Thromboembolism complicates disorders caused by immunoglobulin G (IgG)–containing immune complexes (ICs), but the underlying mechanisms are incompletely understood. Prior evidence indicates that induction of tissue factor (TF) on monocytes, a pivotal step in the initiation, localization, and propagation of coagulation by ICs, is mediated through Fcγ receptor IIa (FcγRIIa); however, the involvement of other receptors has not been investigated in detail. The neonatal Fc receptor (FcRn) that mediates IgG and albumin recycling also participates in cellular responses to IgG-containing ICs. Here we asked whether FcRn is also involved in the induction of TF-dependent factor Xa (FXa) activity by IgG-containing ICs by THP-1 monocytic cells and human monocytes. Induction of FXa activity by ICs containing IgG antibodies to platelet factor 4 (PF4) involved in heparin-induced thrombocytopenia (HIT), β-2-glycoprotein-1 implicated in antiphospholipid syndrome, or red blood cells coated with anti-(α)-Rh(D) antibodies that mediate hemolysis in vivo was inhibited by a humanized monoclonal antibody (mAb) that blocks IgG binding to human FcRn. IgG-containing ICs that bind to FcγR and FcRn induced FXa activity, whereas IgG-containing ICs with an Fc engineered to be unable to engage FcRn did not. Infusion of an α-FcRn mAb prevented fibrin deposition after microvascular injury in a murine model of HIT in which human FcγRIIa was expressed as a transgene. These data implicate FcRn in TF-dependent FXa activity induced by soluble and cell-associated IgG-containing ICs. Antibodies to FcRn, now in clinical trials in warm autoimmune hemolytic anemia to lower IgG antibodies and IgG containing ICs may also reduce the risk of venous thromboembolism.

Lymphangioleiomyomatosis (LAM) is a fatal lung disease associated with germline or somatic inactivating mutations in tuberous sclerosis complex genes (TSC1 or TSC2). LAM is characterized by neoplastic growth of smooth muscle-α-actin–positive cells that destroy lung parenchyma and by the formation of benign renal neoplasms called angiolipomas. The mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin slows progression of these diseases but is not curative and associated with notable toxicity at clinically effective doses, highlighting the need for better understanding LAM's molecular etiology. We report here that LAM lesions and angiomyolipomas overexpress urokinase-type plasminogen activator (uPA). Tsc1^−/− and Tsc2^−/− mouse embryonic fibroblasts expressed higher uPA levels than their WT counterparts, resulting from the TSC inactivation. Inhibition of uPA expression in Tsc2-null cells reduced the growth and invasiveness and increased susceptibility to apoptosis. However, rapamycin further increased uPA expression in TSC2-null tumor cells and immortalized TSC2-null angiomyolipoma cells, but not in cells with intact TSC. Induction of glucocorticoid receptor signaling or forkhead box (FOXO) 1/3 inhibition abolished the rapamycin-induced uPA expression in TSC-compromised cells. Moreover, rapamycin-enhanced migration of TSC2-null cells was inhibited by the uPA inhibitor UK122, dexamethasone, and a FOXO inhibitor. uPA-knock-out mice developed fewer and smaller TSC2-null lung tumors, and introduction of uPA shRNA in tumor cells or amiloride-induced uPA inhibition reduced tumorigenesis in vivo. These findings suggest that interference with the uPA-dependent pathway, when used along with rapamycin, might attenuate LAM progression and potentially other TSC-related disorders.

This study identifies a novel role for the deficiency of plasminogen activator inhibitor (PAI)-1 and resultant unrestricted uPA activity in PASMC remodeling and PH in vitro and in vivo, provides novel mechanistic link from PAI-1 loss through uPA-induced Akt/mTOR and TGFβ-Smad3 upregulation to pulmonary vascular remodeling in PH, and suggests that inhibition of uPA to rebalance the uPA-PAI-1 tandem might provide a novel approach to complement current therapies used to mitigate this pulmonary vascular disease.

Fibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding but is not clinically feasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce “antithrombotic” platelets from CD34+ hematopoietic stem cell–derived in vitro–grown megakaryocytes. CD34+ megakaryocytes internalize and store in alpha granules (α-granules) single-chain uPA (scuPA) and a plasmin-resistant thrombin-activatable variant (uPAT). Both uPAs colocalized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor–related protein 1 (LRP1), and interferon-induced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+ megakaryocytes was mediated, in part, via LRP1 and αIIbβ3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV but not endogenous VWF in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid artery injury model in nonobese diabetic-severe combined immunodeficiency IL2rγnull (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein Ibα) to test whether platelets derived from scuPA- or uPAT-megakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared with NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPA-megakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro–generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet α-granules to sites of vascular injury.

Background: Cardiac fibrosis represents a significant health burden, with endothelial dysfunction and damaged perivascular microenvironment increasingly recognized as key contributors to fibrotic remodeling. The urokinase plasminogen activator receptor (uPAR), a critical component of the urokinase system, plays a pivotal role in vascular remodeling and fibrosis. While prior evidence indicates that uPAR deficiency leads to microvascular dysfunction and perivascular fibrosis, the underlying mechanisms remain poorly defined. This study investigates how uPAR deficiency contributes to fibrotic remodeling of the cardiac perivascular-like microenvironment. Methods: Single-cell RNA sequencing data analysis and immunofluorescence staining on mouse heart cryosections were performed to characterize uPAR expression within the cardiac perivascular microenvironment. To model this microenvironment in vitro, cardiospheres (CSs) were generated from non-myocyte cardiac cells of wild-type and uPAR-knockout mice. CRISPR/Cas9-generated Plaur knockout (KO) 3T3 fibroblasts (FBs) were employed as model stromal cells. Pro-fibrotic activation of FBs was induced by TGFβ1 treatment. Comparative analyses of extracellular matrix (ECM) deposition, fibrotic cell transformation, and comprehensive secretome profiling was conducted using western blotting. Results: Our findings demonstrated that uPAR was expressed by endothelial cells (ECs) and FBs within the cardiac perivascular microenvironment. uPAR deficiency exacerbated profibrotic stimuli in CSs, including elevated active TGFβ1, impaired integrin functions, and altered cell secretome. These alterations collectively disrupt critical cell-cell and cell-matrix interactions, leading to increased ECM deposition, EC loss and decreased cell viability. Using Plaur KO FBs, we demonstrated that uPAR deficiency amplified TGFβ1-mediated Akt signaling pathway and ECM deposition. Conclusions: Our study reveals that uPAR loss drives fibrotic remodeling of the cardiac perivascular-like microenvironment and exacerbates TGFβ1-mediated effects, highlighting its potential as a therapeutic target for cardiac fibrosis.