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. Author manuscript; available in PMC: 2026 Mar 18.
Published in final edited form as: Blood. 2026 Apr 30;147(18):2053–2063. doi: 10.1182/blood.2025029834

Age-associated increases in PAI-1 silenced with siRNA-lipid nanoparticles reduces thrombosis and prolongs lifespan

Francesca Ferraresso 1,2,3, Chad W Skaer 1, Zimu Wei 4, Woosuk S Hur 5, Hongyin Y 1,6, Monica Seadler 1,7, Taylor H Y Chen 1, Wen Dai 1, Manoj Paul 1, Catherine LaPointe 5, Laura M Ketelboeter 1, Hayley Lund 1, Geoffrey G Rodriguez 1, Lih Jiin Juang 2,3, Amy W Strilchuk 2,3, Youjie Zhang 1,8, Pieter R Cullis 2, Mitchell R Dyer 1,7, Allison L Gerras 4, Qizhen Shi 1,6, James P Luyendyk 4, Matthew J Flick 5, Ze Zheng 1,9, Christian J Kastrup 1,2,3,7,8,10,11,*
PMCID: PMC12994142  NIHMSID: NIHMS2150465  PMID: 41587091

Abstract

Plasminogen activator inhibitor 1 (PAI-1) is an inhibitor of fibrinolysis, thereby promoting blood clot stabilization. PAI-1 contributes to thrombosis, diet-induced obesity, and age-associated diseases such as diabetes, cancer, and Alzheimer disease. Circulating PAI-1 increases with age, contributing to the increased thrombotic risk in age-related diseases. In contrast, partial PAI-1 deficiency protects patients from cardiovascular morbidity and extends lifespan. Decreasing circulating PAI-1 levels has both experimental and therapeutic value. RNA gene therapy can regulate the levels of target proteins, including those not amenable to traditional small-molecule or antibody-based therapies. Here, we developed a therapeutic approach to induce long-lasting PAI-1 knockdown in vivo with siRNA-lipid nanoparticles (siPAI-1). One dose of siPAI-1 resulted in 90% knockdown of plasma PAI-1 and lasted 10 days postadministration with no overt toxicity. siPAI-1 decreased thrombus weight following complete ligation of the inferior vena cava (IVC) in young and aged mice, and increased survival in aged mice four days post-IVC ligation. Hepatic PAI-1 mRNA expression in diet-induced obese mice was >10 times higher than in healthy mice and was exponentially correlated with body weight. One dose of siPAI-1 in obese mice resulted in 70% knockdown of circulating PAI-1. Furthermore, siPAI-1 normalized the supraphysiologic concentration of PAI-1 in aged mice, and prolonged lifespan in a fast-aging mouse model. Thus, siRNA-mediated PAI-1 knockdown represents a long-term anti-thrombotic approach and effective strategy to limit pathologic impact of PAI-1 in aging and in age-related diseases.

Graphical Abstract

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INTRODUCTION

Age-related diseases account for over 1.4 million deaths annually in the United States, with thrombosis being a primary cause of death among the elderly, impacting 1 in every 100 individuals over the age of 451–3. An important accelerator of thrombosis is plasminogen activator inhibitor 1 (PAI-1), a circulating protein in the blood that impedes clot degradation (fibrinolysis) by inhibiting tissue and urokinase plasminogen activators (tPA and uPA, respectively),4, 5 both of which mediate the conversion of plasminogen to plasmin5. Upon vascular injury, circulating PAI-1 concentration increases leading to inhibition of fibrinolysis and contributing to clot stabilization but also increases the risk of thrombosis6. Elevated PAI-1 concentrations are correlated with thrombosis risk and are observed in related conditions such as and disseminated intravascular coagulation, which is commonly observed in elderly COVID-19 patients6–9. Notably, in COVID-19 patients, PAI-1 is linked with disease severity and elevated morbidity9. Moreover, PAI-1 acts as a mediator of cellular senescence; its levels rise during aging and the progression of multiple age-associated diseases such as atherosclerosis, obesity, Alzheimer disease, diabetes, and cancer10–14.

