarily secreted by activated myeloid cells with antimicrobial, proinflammatory, and prothrombotic properties. Increased plasma levels of S100A8/A9 in thrombo-inflammatory diseases are associated with thrombotic complications. We assessed the presence of S100A8/A9 in the plasma and lung autopsies from patients with COVID-19 and investigated the molecular mechanism by which S100A8/A9 affects platelet function and thrombosis. S100A8/A9 plasma levels were increased in patients with COVID-19 and sustained high levels during hospitalization correlated with poor outcomes. Heterodimeric S100A8/A9 was mainly detected in neutrophils and deposited on the vessel wall in COVID-19 lung autopsies. Immobilization of S100A8/A9 with collagen accelerated the formation of a fibrin-rich network after perfusion of recalcified blood at venous shear. In vitro, platelets adhered and partially spread on S100A8/A9, leading to the formation of distinct populations of either P-selectin or phosphatidylserine (PS)-positive platelets. By using washed platelets, soluble S100A8/A9 induced PS exposure but failed to induce platelet aggregation, despite GPIIb/IIIa activation and alpha-granule secretion. We identified GPIbα as the receptor for S100A8/A9 on platelets inducing the formation of procoagulant platelets with a supporting role for CD36. The effect of S100A8/A9 on platelets was abolished by recombinant GPIbα ectodomain, platelets from a patient with Bernard-Soulier syndrome with GPIb-IX-V deficiency, and platelets from mice deficient in the extracellular domain of GPIbα. We identified the S100A8/A9-GPIbα axis as a novel targetable prothrombotic pathway inducing procoagulant platelets and fibrin formation, in particular in diseases associated with high levels of S100A8/A9, such as COVID-19.

Basic Mechanisms of Hemolysis-Associated Thrombo-Inflammation and Immune Dysregulation.

Independent of etiology, hemolytic diseases are associated with thrombosis, inflammation and immune dysregulation, all together contributing to organ damage and poor outcome. Beyond anemia and the loss of the anti-inflammatory functions of red blood cells, hemolysis leads to the release of damage-associated molecular patterns including ADP, hemoglobin, and heme, which act through multiple receptors and signaling pathways fostering a hyperinflammatory and hypercoagulable state. Extracellular free heme is promiscuous alarmin capable of triggering oxido-inflammatory and thrombotic events by inducing the activation of platelets, endothelial and innate cells as well as the coagulation and complement cascades. In this review, we discuss the main mechanisms by which hemolysis and, in particular, heme, drive this thrombo-inflammatory milieu and discuss the consequences of hemolysis on the host response to secondary infections.

Post-translational polymodification of ß1-tubulin regulates motor protein localisation in platelet production and function.

In specialised cells, the expression of specific tubulin isoforms and their subsequent post-translational modifications drive and coordinate unique morphologies and behaviours. The mechanisms by which ß1-tubulin, the platelet and megakaryocyte (MK) lineage restricted tubulin isoform, drives platelet production and function remains poorly understood. We investigated the roles of two key post-translational tubulin polymodifications (polyglutamylation and polyglycylation) on these processes using a cohort of thrombocytopenic patients, human induced pluripotent stem cell (iPSC) derived MKs, and healthy human donor platelets. We find distinct patterns of polymodification in MKs and platelets, mediated by the antagonistic activities of the cell specific expression of Tubulin Tyrosine Ligase Like (TTLLs) and Cytosolic Carboxypeptidase (CCP) enzymes. The resulting microtubule patterning spatially regulates motor proteins to drive proplatelet formation in megakaryocytes, and the cytoskeletal reorganisation required for thrombus formation. This work is the first to show a reversible system of polymodification by which different cell specific functions are achieved.

Salmonella-induced thrombi in mice develop asynchronously in the spleen and liver and are not effective bacterial traps.

Thrombosis is a frequent, life-threatening complication of systemic infection associated with multiple organ damage. We have previously described a novel mechanism of inflammation-driven thrombosis induced by Salmonella Typhimurium infection of mice. Thrombosis in the liver develops 7 days after infection, persisting after the infection resolves, and is monocytic cell dependent. Unexpectedly, thrombosis was not prominent in the spleen at this time, despite carrying a similar bacterial burden as the liver. In this study, we show that thrombosis does occur in the spleen but with strikingly accelerated kinetics compared with the liver, being evident by 24 hours and resolving rapidly thereafter. The distinct kinetics of thrombosis and bacterial burden provides a test of the hypothesis that thrombi form in healthy vessels to trap or remove bacteria from the circulation, often termed immunothrombosis. Remarkably, despite bacteria being detected throughout infected spleens and livers in the early days of infection, immunohistological analysis of tissue sections show that thrombi contain very low numbers of bacteria. In contrast, bacteria are present throughout platelet aggregates induced by Salmonella in vitro. Therefore, we show that thrombosis develops with organ-specific kinetics and challenge the universality of immunothrombosis as a mechanism to capture bacteria in vivo.

