Endotheliopathy in CAR T-Cell Therapy: Mechanistic Insights into the VWF/ADAMTS13 Axis and the Angiopoietin-Tie2 Pathway.

Introduction

Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of advanced hematological cancers, offering hope to patients with relapsed or refractory diseases. 1 2 By genetically engineering autologous T lymphocytes to target specific tumor-associated antigens—such as CD19 or B cell maturation antigen (BCMA)—this adoptive immunotherapy redirects cytotoxicity toward malignant cells to induce deep, durable clinical responses. 3 4 Despite its remarkable efficacy, CAR T cell therapy is associated with potentially life-threatening immune-mediated toxicities. The most prominent of these are cytokine-release syndrome (CRS), immune effector cell–associated neurotoxicity syndrome (ICANS), and secondary CAR T cell therapy-related coagulopathy (CARAC), which together represent major contributors to treatment-related morbidity and, in severe cases, mortality. 5 6 7 8 9 10 While tumor-debulking bridging therapies and early supportive care have mitigated some risks, nonrelapse mortality remains significant and varies considerably across different CAR constructs. 11 12

Elucidating how immune activation drives organ injury is a clinical priority, with growing evidence identifying the vascular endothelium as the critical interface between systemic inflammation and microvascular dysfunction during CAR T cell therapy. 13 14 15 16 17 18 19 Far from being a passive barrier, endothelial cells actively coordinate inflammatory and hemostatic responses. Systemic cytokine signaling triggers endothelial activation, accelerating Weibel–Palade body (WPB) exocytosis, the upregulation of adhesion molecules, complement engagement, and neutrophil extracellular trap (NET) formation. Combined, these responses give rise to immunothrombosis, an intravascular defense mechanism that spatially confines inflammatory stimuli within the microvasculature. When inflammatory signaling is sustained or amplified, however, this protective response may evolve into pathological endotheliopathy, marked by barrier disruption, increased vascular permeability, and a prothrombotic endothelial phenotype. 20 21 22 23 24

While these processes are well established in inflammatory conditions such as sepsis, COVID-19, and posttransplant vascular syndromes, their precise contribution to CAR T cell–associated toxicities is only beginning to be uncovered. Within this context, endothelial-derived biomarkers have emerged as promising tools for monitoring and potentially predicting treatment-related complications. Research highlights two interconnected regulatory systems central to this vascular toxicity. The first is the von Willebrand factor (VWF)/ADAMTS13 axis, which regulates platelet adhesion and microvascular thrombosis. The second is the angiopoietin (Ang)–tyrosine kinase receptor Tie2 signaling pathway, which governs endothelial stability and vascular permeability. Dysregulation of these pathways may represent the molecular bridge linking cytokine excess to the vascular manifestations of CRS, ICANS, and CARAC. 16 17 18 19 25 26 27 28 Although these candidate biomarkers remain investigational and lack standardized thresholds, they offer important mechanistic insight and may ultimately support risk-adapted interventions.

This review bridges the gap between separate vascular and immuno-hemostatic models by examining how the VWF/ADAMTS13 axis and the Ang–Tie2 pathway collectively drive CRS, ICANS, and CARAC. By consolidating the biological basis of this shared endotheliopathy, this text outlines biomarker-guided risk stratification and introduces emerging endothelial-targeted strategies to improve clinical outcomes.

Endothelial Activation and Immunothrombosis as Drivers of CAR T Cell Toxicity

Physiological Endothelial Regulation and Immunothrombosis

The innate immune system and hemostasis are tightly interconnected biological networks. Beyond preventing blood loss, the coagulation cascade functions as an effector arm of innate immunity during infection and tissue injury. 20 21 22 23 24 Under physiological conditions, the vascular endothelium maintains this delicate balance through the continuous release of anticoagulant and anti-inflammatory mediators, including nitric oxide (NO), prostacyclin (PGI 2 ), thrombomodulin (TM), tissue-type plasminogen activator, and tissue factor pathway inhibitor (TFPI). 22 29 NO downregulates endothelial adhesion molecules involved in leukocyte rolling and firm adhesion, including P-selectin, E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1), thereby limiting leukocyte recruitment and platelet adhesion, while PGI 2 restrains platelet activation and leukocyte extravasation. Collectively, these mechanisms preserve endothelial quiescence and protect the vasculature from inappropriate thrombus formation. 22 30 31 32

During inflammatory stress, this equilibrium shifts toward a pro-inflammatory and prothrombotic phenotype. Cytokines including interleukin (IL)-1, IL-6, and tumor necrosis factor-α (TNF-α) activate endothelial cells through mitogen-activated protein kinase/nuclear factor kappa-light-chain enhancer of activated B cell (MAPK/NFκB) signaling pathway, a state often referred to as type II endothelial activation. 22 33 One of the earliest responses is the exocytosis of WPBs, which releases ultra-large von Willebrand factor (ULVWF) multimers and P-selectin into the circulation. 29 30 34 At the same time, endothelial cells upregulate tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1), and adhesion molecules such as ICAM-1 and VCAM-1, promoting leukocyte recruitment and local coagulation activation. Disruption of endothelial junctions, reflected by increased platelet endothelial cell adhesion molecule-1 (PECAM-1), further facilitates leukocyte and fluid extravasation. 22 30 33 34 35

