InTraC — CRC 1784

Thromboinflammation represents a pivotal pathophysiological process after ischemic stroke, acting as a mechanistic bridge between local vascular injury and systemic immune dysregulation. Following arterial occlusion and reperfusion, the ischemic neurovascular unit releases a cascade of dan-ger signals that activate platelets, neutrophils, and endothelial cells, leading to the formation of plate-let–neutrophil aggregates, NET release, and contact pathway activation. These events amplify neuroinflammation and simultaneously propagate systemic immune activation, contributing to second-ary vascular comorbidities and increased risk of infection and recurrent stroke.

Despite strong experimental and clinical evidence for these associations, the spatial and temporal dynamics that connect intracranial thromboinflammation to systemic immune responses remain unresolved. It is unknown when and where local vascular inflammation transitions into systemic propagation, which cell types and mediators orchestrate this process, and whether these interactions constitute a continuous and therapeutically accessible cascade.

The central goal of this project is therefore to define the molecular and cellular circuits that drive alarmin-mediated thromboinflammation after stroke and to establish their translational relevance. We will specifically address three interlinked questions: (1) Which brain-derived alarmins initiate post-stroke thromboinflammation? (2) How do these signals propagate from the cerebral vasculature into systemic immune dysregulation? (3) Can therapeutic targeting of these pathways be validated in translational disease models and human stroke cohorts?

Thromboinflammation is a major driver of reperfusion injury and stroke progression in experimental models, but how these findings translate to patients with ischemic stroke remains elusive. Current diagnostics cannot resolve when and in whom thromboinflammatory cascades are active after reperfusion. Previous clinical studies mostly focused on single markers and were cross-sectional, limiting insights into timing and systems-level biology. In preliminary work, we performed proteomics on paired plasma-thrombus samples from acute stroke patients and identified a central role of the interplay of innate immunity with coagulation and platelet activation during thrombus formation: compared to other clinical traits, systemic inflammation (WBC/CRP) correlated with the largest set of thrombus proteins, which were enriched for innate immune pathways and platelet signalling. Using network analysis, we identified Inositol 1,4,5-Trisphosphate Receptor Type 1 (ITPR1) as a hub linking neutrophil and platelet degranulation. In recently published cross-ancestry stroke GWAS, we further identified druggable nodes (e.g., kallikrein B1 [KLKB1], fibrinogen alpha chain [FGA], coagulation factor XI [F11]) across thromboinflammatory pathways. These findings underscore the critical role of thromboinflammation in stroke pathogenesis and progression, which, however, remains clinically inaccessible. To address this challenge, the overarching goal of our project is to deliver clinically meaningful biomarker signatures that detect, monitor and stratify thromboinflammation in acute ischemic stroke. To this end, we will apply a deep, time-resolved molecular phenotyping approach that combines advanced omics technologies in patients with stroke with parallel mechanistic investigations in experimental stroke models to: i) discover multicellular thromboinflammatory signatures in patients with acute ischemic stroke and causally link these to endophenotypes in experimental models, ii) genetically prioritize and (reverse) translate biomarker candidates, and iii) independently validate prioritized biomarkers in two complementary cohorts, thereby paving the way for clinical trials targeting thromboinflammation in stroke and beyond.

The COVID-19 pandemic has taught us that the mechanisms of the close and clinically significant interplay between inflammation and thrombosis are still not fully understood. Both SARS-CoV-2 infection and vaccine-related complications such as vaccine-induced immune thrombotic thrombocytopenia (VITT) caused severe thrombotic events, often resistant to standard therapies. Bruton’s tyrosine kinase (Btk), selectively expressed by immune cells and platelets, has emerged as a central signalling molecule in these processes. As a key mediator in platelets, B cells and myeloid cells, Btk links innate and adaptive immunity to thrombosis, emphasizing the need to explore its potential as a critical therapeutic target. Our previous work showed that Btk is essential for platelet activation elicited by chemokine-IgG immune complexes, as in heparin-induced thrombocytopenia (HIT) and VITT through FcγRIIa signalling. Additionally, the chemokine CXCL12, released from activated platelets, amplifies arterial thrombosis through CXCR4-Btk signalling in an autocrine loop. We found that small molecule Btk inhibitors (BTKi) effectively block Btk-dependent platelet activation in human, pig, and mouse models, enabling translational research across species.