Healthy PAI-1 concentrations in humans range from 21.0 +/− 7.2 ng/mL and with age they typically increase up to 50 ng/mL.15, 16 Patients with complete PAI-1 deficiency (< 1 ng/mL) may experience prolonged bleeding following severe trauma, including spontaneous intracranial, mucocutaneous, joint, and oral bleeding17–19. However, those with partial PAI-1 deficiency (> 1 ng/mL), including heterozygote individuals (SERPINE1 −/+), do not experience excessive risk of bleeding20, but instead have reduced cardiovascular morbidity and evidence of increased lifespan11. Thus, PAI-1 has emerged as a promising therapeutic target for modulating aging and age-associated pathologies, including thrombosis21, 22.

Multiple small-molecule, antibody and nanobody inhibitors for PAI-1 have been developed4, 23–27. These inhibitors have been tested in various mouse and rat disease models of thrombosis, Alzheimer disease, atherosclerosis, and multiple cancer types, in each case showing promising results24, 28. However, PAI-1 inhibitors face specificity challenges due to the inherent conformational plasticity of PAI-1, which exists in active, latent, and cleaved forms. TM5614 is the only orally bioavailable small molecule PAI-1 inhibitor that has reached clinical trials29. In the United States, TM5614 is currently in a phase 2 clinical trial for high-risk patients with severe COVID-19, while in Japan it has recently completed a randomized trial in mild to moderate COVID-1930, 31. In Japan TM5614 is also in a phase 2 clinical trial for the treatment of malignant melanoma32. MDI-2517 is a novel small-molecule inhibitor that has recently entered the clinic and is currently in Phase I to assess safety and tolerability. No PAI-1 inhibitor has yet received FDA approval for therapeutic use5, 33.

RNA gene therapy represents an alternative approach to regulate the levels of target proteins, including those unsuitable for traditional small-molecule or antibody-based approaches34, 35. RNA interference (RNAi) approaches are an alternative strategy to previously explored inhibitors36–39. Small interfering RNA (siRNA) are short, non-coding RNA molecules that can be designed to specifically degrade an mRNA of interest, thereby suppressing the production of specific proteins for long durations (weeks to months)40. Lipid nanoparticles (LNP) are an FDA-approved non-viral nucleic acid delivery system used in the Pfizer/BioNTech/Acuitas and Moderna mRNA COVID-19 vaccines41, 42. siRNA-LNP agents can be used both to degrade hepatic mRNA and to modulate the concentration of its corresponding protein in blood37–39, 43, 44. LNP were first approved in Onpattro, an siRNA targeted for hereditary transthyretin amyloidosis (hATTR)40. Patients that received Onpattro every three weeks for more than six years have maintained improvement in polyneuropathy symptoms without overt toxicity45. LNP preferentially accumulate in the liver, which is a tissue where PAI-1 is synthesized16, 46. PAI-1 is also synthesized in endothelial cells, megakaryocytes, smooth muscle cells, fibroblasts, monocytes, adipocytes, endometrium, peritoneum, mesothelial cells and cardiac myocytes, making it unclear whether an approach based on siRNA-LNP could sufficiently decrease circulating PAI-116. For example, the higher concentrations of circulating PAI-1 in obese patients is attributed to overexpression by adipose tissue13, 47, 48. In this study, we developed a long-lasting siRNA-LNP that decreases circulating PAI-1 levels, providing both an experimental and therapeutic tool to control PAI-1 in age-associated diseases.

METHODS

A detailed description of all the experimental procedures and statistical analysis are available in the supplemental information.

Animals

Murine studies were performed in accordance to approved protocols from the Animal Care and Use Committees of affiliated institutions.

siRNA-LNP formulation

siRNA targeting PAI-1 or Luciferase, were encapsulated in LNP as previously described43.

mRNA quantification and analysis of PAI-1 levels

For mRNA quantification, livers were processed as previously described49. The total PAI-1 concentration was analyzed with a mouse total PAI-1 ELISA kit (IMSPAI1KTT, Innovative Research).

IVC ligation

Complete IVC ligation was performed under isoflurane as described in the Supplementary Material39.

Mouse Models of Bleeding

The bleeding models were lateral vein transection (TVT), tail tip transection (TTT), and a 6-hour tail bleeding test, and were performed using procedures previously described50, 51.

RESULTS

Circulating PAI-1 can be potently knocked down with siRNA-LNP in mice.