Activated neutrophils amplify endothelial activation through formation of NETs, web-like chromatin structures decorated with histones and proteolytic enzymes. NETs provide a scaffold that binds ULVWF multimers and recruits platelets, while also activating factor XII and inhibiting endogenous anticoagulants such as TFPI. In addition, histones released within NETs can directly injure endothelial cells, which further drives thrombo-inflammatory signaling within the microvasculature. 21 23 33 36

The coordinated interaction between endothelial activation, ULVWF multimers release, platelet recruitment, and NET formation results in immunothrombosis, a physiological mechanism that confines inflammatory stimuli within the microvasculature. 23 36 When appropriately regulated, immunothrombosis is protective. However, if inflammatory stimuli persist or intensify, these same pathways lose their spatial restriction and evolve into systemic thrombo-inflammation, a maladaptive state marked by reduced TM, TFPI, and ADAMTS13 activity, heightened TF expression, and progressive endotheliopathy. 22 24 37 38 39 40 41 These vascular alterations therefore represent a key step in the transition from defensive immunothrombosis to pathological thrombo-inflammation ( Fig. 1 ).

VWF/ADAMTS13 and Angiopoietins in Endothelial Dysregulation

VWF is a large multimeric glycoprotein positioned at the interface of hemostasis, inflammation, and vascular integrity. Synthesized by endothelial cells and megakaryocytes and stored in WPBs and platelet α-granules, VWF mediates platelet adhesion to exposed subendothelium under high shear stress and stabilizes circulating factor VIII (FVIII). 42 43 The evolutionary importance of VWF is highlighted by the fact that its quantitative or qualitative defects cause von Willebrand disease, the most common inherited bleeding disorder. 44 Beyond coagulation, however, accumulating evidence demonstrates that VWF regulates leukocyte recruitment, vascular permeability, complement activation, and angiogenesis, identifying it as a multifunctional guardian of vascular homeostasis. 45

The prothrombotic potential of VWF scales with its multimeric length and is strictly regulated by ADAMTS13; this metalloproteinase cleaves highly adhesive ULVWF multimers into smaller, less reactive forms, effectively limiting inappropriate platelet tethering. 46 47 In addition to its proteolytic role, ADAMTS13 exerts anti-inflammatory effects by limiting leukocyte–platelet interactions and dampening cytokine amplification, serving as a vital counter-regulator of endothelial stress. 48 49 50

Inflammatory cytokines profoundly disturb this regulatory balance. IL-6, IL-8, and TNF-α promote WPB exocytosis while simultaneously downregulating ADAMTS13 synthesis and activity, which favors ULVWF persistence. 34 49 50 The resulting imbalance is classically exemplified by thrombotic thrombocytopenic purpura (TTP), 51 and is increasingly recognized across hyperinflammatory states such as sepsis 52 53 and COVID-19. 40 41 54 In these conditions, alterations in the VWF/ADAMTS13 ratio correlate with disease severity and organ dysfunction, establishing this axis as both a mechanistic driver and a biomarker of endothelial stress. Similarly, this thrombo-inflammatory cascade is highly active in allogeneic hematopoietic stem cell transplantation (allo-HSCT) settings, where cytoreductive conditioning uniformly triggers acute endothelial activation, and baseline elevations in functional VWF parameters serve as independent indicators of preexisting endothelial vulnerability to subsequent vascular complications. 55

NETs further potentiate endothelial injury by providing a scaffold for ULVWF multimers and reducing ADAMTS13 activity, thereby stabilizing platelet–VWF aggregates and protecting them from proteolytic cleavage. These effects reinforce platelet recruitment and sustain microvascular thrombosis, creating a self-amplifying NET–VWF–ADAMTS13 circuit that propagates thrombo-inflammatory signaling. 56 57 This integrated pathway has been implicated in complement- and inflammation-driven thrombotic microangiopathies. 58 59

A second key regulatory pathway controlling endothelial stability is the Ang–Tie2 axis. Angs are endothelial growth factor family members that bind the Tie2 receptor, a tyrosine kinase expressed on endothelial cells, and regulate vascular quiescence and barrier integrity. 60 61 Among them, Ang-1 and Ang-2 are the best characterized and exert largely opposing effects on endothelial homeostasis. Under physiological conditions, Ang-1 is constitutively produced by pericytes and vascular smooth muscle cells. Because circulating Ang-1 levels normally exceed those of Ang-2, Tie2 signaling is predominantly driven by Ang-1. Activation of Tie2 stimulates the phosphoinositide-3-kinase–protein kinase B (PI3K–AKT) pathway, which promotes endothelial survival, suppresses adhesion molecule expression such as VCAM-1 and E-selectin, reduces vascular permeability, and strengthens intercellular junctions. 61 62 63 The indispensable nature of Ang-1–Tie2 signaling is illustrated by knockout models, in which loss of either component results in impaired vasculogenesis and embryonic lethality. 63 64 65