We propose that Btk regulates thromboinflammation and its resolution across multiple cell types so that understanding its cell-specific roles and blocking its activity selectively in platelets can provide new strategies to prevent and treat thrombotic complications. Aim 1 focuses on dissecting the cell-specific roles of Btk in thromboinflammation. We will use conditional knockout mouse models to analyse Btk function in platelets (Pf4cre), B cells (CD19cre), and myeloid cells (LyzMcre), along with global knockout approaches (tm1a). Through functional in vitro assays, including NETosis, platelet migration, and using in vivo models of thrombosis and thrombus resolution (arterial, venous), we will determine the relative contributions of Btk in different cell types. Complementary transcriptomic and proteomic analyses of platelets and megakaryocytes (MKs) will elucidate downstream pathways. Blood from patients with X-linked agammaglobulinemia (XLA), carrying Btk mutations, will be used to assess platelet responses, linking human data to murine models. Aim 2 will explore strategies to selectively target Btk in platelets to prevent arterial thrombosis and stent thrombosis in a pig model. We will use a chandler-loop system and a porcine stent implantation model to study BTKi effects in comparison and combination with dual antiplatelet therapy (DAPT). We aim to develop platelet-specific Btk inhibition (Btki) approaches by exploiting the limited protein synthesis capacity of platelets. To this end, we will evaluate different dosing regimens of BTKi and we plan to develop pharmacokinetic profiles. We will explore strategies to block Btk in a platelet-specific manner and will initiate this by investigating RNA-uptake and knockdown mechanisms starting with cultured MK and modulation of a reporter to pave the way for later in vivo RNA interference (RNAi). This will be initiated by exploring RNA delivery to MK in reporter mice through anti-CD41 antibodies conjugated to an RNAi. Aim 3 will investigate the role of Btk in chemokine-mediated signalling, focusing on CXCR4 activation by CXCL12 identifying common and distinct pathways in myeloid cells and platelets. We will use phosphoproteomic profiling to analyse and validate downstream intermediates and specifically test candidates such as Gβγ, Syk, Janus kinases, and p66Shc. We will assess the involvement of Btk and downstream signalling in leukocyte chemotaxis and in platelet locomotion using BTK-deficient mice and inhibitors, probing chemokine-driven thrombosis mechanisms. Given the role of platelet-neutrophil interactions and neutrophil extracellular trap (NET) formation in thromboinflammation, we will evaluate how platelet-derived chemokines (CXCL4, CXCL12, CCL5) and their receptors contribute to NETosis. Approaches such as platelet-specific Btk deletion, BTKi, chemokine heteromer disruption, will be tested for their capacity to interrupt pathological thromboinflammatory cascades.

Thromboembolism remains a leading cause of death worldwide. Atrial fibrillation (AF) markedly increases the risk of cardioembolic stroke and systemic embolism, while deep vein thrombosis (DVT) can progress to pulmonary embolism. Despite their distinct clinical manifestations, AF- and DVT-associated thrombosis share a central pathogenic trigger: local blood stasis. Blood stasis induces local and systemic thromboinflammatory shifts, including procoagulant and proinflammatory reprogramming of endothelial cells (ECs) and circulating immune cells, thereby promoting thrombus formation. However, the molecular and cellular mechanisms driving these stasis-induced EC phenotypes - particularly within the left atrial appendage (LAA), the predominant site of thrombus formation in AF, and in venous stasis - remain poorly understood. We hypothesize that blood stasis induces distinct EC-specific thromboinflammatory programs characterized by the expression and surface presentation of adhesive and procoagulant molecules that shape a local microenvironment promoting immune cell and platelet recruitment and activation. To address this hypothesis, we leverage our unique human LAA and venous tissue biobank obtained during cardiac and vascular surgeries, integrated with complementary mouse models of AF and DVT. Our preliminary single-nucleus RNA sequencing (snRNA-seq) analyses of human LAAs reveal distinct EC activation signatures in AF compared with sinus rhythm (SR), providing a strong foundation for systematic investigation. Building on these data, this project will (Aim 1) define EC-specific thromboinflammatory signatures associated with AF and venous blood stasis across human tissues and relevant animal model systems; (Aim 2) mechanistically dissect the functional relevance of prioritized endothelial pathways using integrated in vivo, in vitro, and ex vivo approaches; and (Aim 3) therapeutically evaluate key endothelial–immune interaction mechanisms as entry points for the selective modulation of thromboinflammation at sites of reduced blood flow in AF and DVT.