We examined whether siRNA designed against PAI-1 mRNA encapsulated in LNP, could achieve knockdown of circulating PAI-1 protein. In silico, six siRNA sequences were designed to target different regions of the PAI-1 mRNA52. Sequences were encapsulated in LNP and screened in mice. The optimal siRNA sequence (siPAI-1) was then selected for future experiments (data not shown). Mice were injected with either siPAI-1 or an siRNA control with no target mRNA in mice (siLuc) at 3 mg of siRNA per kg of body weight (3 mg/kg). PAI-1 protein was measured in plasma and hepatic tissue, and PAI-1 mRNA was measured in hepatic tissue. Three days post-injection, male and female mice injected with siPAI-1 at a dose of 3 mg/kg had significantly lower circulating plasma PAI-1. The concentration of PAI-1 in siLuc control male mice was 2.2 ±0.3 ng/mL compared to 0.3 ±0.1 ng/mL (P<0.0001) in male mice injected with siPAI-1 (Fig. 1a). To evaluate if the lipid nanoparticle itself modulates PAI-1 protein levels, mice were injected with PBS and blood was collected 3 days post-injection. There were similar circulating PAI-1 levels in mice injected with PBS (1.6 ±0.1 ng/mL) or siLuc (2.3 ±0.3 ng/mL, P=0.17). Similarly to male mice, female mice injected with siPAI-1 (1± 0.1 ng/mL) had lower circulating PAI-1 levels 3 days post-injection compared to siLuc treated mice (2 ±0.3 ng/mL, P=0.02) (Fig. 1b). Due to the inter-animal variability in PAI-1 levels in female mice, all subsequent studies used male mice53, 54. The relative amount of mRNA in the liver of mice injected with siPAI-1 (26 ± 6 %) was also significantly decreased compared to mice injected with siLuc (100 ± 37 %, P=0.04) (Fig. 1c). PAI-1 protein concentration was significantly lower in hepatic tissue from siPAI-1 injected mice (4.6 ± 0.6 ng/mL) compared to siLuc treated mice (8.7 ± 1.8 ng/mL, P=0.01) (Fig. 1d).

Figure 1: Circulating PAI-1 can be knocked down with siRNA-lipid nanoparticles in mice.

Figure 1:

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Mice kept with standard animal care and housing were injected with either siPAI-1 or siLuc control. a) Total plasma PAI-1 levels analyzed with an ELISA in male mice. b) Total plasma PAI-1 levels in female mice. c) Hepatic PAI-1 mRNA levels standardized to the siLuc injected male mice. d) Hepatic PAI-1 protein normalized to the concentration of total hepatic protein three days post-injection in male mice. e) Total plasma PAI-1 protein levels analyzed at baseline (2 days before injection) and at 3, 7, 10, 14, 17, and 21 days postinjection in a separate cohort of male mice. f) Fibrinolysis in the presence of tPA assessed with rotational thromboelastometry (ROTEM) of collected blood. g) Cleavage of a fluorescent substrate by plasmin in clotted plasma with added tPA (1 nM). h) Rate of fibrinolysis, determined from data in panel h, where each marker is plasma from an individual mouse. i-k) Coagulation assessed with ROTEM, measuring clot time (i), alpha angle (j), and maximum clot firmness (MCF) (k). N = 3–15; * = P<0.05.

To assess the duration of PAI-1 knockdown upon siPAI-1 administration, mice were injected with siPAI-1 or siLuc and blood sampled at 3-, 7-, 10-, 14-, 17-, and 21- days post-injection. Plasma PAI-1 levels were significantly lower in siPAI-1 injected mice 3-, 7- and 10- days post-injection compared to the siLuc treated control mice at each timepoint (Fig. 1e). Mice injected with siPAI-1 had similar PAI-1 levels to control siLuc mice at 14-, 17- and 21-days post-injection.