In contrast, Ang-2 acts as a context-dependent antagonist or partial agonist of Tie2. Synthesized by endothelial cells and stored in WPBs, Ang-2 is rapidly released during inflammatory stimulation. Excess circulating Ang-2 competitively displaces Ang-1 from the Tie2 receptor, inducing rapid endothelial destabilization and a procoagulant, pro-inflammatory state. 60 61 62 Accordingly, an elevated Ang-2/Ang-1 ratio serves as a reliable clinical hallmark of endotheliopathy in inflammatory conditions such as sepsis and acute respiratory distress syndrome. 61 66 67 Although VWF and Ang-2 are co-stored in WPBs, their release is regulated by distinct signaling pathways, and circulating levels do not necessarily correlate. 60 68 A schematic representation of the Ang–Tie2 ligand–receptor pathway and its opposing regulatory effects on endothelial stability is provided in Fig. 2 .

Despite being described as separate systems, the VWF/ADAMTS13 axis and the Ang-Tie2 pathway are biologically interconnected. VWF is essential for the formation of WPBs, the endothelial storage organelles that also contain Ang-2, linking VWF expression directly to endothelial secretory capacity. 69 70 Experimental data further suggest that VWF deficiency can enhance Ang-2 expression through a Tie2–AKT–FOXO1–dependent feedback mechanism, directly coupling primary hemostatic regulation to angiogenic and permeability signaling. 71

Together, these two interlocking pathways orchestrate the shift from vascular quiescence to inflammatory thrombosis. Early disturbances in either system during CAR T cell therapy likely reflect cytokine-induced endothelial stress and may serve as biomarkers of emerging toxicity.

Endothelial Involvement in CRS and ICANS

Cytokine-Driven Endothelial Activation During CAR T Cell Therapy

CRS arises from robust CAR T cell activation upon tumor-associated antigen recognition. Engaged CAR T-cells secrete perforin, granzymes, and high concentrations of pro-inflammatory cytokines, including interferon-γ (IFN-γ) and TNF-α, thereby inducing pyroptotic tumor cell death. 12 72 This inflammatory cell death releases damage-associated molecular patterns (DAMPs) that stimulate macrophages and dendritic cells via pattern-recognition receptors. This myeloid activation amplifies the cytokine cascade, most prominently IL-6 and IL-1β. In parallel, macrophage inflammasome activation promotes caspase-1 activation and the maturation of IL-1β, sustaining systemic inflammation. 73 74 75 76 77

As cytokine concentrations rise, endothelial activation rapidly intensifies. MAPK and NF-κB signaling accelerates WPB secretion, resulting in brisk release of VWF, Ang-2, and P-selectin. Inflammatory signaling simultaneously upregulates endothelial adhesion molecules and destabilizes intercellular junctions. These changes shift the endothelium from a regulated inflammatory responder to a driver of vascular permeability, leukocyte recruitment, capillary leak, and immunothrombosis. This escalating stress response forms the mechanistic link between systemic cytokine excess and the clinical phenotype of CRS, including secondary coagulopathy. 34 78 79 Among the cytokines implicated, IL-6 plays a particularly prominent role, explaining the clinical efficacy of IL-6 receptor blockade with tocilizumab as a cornerstone of CRS management. 6 78 80

Clinically, CRS is the most frequent early toxicity following CAR T cell infusion, typically emerging within the first 2 weeks. Its presentation ranges from isolated fever to severe systemic inflammation characterized by hypotension, vascular leak, and multiorgan dysfunction. Although CRS is generally reversible, severe cases frequently require intensive care support. 81 82 This timing and potential severity underscore the need to identify early indicators of endothelial stress prior to overt clinical deterioration. 1 6 72 The sequence of cytokine-driven endothelial activation and its downstream vascular consequences is schematically illustrated in Figs. 3 and 4 .