Endothelial cells (ECs) are central in the control of vascular function, inflammation and thrombosis. Previously, we established a role for angiogenesis and endothelial angiogenic signalling in venous thrombus resolution: We identified disease candidates and potential therapeutic targets in the transforming growth factor beta receptor families, that is TGFβ and bone morphogenetic protein (BMP). Our InTraC project now addresses the hypothesis that endothelial receptor signalling mediates thromboinflammation in an organ-specific manner and that lung-specific endothelial responses drive pulmonary thromboinflammation, remodelling after pulmonary embolism and pulmonary hypertension (PH). First, we aim to identify lung-specific endothelial RNA expression signatures in response to deep vein stasis, hypoxia, and angiogenesis inhibition, conditions that induce thromboinflammation and vascular remodelling in the lung. These pulmonary expression profiles will be compared to RNA profiles from systemic ECs to identify lung-specific endothelial responses that drive pulmonary thromboinflammation and remodelling after pulmonary embolism. New targets will be validated in biomaterial (primary ECs, precision cut lung slices, plasma) of patients with PH, with and without thrombosis, as well as in rodent and pig disease models. Second, we will dissect lung endothelial-specific roles of TGFβ and BMP receptors (TGFRB2, BMPR2) during pulmonary thromboinflammation and adverse remodelling.

Thromboinflammation can cause obstruction of both the macro- and microvasculature. While macrovascular thrombosis is amenable to interventional or surgical treatment, microvascular thrombosis often leads to organ failure without effective therapeutic options. A prominent example is myocardial no-reflow, where thrombotic occlusion of the microvasculature persists despite successful reopening of the epicardial vessel after myocardial infarction. Monocytes have emerged as central cellular drivers of thromboinflammation in both microvascular and macrovascular thrombosis. In the myocardial circulation, monocytes and macrophages can promote detrimental microvascular occlusion and worsen outcomes after reperfusion. These observations establish monocytes as key mediators of thrombotic injury.

Beyond this pathogenic role, our recent work uncovered a previously unrecognized functional heterogeneity of monocyte subsets within human thrombi. By constructing a leukocyte atlas of human and murine thrombosis, we identified non-classical, NR4A1high monocytes with pronounced fibrinolytic activity, coexisting with prothrombotic monocyte populations. Reverse translation to mouse models demonstrated that these non-classical monocytes actively remodel the thrombus microenvironment through direct thrombolysis and recruitment of fibrinolytic neutrophils. These findings highlight the dual - protective and harmful - potential of distinct monocyte subsets in microvascular and macrovascular thrombotic disease. However, the mechanisms governing their functional polarization during thrombus resolution remain poorly understood.

We hypothesize that local non-classical monocytes act as central modulators of the micro- and macrovascular thrombotic milieu and actively orchestrate the transition from thromboinflammation to thrombus resolution. We further propose that known inflammatory pathways, highly expressed in thrombus-resolving monocytes, can influence monocyte function beyond their canonical inflammatory roles and determine whether monocytes skew toward prothrombotic or resolving roles. Targeting this balance represents a novel therapeutic strategy to control thromboinflammation and promote immunothrombolysis. To test this hypothesis we will employ a macrovascular model of thrombosis (flow-reduction induced deep vein thrombosis by inferior vena cava stenosis) and a complementary microvascular model of thrombotic occlusion, mimicking clinical no-reflow (induced by myocardial ischemia and reperfusion (MIRI) and investigate the following aims: Aim 1: Characterize monocyte recruitment, expansion, and differentiation cues after micro- or macrovascular thrombotic disease induction. Aim 2: Define the functional consequences of highly expressed thrombus monocyte inflammatory pathways for clot formation and resolution in vitro. Aim 3: Determine the in vivo relevance of highly expressed inflammatory monocyte signalling pathways (Myd88 and TREM1) for macro- and microvascular clot formation and resolution.

Microvascular thrombosis is a hallmark of advanced inflammatory disease and frequently prevents full recovery after thromboinflammatory events. However, safe and effective treatment options for the dissolution of blood clots in the microvasculature are still missing. This is exemplified by fatal disease courses in COVID-19 with this condition, despite conventional coagulation- and platelet-targeting therapies. In addition to platelets and plasmatic coagulation, immune cells substantially contribute to the formation of microvascular thrombi. Own preliminary data show that immune cells actively infiltrate existing microvascular clots to promote thrombus stabilization and expansion. In the proposed project, we aim at establishing novel immune cell-based concepts for microvascular thrombolysis. Here, we will first decipher the mechanisms underlying immune cell-driven persistence and progression of microvascular clots. These studies will generate specific molecular targets, which will enable us to engineer immune cells endowed with i) distinct thrombolytic properties and ii) a molecular repertoire guiding these ‘cellular drugs’ selectively into blood clots. These armed immune cells will serve as novel therapeutics for the targeted dissolution of microvascular thrombi in advanced inflammatory disorders that specifically address thromboinflammation without exhibiting systemic adverse effects such as life-threatening bleeding complications. The ultimate vision behind this concept is an off-the-shelf approach, in which such strategy would be readily available and applicable to the patients in need.