Lymphatic blood filling in CLEC-2-deficient mouse models.

C-type lectin-like receptor 2 (CLEC-2) is considered as a potential drug target in settings of wound healing, inflammation, and infection. A potential barrier to this is evidence that CLEC-2 and its ligand podoplanin play a critical role in preventing lymphatic vessel blood filling in mice throughout life. In this study, this aspect of CLEC-2/podoplanin function is investigated in more detail using new and established mouse models of CLEC-2 and podoplanin deficiency, and models of acute and chronic vascular remodeling. We report that CLEC-2 expression on platelets is not required to maintain a barrier between the blood and lymphatic systems in unchallenged mice, post-development. However, under certain conditions of chronic vascular remodeling, such as during tumorigenesis, deficiency in CLEC-2 can lead to lymphatic vessel blood filling. These data provide a new understanding of the function of CLEC-2 in adult mice and confirm the essential nature of CLEC-2-driven platelet activation in vascular developmental programs. This work expands our understanding of how lymphatic blood filling is prevented by CLEC-2-dependent platelet function and provides a context for the development of safe targeting strategies for CLEC-2 and podoplanin.

Stimulation with FITC-labeled antigens confers B cells with regulatory properties.

B cells with regulatory properties (Bregs) were identified in human and in mice among different B-cell subsets. Their regulatory properties rely mainly on the production of anti-inflammatory cytokines, in particular IL10, IL-35 and TGFß, and were extensively studied in mouse models of autoimmune and inflammatory diseases. However, the exact nature of the stimulatory signals conferring regulatory properties to B cells is still not clear. We serendipitously observed that fluorescein isothiocyanate (FITC) binds to a significant proportion of naïve mouse B cells. Binding of FITC to the B-cell surface implicated at least in part the B-cell receptor. It triggered IL-10 production and allowed the endocytosis of FITC-coupled antigens followed by their presentation to CD4+ T cells. In particular, B cells incubated with FITC-OVA polarized OTII T cells towards a Tr1/Th2 phenotype in vitro. Further, the adoptive transfer of B cells incubated with FITC-labeled myelin oligodendrocyte glycoprotein peptide protected mice from experimental autoimmune encephalomyelitis, a T-cell-dependent autoimmune model. Together, the data show that FITC-stimulated B cells polarize immune responses towards Tr1/Th2 and acquire immuno-modulatory properties.

Endothelial cells: source, barrier, and target of defensive mediators.

Endothelium is strategically located at the interface between blood and interstitial tissues, placing thus endothelial cell as a key player in vascular homeostasis. Endothelial cells are in a dynamic equilibrium with their environment and constitute concomitantly a source, a barrier, and a target of defensive mediators. This review will discuss the recent advances in our understanding of the complex crosstalk between the endothelium, the complement system and the hemostasis in health and in disease. The first part will provide a general introduction on endothelial cells heterogeneity and on the physiologic role of the complement and hemostatic systems. The second part will analyze the interplay between complement, hemostasis and endothelial cells in physiological conditions and their alterations in diseases. Particular focus will be made on the prototypes of thrombotic microangiopathic disorders, resulting from complement or hemostasis dysregulation-mediated endothelial damage: atypical hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. Novel aspects of the pathophysiology of the thrombotic microangiopathies will be discussed.

Prevention of the anti-factor VIII memory B-cell response by inhibition of Bruton tyrosine kinase in experimental hemophilia A.

Hemophilia A is a rare hemorrhagic disorder caused by the lack of functional pro-coagulant factor VIII. Factor VIII replacement therapy in patients with severe hemophilia A results in the development of inhibitory anti-factor VIII IgG in up to 30% of cases. To date, immune tolerance induction, with daily injection of large amounts of factor VIII, is the only strategy to eradicate factor VIII inhibitors. This strategy is, however, efficient in only 60-80% of patients. We investigated whether blocking B-cell receptor signaling upon inhibition of Bruton tyrosine kinase prevents anti-factor VIII immune responses in a mouse model of severe hemophilia A. Factor VIII-naïve and factor VIII-sensitized factor VIII-deficient mice were fed with the selective inhibitor of Bruton tyrosine kinase, (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxyl] phenyl)-1H pyrazole-4-carboxamide (PF-06250112), to inhibit B-cell receptor signaling prior to challenge with exogenous factor VIII. The consequences on the anti-factor VIII immune response were studied. Inhibition of Bruton tyrosine kinase during the primary anti-factor VIII immune response in factor VIII-naïve mice did not prevent the development of inhibitory anti-factor VIII IgG. In contrast, the anti-factor VIII memory B-cell response was consistently reduced upon treatment of factor VIII-sensitized mice with the Bruton tyrosine kinase inhibitor. The Bruton tyrosine kinase inhibitor reduced the differentiation of memory B cells ex vivo and in vivo following adoptive transfer to factor VIII-naïve animals. Taken together, our data identify inhibition of Bruton tyrosine kinase using PF-06250112 as a strategy to limit the reactivation of factor VIII-specific memory B cells upon re-challenge with therapeutic factor VIII.