To test if PAI-1 knockdown in mice altered coagulation and fibrinolysis, rotational thromboelastometry (ROTEM) was performed on whole blood from mice injected with siPAI-1 or siLuc. To assess fibrinolysis ex vivo, human tissue plasminogen activator (tPA, 500 ng/mL) was added. Blood from mice injected with siPAI-1 displayed faster fibrinolysis compared to siLuc seven days post-injection. Measuring the percentage of clot remaining at 30 and 45 minutes after clot initiation (LI30 and LI45), siPAI-1 injected mice had an LI30 of 58 ± 1 % and an LI45 of 25 ± 9 %, whereas, mice injected with siLuc had an LI30 of 79 ± 5 % (P=0.02) and an LI45 of 55 ± 3 % (P=0.002) (Fig. 1f). To determine the impact on plasmin generation, a plasmin generation assay was performed using clotted blood plasma and measuring the cleavage of a fluorescent substrate for plasmin. Mice injected with siPAI-1 had significantly higher plasmin activity (2.1 ± 0.2 ΔRFU/min) compared to siLuc injected mice (1.5 ±0.1 ΔRFU/min; P=0.04) (Fig. 1g–h). The blood coagulation profile was assessed for potential risk of altered coagulation following PAI-1 knockdown, including alpha angle, clot time, and maximum clot firmness using ROTEM. No significant differences were observed between siPAI-1 and siLuc injected mice in these parameters. (Fig. 1h–k).

siPAI-1 decreases venous thrombosis in both young adult and aged mice and increases survival rates in aged mice in a model of deep vein thrombosis.

Due to the ability of siPAI-1 to enhance fibrinolysis, we assessed if siPAI-1 could decrease thrombus formation. In both young adult and aged mice (9–10 and 80–82 weeks old, respectively), the IVC was completely ligated 3 days after infusion of siPAI-1 or siLuc. The clot and the IVC segment containing the clot were collected 4 days post-ligation (7 days post-infusion) (Fig. 2a). Young adult mice injected with siPAI-1 had significantly smaller thrombi (3± 1 mg of clot and affected IVC segment) compared to siLuc injected mice (45 ±10, P<0.001) (Fig. 2b) and significantly lower plasma PAI-1 concentrations at the endpoint (4 ±1 ng/mL) compared to siLuc injected mice (10 ±0.4 ng/mL, P=0.03) (Fig. 2c). Similarly, if only the clot was weighted, siPAI-1 (2 ±2 mg) had significantly smaller thrombi compared to siLuc treated mice (16 ±2 mg, P<0.001) (Fig. 2d). Following extraction of the thrombi, the IVC were visualized with trichrome H&E staining. Mice injected with siPAI-1 had little-to-no visual clots within the vessel (Fig. 2e). No mortality occurred in young adult mice in the IVC thrombosis model.

Figure 2: siPAI-1 decreases thrombosis in both young and aged mice and increases survival in aged mice in the inferior vena cava (IVC) stasis model of venous thrombosis.

Figure 2:

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a) Workflow of the experiment, where mice were injected with either siPAI-1 or siLuc and then their IVC were completely ligated three days later. b) Weight of the clot and IVC segment where the clot was present in young mice (8–10 weeks old) four days post-ligation. c) Total plasma PAI-1 protein levels four days post-ligation of young adult mice. d) Weight of the clot in young mice (8–10 weeks old) four days post-ligation. e) Representative trichrome hematoxylin and eosin (H&E) cross-sectional stains of histological sections of the IVC four days post-ligation. f) Survival of aged mice (80–81 weeks old). g) Clot weight for aged mice four days post-ligation (black dot) and clot weight for aged mice that died prior to four days (red cross). N = 5–8; * = P<0.05; error bars represent mean ± SEM.

In the aged mice, the siLuc injected mice had a significantly lower chance of survival at 4 days following IVC ligation (33%) compared to mice injected with siPAI-1 (83%, P=0.02) (Fig. 2f). Four of the six siLuc injected mice died between day 2 and 3 post-ligation, while one of six siPAI-1 injected mice died 3 days post-ligation. Due to the high-mortality rate, blood samples were not collected for evaluation of PAI-1 knockdown. Thrombi were collected 4 days post-ligation for mice that survived to the study endpoint and within 6 hours from death for mice that died prior to the study endpoint. siPAI-1 injected mice developed smaller clots (16 ± 5 mg) than mice injected with siLuc (66 ± 16 mg, P=0.001) (Fig. 2g).

siPAI-1 normalizes pathologically high PAI-1 levels in obese and hypercholesteremic mice

