Coagulation factor XI regulates endothelial cell permeability and barrier function in vitro and in vivo.

Loss of endothelial barrier function contributes to the pathophysiology of many inflammatory diseases. Coagulation factor XI (FXI) plays a regulatory role in inflammation. While activation of FXI increases vascular permeability in vivo, the mechanism by which FXI or its activated form FXIa disrupts endothelial barrier function is unknown. We investigated the role of FXIa in human umbilical vein endothelial cell (HUVEC) or human aortic endothelial cell (HAEC) permeability. The expression patterns of vascular endothelial (VE)-cadherin and other proteins of interest were examined by Western blot or immunofluorescence. Endothelial cell permeability was analyzed by transwell assay. We demonstrate that FXIa increases endothelial cell permeability by inducing cleavage of the VE-cadherin extracellular domain, releasing a soluble fragment. The activation of a disintegrin and metalloproteinase 10 (ADAM10) mediates the FXIa-dependent cleavage of VE-cadherin, as adding an ADAM10 inhibitor prevented the cleavage of VE-cadherin induced by FXIa. The binding of FXIa with plasminogen activator inhibitor 1 and very low-density lipoprotein receptor on HUVEC or HAEC surfaces activates vascular endothelial growth receptor factor 2 (VEGFR2). The activation of VEGFR2 triggers the MAPK signaling pathway and promotes the expression of active ADAM10 on the cell surface. In a pilot experiment using an established baboon model of sepsis, the inhibition of FXI activation significantly decreased the levels of soluble VE-cadherin to preserve barrier function. This study reveals a novel pathway by which FXIa regulates vascular permeability. The effect of FXIa on barrier function may be another way by which FXIa contributes to the development of inflammatory diseases.

Cytoprotective E-WE thrombin reduces disease severity in a murine model of relapsing-remitting multiple sclerosis.

The blood-brain barrier is composed of microvascular endothelial cells, immune cells, and astrocytes that work in concert with the coagulation cascade to control inflammation and immune cell infiltration into the central nervous system. Endothelial cell dysfunction leading to increased permeability and compromised barrier function are hallmarks of neuroinflammatory and autoimmune disorders, including multiple sclerosis (MS). Therapeutic strategies that improve or protect endothelial barrier function may be beneficial in the treatment or prevention of neuroinflammatory diseases. We therefore tested the hypothesis that biasing thrombin toward anticoagulant and cytoprotective activities would provide equivalent or even additive benefit compared with standard-of-care therapeutic strategies, including corticosteroids. In a mouse model of relapsing-remitting MS, treatment with the thrombin mutant, E-WE thrombin, an engineered thrombin mutant with cytoprotective activities that is biased toward anticoagulant and cytoprotective activity, reduced neuroinflammation and extracellular fibrin formation in SJL mice inoculated with proteolipid protein (PLP) peptide. When administered at the onset of detectable disease, E-WE thrombin significantly improved the disease severity of the initial attack as well as the relapse and delayed the onset of relapse to a similar extent as observed with methylprednisolone. Both methylprednisolone and E-WE thrombin reduced demyelination and immune cell recruitment. These results provide rationale for considering engineered forms of thrombin biased toward anticoagulant and cytoprotective activity as a therapeutic strategy and perhaps an effective alternative to high-dose methylprednisolone for the management of acute relapsing MS attacks.NEW & NOTEWORTHY There are limited treatment options for mitigating acute relapsing attacks for patients with multiple sclerosis. We tested the hypothesis that harnessing the cytoprotective activity of the blood coagulation enzyme, thrombin, would provide benefit and protection against relapsing disease in a mouse model of MS. Our results provide rationale for considering engineered forms of thrombin biased toward cytoprotective activity as a therapeutic strategy and perhaps an alternative to steroids for the management of relapsing MS attacks.

Safety and tolerability of the protein C activator AB002 in end-stage renal disease patients on hemodialysis: a randomized phase 2 trial.