Platelet glycoprotein VI and C-type lectin-like receptor 2 deficiency accelerates wound healing by impairing vascular integrity in mice.

Platelets promote wound healing by forming a vascular plug and by secreting growth factors and cytokines. Glycoprotein (GP)VI and C-type lectin-like receptor (CLEC)-2 signal through a (hem)-immunoreceptor tyrosine-based activation motif, which induces platelet activation. GPVI and CLEC-2 support vascular integrity during inflammation in the skin through regulation of leukocyte migration and function, and by sealing sites of vascular damage. In this study, we investigated the role of impaired vascular integrity due to GPVI and/or CLEC-2 deficiency in wound repair using a full-thickness excisional skin wound model in mice. Transgenic mice deficient in both GPVI and CLEC-2 exhibited accelerated skin wound healing, despite a marked impairment in vascular integrity. The local and temporal bleeding in the skin led to greater plasma protein entry, including fibrinogen and clotting factors, was associated with increased fibrin generation, reduction in wound neutrophils and M1 macrophages, decreased level of tumor necrosis factor (TNF)-α, and enhanced angiogenesis at day 3 after injury. Accelerated wound healing was not due to developmental defects in CLEC-2 and GPVI double-deficient mice as similar results were observed in GPVI-deficient mice treated with a podoplanin-blocking antibody. The rate of wound healing was not altered in mice deficient in either GPVI or CLEC-2. Our results show that, contrary to defects in coagulation, bleeding following a loss of vascular integrity caused by platelet CLEC-2 and GPVI deficiency facilitates wound repair by increasing fibrin(ogen) deposition, reducing inflammation, and promoting angiogenesis.

Origin-Specific Adhesive Interactions of Mesenchymal Stem Cells with Platelets Influence Their Behavior After Infusion.

We investigated the adhesive behavior of mesenchymal stem cells (MSC) in blood, which might influence their fate when infused as therapy. Isolated human bone marrow MSC (BMMSC) or umbilical cord MSC (UCMSC) adhered efficiently from flow to the matrix proteins, collagen, or fibronectin, but did not adhere to endothelial selectins. However, when suspended in blood, BMMSC no longer adhered to collagen, while UCMSC adhered along with many aggregated platelets. Neither MSC adhered to fibronectin from flowing blood, although the fibronectin surface did become coated with a platelet monolayer. UCMSC induced platelet aggregation in platelet rich plasma, and caused a marked drop in platelet count when mixed with whole human or mouse blood in vitro, or when infused into mice. In contrast, BMMSC did not activate platelets or induce changes in platelet count. Interestingly, isolated UCMSC and BMMSC both adhered to predeposited platelets. The differences in behavior in blood were attributable to expression of podoplanin (an activating ligand for the platelet receptor CLEC-2), which was detected on UCMSC, but not BMMSC. Thus, platelets were activated when bound to UCMSC, but not BMMSC. Platelet aggregation by UCMSC was inhibited by recombinant soluble CLEC-2, and UCMSC did not cause a reduction in platelet count when mixed with blood from mice deficient in CLEC-2. We predict that both MSC would carry platelets in the blood, but their interaction with vascular endothelium would depend on podoplanin-induced activation of the bound platelets. Such interactions with platelets might target MSC to damaged tissue, but could also be thrombotic. Stem Cells 2018;36:1062-1074.

Podoplanin: An emerging cancer biomarker and therapeutic target.

Podoplanin (PDPN) is a transmembrane receptor glycoprotein that is upregulated on transformed cells, cancer associated fibroblasts and inflammatory macrophages that contribute to cancer progression. In particular, PDPN increases tumor cell clonal capacity, epithelial mesenchymal transition, migration, invasion, metastasis and inflammation. Antibodies, CAR-T cells, biologics and synthetic compounds that target PDPN can inhibit cancer progression and septic inflammation in preclinical models. This review describes recent advances in how PDPN may be used as a biomarker and therapeutic target for many types of cancer, including glioma, squamous cell carcinoma, mesothelioma and melanoma.