The role of platelet-neutrophil interplay in pancreatitis as mediator of inflammation and splanchnic and portal vein thrombosis, though critically involved in disease progression and severity, remains largely unexplored. This project aims to define the thromboinflammatory mechanisms involved in pancreatitis and, specifically, pancreatitis-associated splanchnic and portal vein thrombosis formation in mice and patients. We hypothesize that the delicate balance between thrombus formation and prevention of bleeding in pancreatitis is shaped by two interlinked mechanisms: (i) the excessive extracellular activation of serine proteases activating the coagulation cascade, and (ii), reciprocal activation of platelets and neutrophils during an overwhelming systemic inflammatory response. Both mechanisms, though obviously crucial and amenable to treatment, have not been studied in pancreatitis and associated splanchnic or portal vein thrombosis.

Our focus will be on the role of platelets and platelet-neutrophil interplay as critical contributors to pancreatitis-associated thromboinflammation. We will perform functional phenotyping of platelet and neutrophil activation in mice and patients with pancreatitis and correlate our findings with the incidence of pancreatitis-associated thromboinflammation. Specifically, using ex vivo tissue analysis and contrast-enhanced CT angiography, we will comprehensively assess local and systemic thrombotic complications during pancreatitis and correlate their occurrence with platelet and immune phenotypes derived from multiOmics profiling. Further, with respect to our preliminary experiments, we will investigate the role of acid sphingomyelinase (ASM/SMPD1) in activation of platelets and neutrophils and concurrent disease progression of pancreatitis as amenable treatment target. Based on these findings, we will use targeted nanoparticles for cell-specific delivery of SMPD1 inhibitors in a novel therapeutic, anti-inflammatory approach for pancreatitis.

Activation of endothelial cells (ECs) is a key initiator of immunothrombosis and thromboinflammation. It leads to platelet and leukocyte recruitment, which are key cellular factors in the interplay of thrombosis and inflammation and drive deep venous thrombosis (DVT). In parallel, activation alters the effect of ECs on the coagulation system, which further exacerbates thrombotic states. Thus, selective targeting of activated ECs represents an attractive strategy for therapeutic and preventive measures in thromboinflammatory diseases. However, the upstream factors activating ECs and their impact on the transition from immunothrombosis to thromboinflammation are poorly understood. In preliminary work, we identified a central role for IgM-EC interaction in initiating DVT under conditions of reduced blood flow. This relies on the binding of IgM to the endothelial IgM receptors FcµR and polymeric Immunoglobulin receptor (pIgR). As a consequence, ECs are activated and mobilise P-selectin and von Willebrand Factor (vWF) to their surface. This initiates leukocyte and platelet recruitment to the vessel wall. Combined inhibition of pIgR and FcµR completely prevents experimental DVT in a mouse model. Despite these findings, the mechanisms of IgM mediated endothelial activation are largely unknown and will be dissected in this proposal. In addition, we will evaluate whether disruption of IgM-endothelium interaction could be exploited for pharmacologic intervention in thromboinflammation. We will employ a targeted drug delivery approach aiming to specifically deliver siRNA to activated ECs for gene silencing of endothelial IgM receptors. This is based on our expertise in the generation of polybetaaminoester nanoparticles (PBAE-NC), which are targeted through ligand peptides to specific receptors. Moreover, they can be loaded with siRNA for gene silencing in their target cell. Based on this, we will develop and optimize EC-targeted siRNA PBAE-NC in vitro (aim 1). In aim 2, we will perform comprehensive pharmacodynamic and -kinetic profiling to investigate how siRNA-loaded nanoparticles can be targeted to inflamed ECs in vivo to downregulate pIgR and FcµR by post-transcriptional gene silencing. In a translational approach, we will determine how gene silencing can be exploited for the inhibition of experimental DVT. In aim 3, we will dissect the signalling of IgM receptors resulting in endothelial activation to identify additional targets for siRNA development.