Even though ICANS is regarded as a distinct clinical entity, it frequently co-occurs with CRS, sharing a common pathophysiological basis in therapy-related endotheliopathy that extends beyond the systemic circulation to specialized vascular beds. Within the neurovascular unit, damaged endothelial cells and pericytes compromise blood–brain barrier (BBB) integrity, permitting circulating cytokines and immune cells to infiltrate the central nervous system (CNS). 72 73 83 Myeloid-derived cytokines (such as IL-1 and IL-6) drive standard endothelial activation and decrease tight junction expression, but recent single-cell sequencing has additionally unveiled a critical “on-target, off-tumor” mechanism targeting pericytes. 84 85 Human brain pericytes, which position themselves at endothelial tight junctions to maintain neurovascular integrity, express CD19 and its obligatory chaperone CD81 throughout adulthood. Consequently, CD19-directed CAR T-cells directly target and deplete these cells, provoking the local release of IL-6 and vascular endothelial growth factor (VEGF). These mediators activate adjacent endothelial cells, amplifying neurovascular injury via a positive feedback loop. 84 86 Although the relative contribution of direct pericyte targeting versus systemic cytokine-mediated injury remains debated, this off-target vulnerability helps explain why ICANS occurs with a higher frequency and severity in CD19-targeted therapies compared with constructs targeting alternative antigens (e.g., CD22 or BCMA).

In addition to direct pericyte injury, inflammatory mediators further compromise BBB integrity. Cytokines released during CAR T cell activation, most prominently IFN-γ from CAR T-cells and colony-stimulating factors from monocytes and macrophages, induce endothelial adhesion molecules such as ICAM-1 and VCAM-1, thereby facilitating leukocyte adhesion and transmigration across the compromised barrier. 81 86 Within the CNS, infiltrating cytokines activate microglia and astrocytes, amplifying local inflammatory circuits and contributing to the characteristic neurological manifestations of ICANS. 77 87 Clinical correlates of this neurovascular breakdown include elevated cerebrospinal fluid (CSF) protein, increased CSF leukocyte counts, and in some cases detectable CAR T-cells, although the precise contribution of intracerebral CAR T cell infiltration to neurotoxicity remains uncertain. 6 18 25 73

This systemic-to-central trafficking is further influenced by therapeutic interventions. Interestingly, while IL-6 receptor blockade with tocilizumab effectively controls systemic CRS symptoms, it prevents receptor binding and clearance, leading to a temporary surge in circulating peripheral IL-6 levels. 18 88 89 Whether this sudden spike crosses a compromised BBB and contributes to subsequent neuroinflammation or delayed ICANS remains a subject of ongoing clinical debate. This limitation underscores preclinical observations highlighting the potential primacy of myeloid-derived IL-1 in ICANS pathogenesis. Indeed, upstream receptor blockade with anakinra, unlike tocilizumab, has demonstrated protection against delayed lethal neurotoxicity and BBB disruption in preclinical models. 89 90

ICANS typically emerges within 4 to 10 days after CAR T cell infusion, usually in temporal association with CRS. Higher CRS grades strongly correlate with the risk of severe neurotoxicity following CD19-directed CAR T cell therapy. 5 6 Clinically, ICANS spans a broad spectrum from mild confusion and expressive aphasia to seizures and, in rare instances, cerebral edema. 9 18 25 74 76 77

Endothelial Biomarkers and Clinical Manifestations

Composite Indices

Given the pivotal role of endothelial injury in the pathogenesis of CRS and ICANS, considerable effort has been directed toward identifying biomarkers that reflect early endothelial stress. Conventional laboratory parameters such as elevated C-reactive protein (CRP), ferritin, D-dimer levels, and thrombocytopenia reflect systemic inflammation and microvascular activation but lack specificity for endotheliopathy. 88 91 To address this limitation, composite indices have been developed.

The Endothelial Activation and Stress Index (EASIX), calculated as (LDH × creatinine)/platelet count, was originally validated in allo-HSCT to predict a continuum of hyperinflammatory endothelial complications, including sinusoidal obstruction syndrome, transplant-associated thrombotic microangiopathy (TMA), engraftment syndrome, and acute graft-versus-host disease. 92 93 94 95 96 97 Recent European Society for Blood and Marrow Transplantation consensus guidelines emphasize that this spectrum of vascular toxicities and coagulation disorders extends directly to advanced cellular therapies. 98 Elevated preinfusion EASIX scores have since been associated with the development and severity of CAR T cell–related toxicities. 27 28 99 Several adaptations aim to improve clinical applicability. The modified EASIX (mEASIX), which replaces creatinine with CRP, has demonstrated strong associations with severe CRS, ICANS, and consumptive CARAC. 26 27 100 101 102 A simplified version (sEASIX), excluding creatinine entirely, has likewise shown predictive potential, although available data remain limited to specific cohorts. 26 99

Circulating Endothelial Biomarkers

Specific endothelial biomarkers provide additional mechanistic insight. Retrospective and prospective data demonstrate that endothelial vulnerability often precedes CAR T cell infusion. Compared with healthy controls, patients showed reduced baseline ADAMTS13 activity and elevated VWF antigen, suggesting preexisting endothelial activation or exhaustion characterized by the depletion of intracellular storage pools. 16 87 These baseline abnormalities persisted postinfusion and correlated with toxicity. Patients who later developed CRS grade ≥ 2 exhibited lower baseline ADAMTS13 activity. Similar patterns were observed in ICANS, with reduced ADAMTS13 activity and higher VWF levels compared with patients without neurotoxicity. Notably, higher baseline ADAMTS13 activity was associated with better treatment response, suggesting that the VWF/ADAMTS13 balance may reflect broader endothelial fitness relevant to therapeutic efficacy. 87 Thus, dysregulation of this axis may precede the cytokine surge as an intrinsic vulnerability rather than a secondary inflammatory consequence.