PAI-1 levels are elevated in obesity, as is the risk of thrombosis. Thus, we explored if siPAI-1 could knock down PAI-1 in obese mice, effectively examining the role of hepatic-derived PAI-1 in obesity. Circulating PAI-1 protein levels in wildtype mice fed a high-fat diet were significantly higher compared to low-fat diet fed mice (5.8 ± 1 versus 1.2 ± 0.1 ng/mL, respectively, P=0.003) (Fig. 3a). Hepatic mRNA PAI-1 levels were also significantly higher in high fat diet mice when compared relative to low fat diet mice (961 ± 229 %, P<0.001) (Fig. 3b). The hepatic mRNA PAI-1 levels in high-fat diet fed mice were exponentially correlated to the mouse body weight (R2:0.82) (Fig. 3c). As the mRNA expression and circulating PAI-1 protein levels are significantly higher in obese mice, we tested if siPAI-1 could still knockdown circulating PAI-1 protein. Obese mice injected with siPAI-1 had significantly lower circulating PAI-1 levels (2.7 ± 0.3 ng/mL) compared to siLuc injected mice (6.7 ± 3 ng/mL, P=0.01) (Fig. 3d).

Figure 3: Hepatic PAI-1 mRNA expression is elevated in obese mice and can be normalized with siPAI-1.

Figure 3:

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a) Total plasma PAI-1 levels of mice on a high fat diet (HFD) or low fat diet (LFD). b) Hepatic PAI-1 mRNA expression levels of mice from panel a. c) Hepatic PAI-1 mRNA expression with respect to mouse body weight at endpoint. d) Total plasma PAI-1 levels of WT obese mice on a HFD 7 days after receiving siLuc or siPAI-1. e) Total plasma PAI-1 levels at Day 3 and 10 of ApoE−/− mice injected with siPAI-1 or siLuc twice (injections on day 0 and 7). Grey dotted line represents the initiation of a HFD (N=6). f) Cholesterol levels of ApoE−/− mice 3 days after receiving siLuc or siPAI-1. Shaded blue area represents normal range of PAI-1.58 N = 3–12; * = P<0.05; error bars represent mean ± SEM.

Due the ability of siPAI-1 to reduce circulating PAI-1 in obese mice, we examined the role of hepatic PAI-1 expression in pro-atherosclerotic mice (ApoE−/−) to assess the siPAI-1 impact on cholesterol levels. High cholesterol levels (> 240 mg/dL) is a risk factor for thrombosis and other heart diseases, and a reduction in circulating cholesterol of ~15 % significantly decreases the risk of cardiovascular diseases55. Mice were injected twice, seven days apart, with either siPAI-1 or siLuc, and plasma was collected 3 days following each injection. LNP require apoE to transfect hepatocytes, so due to the apoE deficiency in these mice, LNP were incubated with human recombinant apoE prior to injection. Three days post the first injection, mice were switched to a high-fat and high-cholesterol diet for the remaining of the study to induce the pro-atherosclerotic hypercholesteremic phenotype that includes elevated cholesterol. Ten days after the first injection, mice injected with siPAI-1 had significantly lower circulating PAI-1 levels (0.7 ± 0.3 ng/mL) compared to siLuc injected mice (4.3 ± 0.4 ng/mL, P<0.001) (Fig. 3e). There were significantly lower (14 %) cholesterol levels in the siPAI-1 injected mice compared to siLuc injected mice (246 ± 14 versus 287 ± 5 mg/dL, P=0.02) (Fig. 3f).

siPAI-1 sustainably knocks down elevated PAI-1 levels in aged mice and prolongs lifespan

To assess the feasibility of repeated injections of siPAI-1 and knockdown over time, aged mice (80–82 weeks old) were injected every seven days for one month and plasma and serum were collected 3 days post each injection. siPAI-1 injected mice displayed consistently significantly lower PAI-1 concentrations compared to siLuc injected mice. Throughout the month, mice injected with siLuc exhibited higher PAI-1 levels relative to siPAI-1 treated mice (Fig. 4a)56. In mice treated with siPAI-1, mean PAI-1 levels ranged from 1.22 ± 0.10 ng/mL to 1.81 ± 0.30 ng/mL, whereas in mice treated with siLuc, levels varied from 2.50 ± 0.20 ng/mL to 3.80 ± 1.10 ng/mL. Serum was assessed for potential off-target effects and immune activation. No differences were observed in IL-6 serum levels (Fig. 4b) as well as the other 9 cytokine levels tested between siPAI-1 and siLuc treated mice (data not shown).