Inhibitor Formation in Congenital Hemophilia A: an Immunological Perspective.

The immunogenicity of therapeutic factor VIII (FVIII) in patients with hemophilia A has been puzzling scientific and clinical communities for more than 3 decades. Indeed, the development of inhibitory antibodies to FVIII remains a major clinical challenge and is associated with enormous societal costs. Thus, the reasons for which a presumably innocuous, short-lived, intravenously administered glycoprotein triggers such a deleterious, long-lasting neutralizing immune response is an enigma. This review does not pretend to bring an answer to this challenging question. It will however summarize the latest findings regarding the molecular interactions at play in the recognition of FVIII by the immune cells, the validity of the proposed risk factors for FVIII alloimmunization, and the different solutions that allow induction of FVIII-specific tolerance in preclinical models of hemophilia A.

Complement C3 is a novel modulator of the anti-factor VIII immune response.

Development of neutralizing antibodies against therapeutic Factor VIII (FVIII) is the most serious complication of the treatment of hemophilia A. There is growing evidence to show the multifactorial origin of the anti-FVIII immune response, combining both genetic and environmental factors. While a role for the complement system on innate as well as adaptive immunity has been documented, the implication of complement activation on the onset of the anti-FVIII immune response is unknown. Here, using in vitro assays for FVIII endocytosis by human monocyte-derived dendritic cells and presentation to T cells, as well as in vivo complement depletion in FVIII-deficient mice, we show a novel role for complement C3 in enhancing the immune response against therapeutic FVIII. In vitro, complement C3 and its cleavage product C3b enhanced FVIII endocytosis by dendritic cells and presentation to a FVIII-specific CD4+ T-cell hybridoma. The C1 domain of FVIII had previously been shown to play an important role in FVIII endocytosis, and alanine substitutions of the K2092, F2093 and R2090 C1 residues drastically reduce FVIII uptake in vitro Interestingly, complement activation rescued the endocytosis of the FVIII C1 domain triple mutant. In a mouse model of severe hemophilia A, transient complement C3 depletion by humanized cobra venom factor, which does not generate anaphylatoxin C5a, significantly reduced the primary anti-FVIII immune response, but did not affect anti-FVIII recall immune responses. Taken together, our results suggest an important adjuvant role for the complement cascade in the initiation of the immune response to therapeutic FVIII.

Platelet CLEC-2 protects against lung injury via effects of its ligand podoplanin on inflammatory alveolar macrophages in the mouse.

There is no therapeutic intervention proven to prevent acute respiratory distress syndrome (ARDS). Novel mechanistic insights into the pathophysiology of ARDS are therefore required. Platelets are implicated in regulating many of the pathogenic processes that occur during ARDS; however, the mechanisms remain elusive. The platelet receptor CLEC-2 has been shown to regulate vascular integrity at sites of acute inflammation. Therefore the purpose of this study was to establish the role of CLEC-2 and its ligand podoplanin in a mouse model of ARDS. Platelet-specific CLEC-2-deficient, as well as alveolar epithelial type I cell (AECI)-specific or hematopoietic-specific podoplanin deficient, mice were established using cre-loxP strategies. Combining these with intratracheal (IT) instillations of lipopolysaccharide (LPS), we demonstrate that arterial oxygen saturation decline in response to IT-LPS in platelet-specific CLEC-2-deficient mice is significantly augmented. An increase in bronchoalveolar lavage (BAL) neutrophils and protein was also observed 48 h post-IT-LPS, with significant increases in pro-inflammatory chemokines detected in BAL of platelet-specific CLEC-2-deficient animals. Deletion of podoplanin from hematopoietic cells but not AECIs also reduces lung function and increases pro-inflammatory chemokine expression following IT-LPS. Furthermore, we demonstrate that following IT-LPS, platelets are present in BAL in aggregates with neutrophils, which allows for CLEC-2 interaction with podoplanin expressed on BAL inflammatory alveolar macrophages. Taken together, these data suggest that the platelet CLEC-2-podoplanin signaling axis regulates the severity of lung inflammation in mice and is a possible novel target for therapeutic intervention in patients at risk of developing ARDS.

Platelet GPVI repairs its own damage.

In this issue of Blood, Gros et al report that glycoprotein VI (GPVI) promotes the proinflammatory role of platelets by increasing neutrophil secretion and toxicity while at the same time repairing the vascular damage inflicted by neutrophil activation, thereby maintaining vascular integrity. Significantly, this effect is independent of hemostasis.