Imbalance within the VWF/ADAMTS13 axis becomes increasingly pronounced during CRS and ICANS. In advanced ICANS, serum VWF levels may rise four- to fivefold above normal, accompanied by elevated IL-8 levels consistent with WPB release. 16 18 Functional assays demonstrate that serum obtained 3–5 days after infusion from patients with early neurotoxicity induces abundant VWF–platelet string formation on cultured endothelial cells. Conversely, in advanced ICANS, string formation paradoxically decreases despite elevated circulating VWF concentrations. This phase coincides with the depletion of high-molecular-weight VWF multimers and a significantly reduced ADAMTS13/VWF ratio (26.4% vs. 35.2%; p = 0.007), suggesting impaired proteolytic regulation of VWF at peak toxicity. 18

Alterations in the Ang pathway further track endothelial destabilization across the treatment timeline. Compared with healthy controls, CAR T cell recipients exhibit elevated Ang-2 levels at baseline, which rise further postinfusion. Importantly, increases in Ang-2, the Ang-2/Ang-1 ratio, and soluble endothelial adhesion molecules (sE-selectin, sVCAM-1, sICAM-1) are detectable days before clinical CRS onset, differentiating mild from severe CRS within the first 72 h. 16 17 18 Predictive models incorporating sVCAM-1 and the Ang-2/Ang-1 ratio show high specificity for identifying patients at risk of severe toxicity, 16 17 while early Ang-2 elevation similarly associates with ICANS severity. 18 At peak toxicity, severe CRS and ICANS are consistently characterized by marked increases in Ang-2 and soluble adhesion molecules, alongside a decline in Ang-1 concentrations. This coordinated shift toward an elevated Ang-2/Ang-1 ratio drives a pro-thrombotic endothelial phenotype across various CAR constructs—including CD19- and CD28-based products—with the reduction in Ang-1 likely exacerbated by concurrent thrombocytopenia. 17 19 25 100 These trajectories are summarized in Table 1 .

Beyond diagnostic correlation, early Ang kinetics offer significant risk-stratification potential. Ang-2 elevations within the first 24 hours postinfusion forecast the subsequent need for intensive care support. A multivariable model integrating Ang-2, soluble ST2, and NET markers showed good predictive accuracy (AUC: 0.80), while a broader biomarker panel including Ang-2, NETs, sC5b-9, VWF, and PAI-1 reliably distinguished severe CAR-related toxicity from sepsis. 16 Elevated endothelial activation markers have similarly been associated with early renal and hepatic dysfunction. 17 Nonetheless, biomarker patterns across studies are not uniformly consistent; a prospective pediatric and young adults cohort found no significant differences in Ang kinetics in relation to ICANS, potentially reflecting age-related biological variation or confounding by concurrent CRS. 103

From a clinical perspective, the utility of endothelial biomarkers appears to depend strongly on timing. Baseline abnormalities may identify patients with preexisting endothelial vulnerability, whereas early postinfusion changes may help recognize individuals at increased risk of severe toxicity. In contrast, peak biomarker elevations largely reflect the severity of established CRS or ICANS and therefore have greater prognostic than predictive value. At present, endothelial biomarkers should be regarded as complementary to clinical risk assessment and composite indices such as EASIX rather than as standalone decision-making tools. Key clinical studies evaluating VWF, ADAMTS13, and Angs in CAR T cell-associated toxicities are summarized in Table 2 .

Sources of Heterogeneity and Methodological Limitations

When interpreting these biomarker trajectories, several sources of clinical and biological heterogeneity should be considered. First, costimulatory domains influence kinetics: CD28-costimulated constructs (e.g., axicabtagene ciloleucel) frequently exhibit more rapid in vivo expansion and earlier cytokine peaks than 4–1BB-based products (e.g., tisagenlecleucel), driving earlier CRS onset and higher rates of neurotoxicity. 104 105 106 Second, discordant findings between adult and pediatric cohorts suggest that age-related differences and baseline vascular fitness may influence endothelial responses. Adult patients often present with extensive prior treatment exposure, vascular comorbidities, and preexisting neurovascular risk factors, whereas pediatric patients may possess a more resilient endothelial baseline. Consistent with this concept, variations in endothelial biomarkers have been detected before lymphodepletion and at the time of infusion. 18 81 Finally, the contribution of lymphodepleting chemotherapy should be considered. Fludarabine- and cyclophosphamide-based conditioning may itself induce endothelial injury and perturbations in the VWF/ADAMTS13 axis prior to CAR T cell expansion. Consequently, early biomarker abnormalities may partly reflect chemotherapy-induced vascular stress rather than exclusively CAR T cell-mediated toxicity. 12 13 18 81