Figure 4: Repeated injections of siPAI-1 consistently knocks down circulating PAI-1 levels and prolongs lifespan in a fast-aging mouse model.

Figure 4:

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a-b) Aged WT mice (80–82 weeks old) administered siPAI-1 or siLuc every 7 days. Total plasma PAI-1 (a) serum IL-6 (b) 3 days following each injection. c) Probability of survival of fast-aging Klotho−/− mice injected every 7 days starting at 3 weeks of age. d-e) Aged WT mice (starting at 70 week old) injected every 7 days for 6 months. Images are H&E stains of the heart and liver at endpoint, where each column is a separate mouse (d). Heat map is of representative serum cytokine levels standardized to the PBS injected mice (e). N = 4–6, * = P<0.05; error bars represent mean ± SEM. Shaded blue area represents normal range of PAI-1.58 N = 4–6, ns=not significant; * = P<0.05; error bars represent mean ± SEM.

Based on the ability of siPAI-1 to induce several beneficial changes on age-associated factors, such as thrombosis and cholesterol, and based on the known extended lifespan of partial PAI-1 deficient mice and humans, we assessed the potential of PAI-1 to prolonging lifespan in Klotho−/− mice, a fast-aging mouse model6, 7, 10, 11. Klotho−/− mice exhibit accelerated degeneration of multiple age-sensitive traits similar to humans such as ectopic calcification, skin atrophy, muscle atrophy, osteoporosis, arteriosclerosis and pulmonary emphysema.57 Klotho−/− mice were injected with siPAI-1 every 7 days starting at 3 weeks old. Mice injected with siPAI-1 had a 20 % longer lifespan (P<0.05) compared to mice injected with siLuc (Fig. 4c).

To assess long-term toxicity, mice were injected weekly for six months with siPAI-1 or PBS as control. At study endpoint, serum was collected to measure the concentration of 10 cytokines, and the heart and liver were collected for histological staining. A veterinary pathologist examined the histological stains of the heart and liver of mice treated with siPAI-1 and PBS and no pathological abnormalities were observed between treatment groups (Fig. 4d). There were no significant differences in the cytokine levels between siPAI-1 and PBS treated mice (Fig. 4e). siPAI-1 treated mice had overall lower cytokine levels compared to PBS treated mice. No cardiac fibrosis was observed in the heart samples collected from siPAI-1 treated mice.

siPAI-1 does not elicit overt toxicity in vivo.

We examined acute and fundamental markers of toxicity to begin understanding the therapeutic potential of siPAI-1. To assess acute toxicity, we analyzed the levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) 5 hours post-injection in young adult mice. No significant changes were observed between siPAI-1 and PBS injected mice (Fig. 5a–d, P=0.42, P=0.26, P=0.17, P=0.89, respectively). Although AST and ALT levels of some mice were comparatively higher than control mice receiving PBS, they were within or near normal ranges52. There were no increases in other toxicity markers such as albumin, bile acids, creatinine (CREA), total bilirubin (TBIL), blood urea nitrogen (BUN), gamma-glutamyl transferase (GGT), globulin, and creatinine, suggesting no overt toxicity (data not shown). To assess hepatocellular injury, ALT levels were assessed 1-, 3- and 7- days post-injection. No significant changes were observed between siPAI-1 and siLuc injected mice and ALT levels remained within normal ranges (Fig. 5e)58. White blood cell count (WBC), red blood cell count (RBC), and platelet count were analyzed 7 days post-injection and were all within normal range and comparable to PBS injected mice (Fig. 5 f–h). To assess the biodistribution of PAI-1 knockdown, PAI-1 protein was measured in several non-hepatic tissues that express PAI-1, the heart, vein (IVC) a large vein (IVC), and white fat. Mice treated with siPAI-1 (11±2 ng/mL PAI-1 per mg total protein) had significantly lower PAI-1 levels in the IVC compared to siLuc (23 ± 5 ng/mL PAI-1 per mg total protein, P=0.002) treated mice, but no changes in the heart and adipose tissue (Fig. 5i). Low to undetectable PAI-1 levels were observed in adipose tissue across both treatment groups, likely due to the young age and low body weight of the mice.