BACKGROUND: The protein C system regulates blood coagulation, inflammation, and vascular integrity. AB002 is an injectable protein C activating enzyme under investigation to safely prevent and treat thrombosis. In preclinical models, AB002 is antithrombotic, cytoprotective, and anti-inflammatory. Since prophylactic use of heparin is contraindicated during hemodialysis in some end-stage renal disease (ESRD) patients, we propose using AB002 as a short-acting alternative to safely limit blood loss due to clotting in the dialysis circuit. METHODS: This phase 2, randomized, double-blind, placebo-controlled, single-dose study evaluates the safety and tolerability of AB002 administered into the hemodialysis line of ESRD patients during hemodialysis at one study center in the United States (ClinicalTrials.gov: NCT03963895). In this study, 36 patients were sequentially enrolled into two cohorts and randomized to AB002 or placebo in a 2:1 ratio. In cohort 1, patients received 1.5 µg/kg AB002 (n = 12) or placebo (n = 6); in cohort 2, patients received 3 µg/kg AB002 (n = 12) or placebo (n = 6). Patients underwent five heparin-free hemodialysis sessions over 10 days and were dosed with AB002 or placebo during session four. RESULTS: Here we show that AB002 is safe and well-tolerated in ESRD patients, with no treatment-related adverse events. Clinically relevant bleeding did not occur in any patient, and the time to hemostasis at the vascular access sites is not affected by AB002. CONCLUSIONS: As far as we are aware, this proof-of-concept study is the first clinical trial assessing the therapeutic potential of protein C activation. The results herein support additional investigation of AB002 to safely prevent and treat thrombosis in at-risk populations.

Some people with kidney disease require hemodialysis, a process in which a machine filters the blood to remove waste products. The process of hemodialysis can trigger blood clotting in the hemodialysis circuit. Therefore, the blood-thinner heparin is commonly used to prevent blood from clotting. However, some patients cannot tolerate heparin. Here we describe a clinical trial in which we tested whether a drug called AB002 is safe and can reduce hemodialysis circuit clotting in people with permanent kidney disease (end-stage renal disease) undergoing hemodialysis. AB002 appears to be safe and well-tolerated, and we observed reduced clotting without any signs of increased bleeding. Further studies are required in more patients to determine whether AB002 can be used routinely during hemodialysis to safely prevent or treat blood clots.

Pharmacologic targeting of coagulation factors XII and XI by monoclonal antibodies reduces thrombosis in nitinol stents under flow.

BACKGROUND: Cardiovascular implantable devices, such as vascular stents, are critical for the treatment of cardiovascular diseases. However, their success is dependent on robust and often long-term antithrombotic therapies. Yet, the current standard-of-care therapies often pose significant bleeding risks to patients. Coagulation factor (F)XI and FXII have emerged as potentially safe and efficacious targets to safely reduce pathologic thrombin generation in medical devices. OBJECTIVES: To study the efficacy of monoclonal antibody-targeting FXII and FXI of the contact pathway in preventing vascular device-related thrombosis. METHODS: The effects of inhibition of FXII and FXI using function-blocking monoclonal antibodies were examined in a nonhuman primate model of nitinol stent-related thrombosis under arterial and venous flow conditions. RESULTS: We found that function-blocking antibodies of FXII and FXI reduced markers of stent-induced thrombosis in vitro and ex vivo. However, FXI inhibition resulted in more effective mitigation of thrombosis markers under varied flow conditions. CONCLUSION: This work provides further support for the translation of contact pathway of coagulation inhibitors for their adjunctive clinical use with cardiovascular devices.

Activation of coagulation FXI promotes endothelial inflammation and amplifies platelet activation in a nonhuman primate model of hyperlipidemia.

Background: Hyperlipidemia is associated with chronic inflammation and thromboinflammation. This is an underlying cause of several cardiovascular diseases, including atherosclerosis. In diseased blood vessels, rampant thrombin generation results in the initiation of the coagulation cascade, activation of platelets, and endothelial cell dysfunction. Coagulation factor (F) XI represents a promising therapeutic target to reduce thromboinflammation, as it is uniquely positioned at an intersection between inflammation and thrombin generation. Objectives: This study aimed to investigate the role of FXI in promoting platelet and endothelial cell activation in a model of hyperlipidemia. Methods: Nonhuman primates (NHPs) were fed a standard chow diet (lean, n = 6) or a high-fat diet (obese, n = 8) to establish a model of hyperlipidemia. Obese NHPs were intravenously administered a FXI blocking antibody (2 mg/kg) and studied at baseline and at 1, 7, 14, 21, and 28 days after drug administration. Platelet activation and inflammatory markers were measured using fluorescence-activated cell sorting or enzyme-linked immunosorbent assay. Molecular imaging was used to quantify vascular cell adhesion molecule 1 (VCAM-1) expression at the carotid bifurcation. Results: Obese NHPs demonstrated increased sensitivity for platelet P-selectin expression and phosphatidylserine exposure in response to platelet GPVI or PAR agonists compared with lean NHPs. Obese NHPs exhibited elevated levels of C-reactive protein, cathepsin D, and myeloperoxidase compared with lean NHPs. Following pharmacological inhibition of FIX activation by FXIa, platelet priming for activation by GPVI or PAR agonists, C-reactive protein levels, and endothelial VCAM-1 levels were reduced in obese NHPs. Conclusion: FXI activation promotes the proinflammatory phenotype of hyperlipidemia by priming platelet activation and inciting endothelial cell dysfunction.