Compounding these biological differences, current evidence remains limited by relatively small cohorts and substantial methodological variability. Assays for VWF and Angs lack cross-platform standardization. Studies variably report VWF antigen levels without assessing functional activity or multimer distribution, potentially obscuring clinically relevant qualitative differences. Similarly, Ang-1 measurements are highly sensitive to preanalytical conditions, particularly platelet activation during sample collection. Importantly, clinically validated thresholds remain unavailable, precluding routine biomarker-guided decision-making. Consequently, these markers should currently be regarded as candidate biomarkers and pathophysiological correlates rather than established clinical tools.

In conclusion, current evidence supports a model in which endothelial injury acts as a potential bridge linking CAR T cell–mediated immune hyperactivation to vascular leak, dysregulated hemostasis, and neurovascular injury. Despite biological and methodological heterogeneity across studies, observed perturbations in the VWF/ADAMTS13 axis, the Ang-2/Ang-1 balance, and endothelial adhesion molecules frequently correlate with toxicity severity, lending support to the concept of CRS and ICANS as therapy-related endotheliopathies.

CAR T Cell Therapy-related Coagulopathy: A Consequence of Endotheliopathy

Coagulation abnormalities following CAR T cell infusion occur in the context of systemic inflammation and are best interpreted as the hematologic expression of cytokine-driven vascular injury rather than a primary coagulation disorder. Laboratory-defined coagulopathy has been reported in up to 60% of treated patients, with the highest incidence observed in multiple myeloma cohorts receiving BCMA-directed products. Risk factors include high tumor burden, extensive prior therapy, low baseline platelet counts, and, most prominently, the presence and severity of CRS. 107 108 109 110 111

These abnormalities evolve in parallel with the inflammatory cascade characteristic of CRS. Rising IL-6, CRP, and IFN-γ levels coincide with increasing D-dimer concentrations, prolongation of PT and aPTT, and enhanced thrombin generation. Platelet counts and fibrinogen levels decline proportionally to CRS severity, with hypofibrinogenemia becoming particularly pronounced in severe CRS or immune effector cell–associated hemophagocytic syndrome (IEC-HS), where it may serve as a diagnostic criterion. 88 107 108 109 110 112 113 114 Consistent with this inflammatory–hemostatic interplay, elevated mEASIX scores during CRS grade ≥ 2 correlate with increased VWF antigen, FVIII, and D-dimer levels, together with PT/aPTT prolongation, underscoring the convergence of vascular injury and hemostatic stress. 100

Clinically, CARAC typically begins around day 4 postinfusion, peaks between days 8 and 9, and gradually resolves as cytokine levels normalize. The steep fall in fibrinogen during peak CRS, occasionally accentuated after IL-6 receptor blockade with tocilizumab, illustrates the tight coupling between inflammatory signaling and hepatic acute-phase regulation. 25 107 108 109 110 112 115 116

Mechanistically, inflammatory cytokines promote a procoagulant state characterized by enhanced thrombin generation, impaired fibrinolysis, and consumption of platelets and clotting factors. Suppression of endogenous anticoagulant pathways (antithrombin, protein C, protein S), together with elevated PAI-1, further destabilizes hemostatic balance. The resulting phenotype resembles a consumptive coagulopathy with TMA–like features, typified by thrombocytopenia, clotting factor depletion, fibrinogen consumption, and secondary fibrinolysis. 12 107 108 109 110 112 113 115 116 117 118 119 120 Supporting the link between endothelial injury and hemostatic derangements, patients who developed CARAC in a phase I anti-CD22 CAR T cell study exhibited significantly higher Ang-2 levels and Ang-2/Ang-1 ratios. 114 The principal laboratory abnormalities observed during CARAC are summarized in Table 3 .

In severe cases, the laboratory profile may approach criteria for disseminated intravascular coagulation (DIC), although fulminant DIC remains uncommon. Parallels with macrophage activation syndrome and hemophagocytic lymphohistiocytosis suggest that hypofibrinogenemia during CRS may partly reflect primary hyperfibrinolysis, potentially driven by macrophage-derived plasminogen and cytokines such as IL-1β and TNF-α. 111 112 These insights position CARAC within a broader continuum of inflammation-mediated thrombo-inflammatory syndromes rather than as a discrete coagulation disorder.