Figure 5: Knockdown of PAI-1 with siPAI-1 does not elicit overt toxicity or increased bleeding in mice.

Figure 5:

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a-d) Mice receiving an injection of siPAI-1 or PBS as a control, measuring serum levels of ALP (a) AST (b) ALT (c), and BUN (d) 5 hours post-injection. e) Serum ALT levels of mice injected with siPAI-1 or siLuc 1-, 3-, and 7-days post-injection. f-h) Blood cell counts of mice that received an injection of siPAI-1 or siLuc at either 1mg/kg or 3 mg/kg, including white blood cells (WBC) (f), red blood cells (RBC) (g), and platelets (h) 5 hours post-injection.. i) The relative amount of PAI-1 protein in a vein (IVC), heart, and adipose tissue, normalized to the concentration of total hepatic protein three days post-injection j-m) Bleeding assessments in mice treated with siPAI-1 or siLuc 3 days post-injection. Primary and rechallenge bleeding time of mice subjected to TTT (j) and TVT (k). Bleeding time (l) and percentage of hemoglobin remaining (m) following a 6-hour tail bleed injury model. Shaded blue area represents normal range of biomarker and shaded red area represents toxic range. N = 5–7, * = P<0.05; ns = not significant; error bars represent mean ± SEM.

To assess the bleeding risk associated with knocking down PAI-1 in mice, three bleeding injury models were performed: lateral vein transection (TVT) injury, tail tip transection (TTT) and a 6-hour tail bleeding test. In both the TVT and TTT injury models, there were no significant differences in blood loss or bleeding time between siPAI-1 and siLuc treated mice during either the primary bleed or a rechallenge bleed (Fig. 5j–k). In the 6-hour tail bleeding challenge there was no statistical difference in bleeding time between siPAI-1 (0.9 ±0.3 hours) and siLuc (0.6 0.1 hours, P=0.35) treated mice. As well, there was no significant difference in the change in circulating hemoglobin concentration. siPAI-1 treated mice had 68 ±9 % of their baseline hemoglobin at the end of the 6-hour tail bleeding challenge and siLuc treated mice had 74 3 % (P=0.56) (Fig. 5l–m).

DISCUSSION

These results demonstrate that following an intravenous injection of siPAI-1, circulating PAI-1 concentrations significantly decrease with no signs of overt toxicity. In the last two decades, many attempts have been made to develop an in vivo PAI-1 inhibitor5. siPAI-1 represents the first approach that provides feasible long-term in vivo inhibition of PAI-1. Knocking down PAI-1 at the RNA level resulted in reduced circulating PAI-1 activity and antigen concentrations. siPAI-1 can therefore be applied to study the role of PAI-1 in various PAI-1 related diseases. siPAI-1’s impact on fibrinolysis was evaluated using ROTEM with whole blood and a plasmin generation assay with plasma. In the ROTEM experiments, supraphysiological concentrations of tPA (500 ng/mL) were used to generate a clot and subsequently achieve fibrinolysis. Consistent with these results, the plasmin generation assay performed on platelet-poor plasma using physiologically relevant concentrations of tPA also confirmed a difference in fibrinolysis between treatment groups. siRNA-LNP agents have the advantage that they primarily deliver their cargo to the liver, thus enabling the study of hepatic PAI-1 contribution59. We observed lower PAI-1 levels in the IVC of siPAI-1 treated mice, but we do not believe this reflects direct lipid nanoparticle delivery to the vessel and inhibition of PAI-1 synthesis in endothelial cells. The reduction in protein content in the IVC may be due to reduced scavenging of circulating PAI-1 that was produced in hepatocytes. Circulating PAI-1 can complex with urokinase plasminogen activator (uPA), which is internalized via the uPA receptor (uPAR) on endothelial cells60. However, future studies will be required to investigate this mechanism. Aside from the IVC vessel, other non-hepatic tissues known to synthesize PAI-1, such as the adipose tissue and the heart showed no reduction in PAI-1 protein levels following siPAI-1 administration. Although we do not anticipate significant changes in biodistribution over time following administration compared to siRNA-LNP that are used clinically61, additional studies will be required to characterize siRNA clearance and biodistribution pharmacokinetic profiles. The relationship between the site of PAI-1 synthesis and disease is unknown62. One aspect of our study examined the relationship between hepatic PAI-1 and diet-induced obesity, prevalent in older populations. The increase in PAI-1 expression in obesity has been assumed to be due to expression in adipose tissue, although hepatic PAI-1 concentrations in obese mice were statistically higher compared to normal mice. The hepatic PAI-1 mRNA expression was correlated to the mouse body weight, thus implicating a hepatic PAI-1 contribution to the increased circulating levels of PAI-1 in obesity. These results are alike humans in which PAI-1 mRNA expression from liver biopsies are correlated with the person’s BMI but not with the PAI-1 mRNA expression in the adipose tissue63. However, the adipose tissue could still contribute to the rise in PAI-1 concentrations in obesity as the knockdown in obese mice is lower than in young healthy mice.