Despite frequent laboratory abnormalities, overt thrombotic and bleeding events affect a minority of patients but carry significant clinical weight. A comprehensive narrative review highlighted that while VTE rates remain low in highly selected trial populations (0.48–3.26%), real-world observational cohorts report substantially higher VTE rates ranging from 2.1% to 10.8%. This discrepancy is mirrored in bleeding complications, which range from 0% to 1.85% in clinical trials up to 2.8% to 12.5% in real-world registries, with gastrointestinal and mucosal sites predominating. 111 113 117 121 122 123 Further quantifying this risk, a large-scale meta-analysis of 24 studies encompassing nearly 3000 patients established a pooled VTE proportion of 6.16% (adjusting up to 7.82% after accounting for publication bias), with most events clustering within the first 50 days postinfusion. Crucially, this analysis confirmed that severe immune toxicities dramatically compound this risk, with grade >2 CRS (RR = 4.12) and ICANS (RR = 3.43) serving as powerful independent predictors of VTE. 124

These systematic findings align closely with a recent multicenter Canadian cohort of 561 CD19 CAR T recipients that reported a 90-day cumulative incidence of 6.7% for VTE and 6.5% for clinically relevant bleeding. While no fatal pulmonary embolism occurred, two fatal intracranial hemorrhages were observed in thrombocytopenic patients not receiving thromboprophylaxis. 125 These findings demonstrate that thrombotic and hemorrhagic complications, although affecting a minority of patients, are clinically meaningful and closely linked to severe CRS, multiorgan dysfunction, and the need for intensive care support. Nevertheless, granular prospective data directly mapping the real-time kinetics of specific coagulation and fibrinolysis biomarkers alongside clinical thrombotic events remain scarce in current literature.

Compounding this scarcity of data, terminology in this field remains inconsistent. The term CARAC has been used to describe a spectrum ranging from isolated laboratory abnormalities to TMA-like presentations and, rarely, overt DIC. Such heterogeneity complicates comparison across studies and impedes clinical interpretation. Recent commentaries therefore emphasize the need for standardized definitions and grading systems, analogous to those used for CRS and ICANS, to distinguish transient endothelial activation from clinically significant consumptive coagulopathy. 111 126 Addressing this need, recently proposed consensus criteria by Schoettler et al define TMA in the setting of cellular therapies as the presence of microangiopathic hemolytic anemia (characterized by schistocytes and elevated LDH), persistent or consumptive thrombocytopenia, and concurrent renal or neurological dysfunction, often accompanied by markers of terminal complement activation such as elevated soluble C5b-9. 127 This distinction is clinically important, as true TMA may require targeted intervention beyond standard supportive care.

Overall, current data support the concept that CARAC represents the hematologic correlate of cytokine-mediated endothelial injury. Integrating this vascular perspective into clinical practice may improve risk stratification and guide interventions aimed at maintaining endothelial stability.

Clinical Implications and Future Perspectives

Recognition of endotheliopathy as a central driver of CRS, ICANS, and CARAC has prompted interest in vascular-targeted interventions. Yet, despite growing mechanistic evidence supporting a central role for endothelial injury in CAR T cell–related toxicities, no endothelial-specific therapy is currently approved in this setting. At present, management remains centered on supportive care and control of the underlying inflammatory response, highlighting a substantial gap between preclinical mechanistic understanding and clinical translation.

Established Therapeutic Approaches

Interventions for CAR T cell–related toxicities primarily target the inflammatory cascade driving endotheliopathy. Supportive care, corticosteroids, and IL-6 receptor blockade with tocilizumab remain the cornerstones of CRS management and have substantially improved clinical outcomes. 7 9 76 81 Although these interventions do not directly target the endothelium, their efficacy indirectly underscores the central contribution of vascular injury to the pathogenesis of CRS and related complications. 89 128 The resolution of thrombotic and bleeding complications after CAR T cell therapy follows established hematological principles but should be individualized according to the evolving balance between thrombosis risk, thrombocytopenia, and coagulopathy, particularly during the first weeks after infusion. 113 116 123

Investigational Endothelial-Protective Strategies

Several endothelial-protective approaches have been proposed and are supported by preclinical, translational, or early clinical evidence. Agents with immunomodulatory and endothelial-stabilizing properties, including statins and defibrotide, have attracted particular interest because of their established role in other endothelial injury syndromes. Statins reduce pro-inflammatory cytokine signaling and leukocyte adhesion, whereas defibrotide promotes endothelial protection through increased NO bioavailability and reduced oxidative stress. Though neither strategy has been systematically evaluated in CAR T cell recipients, both represent promising options for mitigating therapy-related endotheliopathy. 12 129 130 Likewise, targeting upstream inflammatory mediators may indirectly attenuate vascular injury. Given the central role of IL-1β and TNF-α in driving endotheliopathy during CAR T cell therapy, cytokine-directed approaches have emerged as compelling therapeutic strategies. Among these, IL-1 blockade with anakinra has gained increasing clinical acceptance for the management of refractory CRS, ICANS, and IEC-HS, 89 90 whereas TNF-α inhibition with agents such as adalimumab remains at an earlier stage of clinical development. Early clinical and translational studies suggest that these approaches may limit therapy-related vascular injury without compromising antitumor efficacy. 81 131 132 Nevertheless, clinical experience remains limited, and prospective studies are required before endothelial-targeted cytokine blockade can be safely incorporated into routine practice.

Experimental Pathway-Directed Approaches

Beyond general endothelial protection, several therapeutic concepts seek to directly modulate the endothelial pathways discussed in this review. Currently, these strategies remain strictly experimental and are mainly extrapolated from thrombotic microangiopathies, sepsis, and other forms of systemic endothelial injury. Within the VWF/ADAMTS13 axis, potential interventions include recombinant ADAMTS13 administration to cleave excessive ULVWF multimers, 133 and VWF-directed therapies such as caplacizumab to reduce pathological platelet–VWF interactions. 134 Recombinant ADAMTS13 has been shown ex vivo to normalize VWF multimer distribution in plasma obtained from patients with severe COVID-19, providing proof-of-concept that restoration of the VWF/ADAMTS13 balance may be therapeutically feasible in inflammatory endotheliopathies. 135 However, translating these anti-VWF strategies into the CAR T cell arena poses significant clinical challenges. The use of caplacizumab, an anti-VWF nanobody, carries a profound risk of inducing severe, life-threatening hemorrhage. 134 This risk is exponentially amplified in CAR T cell recipients, who frequently experience deep, prolonged cytopenias and consumptive coagulopathies. 113 116 Consequently, defining whether the benefit of preventing microvascular thrombosis outweighs the substantial bleeding risk remains a critical, unanswered question that has yet to be evaluated in prospective trials.

Another emerging avenue centers on modulation of the Ang-Tie2 axis. Experimental studies consistently show that enhancing Tie2 signaling stabilizes endothelial junctions, reduces vascular leakage, and improves survival in inflammatory injury models. Gene-based Ang-1 delivery has improved vascular integrity in sepsis and organ injury models. 136 137 138 139 Vasculotide, a synthetic Tie2 agonist, reduces vascular permeability and mortality across multiple preclinical inflammatory settings, 136 140 141 whereas the Ang-1 mimetic MAT.Ang-1 similarly decreases vascular leakage without hemodynamic compromise. 142 Conversely, Ang-2 inhibition reduces vascular leak and organ injury; notably, combining Ang-2 blockade and Tie2 activation appears more effective than either strategy alone in preserving glycocalyx integrity and improving survival. 61 67 136 Although these findings provide compelling proof-of-concept for endothelial-targeted interventions, their applicability to CAR T cell–associated toxicities remains entirely untested. The principal endothelial-targeted therapeutic approaches discussed in this review, together with their targets and current level of evidence, are summarized in Table 4 .

Future Directions

Future research priorities include prospective multicenter validation of endothelial candidate biomarkers such as the VWF/ADAMTS13 ratio, ADAMTS13 activity, and Ang profiles, as well as the establishment of clinically actionable thresholds for risk stratification. Importantly, a distinction should be made between biomarkers that identify patients at increased risk of toxicity before treatment and those that reflect evolving toxicity severity after CAR T cell infusion. Baseline assessment of endothelial status prior to lymphodepletion may help identify patients with preexisting endothelial vulnerability, whereas dynamic biomarker changes during therapy may provide prognostic information regarding disease trajectory and treatment response. Ultimately, clinical trials integrating biomarker-guided stratification with mechanistically targeted interventions will be required to determine whether modulation of the VWF/ADAMTS13 axis or Ang–Tie2 pathway can safely alter toxicity trajectories without compromising antitumor efficacy.

Conclusion

CAR T cell therapy has fundamentally reshaped the treatment landscape of hematologic malignancies, yet its clinical impact is increasingly shaped by vascular toxicities driven by endothelial injury and dysregulated hemostasis. Growing evidence identifies cytokine-driven endotheliopathy as a unifying mechanism linking immune hyperactivation to immunothrombosis, vascular instability, and organ dysfunction, mediated in part through perturbations of the VWF/ADAMTS13 axis and the Ang-2–Tie2 signaling pathway.

Reframing CAR T cell–related toxicities as manifestations of underlying endothelial vulnerability provides a coherent biological framework and highlights opportunities for earlier risk stratification. Endothelial biomarkers may help identify patients at increased risk of toxicity and improve understanding of the mechanisms underlying CRS, ICANS, and CARAC. However, a major translational challenge remains the absence of standardized assays, validated thresholds, and external prospective validation.

Future studies should focus on defining the clinical utility of baseline endothelial assessment and early postinfusion biomarker monitoring, while integrating these approaches into prospective risk-stratification strategies. Although these biomarkers remain investigational, continued refinement of endothelial profiling may ultimately contribute to safer and more personalized CAR T cell therapy.

Footnotes

Conflict of Interest The authors declare that they have no conflict of interest.

Contributors’ Statement S.D.B.: visualization and writing—original draft, review, and editing. I.V. and A.G.: conceptualization and writing—review and editing. S.A.: writing—review and editing.

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