As atherosclerosis is an age-associated disease closely related with obesity, we examined whether siPAI-1 normalizes circulating PAI-1 levels in a proatherosclerotic mouse model, ApoE−/− mice on a western diet. The potential benefits of lowering PAI-1 levels in proatherosclerotic mice were observed in circulating cholesterol levels as siPAI-1 injected mice had significantly lower cholesterol levels compared to controls. These findings are consistent with the mechanism that PAI-1 sequesters tPA in hepatocytes, thus permitting lipidation of apolipoprotein B (apoB) and secretion of very-low-density lipoprotein (VLDL) in the blood which in turn increase cholesterol levels64.

This study applied siPAI-1 to determine the role of PAI-1 in an adult and aged mouse IVC thrombosis model four days post-ligation. Similar to ferric chloride thrombosis models previously tested with oral PAI-1 inhibitor,54 the clots were smaller in adult mice injected with siPAI-1 compared to the controls65. Future studies will need to determine the temporal role of PAI-1 in clot resolution in the context of partial PAI-1 knockdown. In aged mice, the survival of the mice post-ligation was significantly improved with PAI-1 knockdown. Two days post-ligation is the transition point between acute and chronic thrombosis, when the thrombus burden is the highest. All the mice that died prior to the study endpoint, died during this transition period. This transition causes an increase in inflammation that may have contributed to the early death of the mice. The relatively smaller thrombus burden combined with the inhibition of PAI-1 driven expression of inflammatory mediators potentially contributed to the increased survival of the siPAI-1 injected mice. As the control mice had significantly decreased survival post-ligation, there is a potential for survivor bias when assessing the thrombosis measurements. The magnitude of thrombus reduction observed by siPAI-1 in our study is greater than that reported in previous studies using PAI-1 knockout mice. These differences may reflect compensatory mechanisms that develop in germline knockouts, particularly given PAI-1 is synthesized by various cell types. By specifically targeting hepatic PAI-1 with siPAI-1, we preserve contributions from other sources of PAI-1, which may be acting through distinct mechanisms. Additionally, IVC ligation can alter hepatic blood flow, potentially impacting coagulation and fibrinolytic gene expression and thereby influencing clot resolution, which may differ between complete PAI-1 knockout and partial hepatic PAI-1 knockdown. Future studies will have to investigate mechanistic differences between genetic knockout of PAI-1 and partial PAI-1 knockdown. Furthermore, it remains unclear whether platelets acquire PAI-1 exclusively from megakaryocytes during their formation or also scavenge it from the circulation. Platelet-associated PAI-1 levels vary substantially across species, and many mouse strains, including C57BL/6J, exhibit significantly lower levels compared to humans66. Future investigations in large animal models will be required to define the contribution and regulation of platelet-derived PAI-1 following siPAI-1 administration. As PAI-1 plays a role in senescence and proliferation, humans who have low levels of PAI-1 on average live 10 % longer and are protected from cardiovascular morbidity. The potential of siPAI-1 was evaluated in a fast-aging mouse model, Klotho−/− mice10–12. siPAI-1 injected mice lived 20 % longer compared to control mice, thus providing proof-of-concept that siPAI-1 has an effect in aging. PAI-1 is a key mediator of p53 and IL6 signaling, implicating it in the regulation of senescence and age-associated inflammation12. However, additional studies will be required to elucidate the specific mechanisms by which siPAI-1 contributes to extending lifespan.

Similar to humans, mice have significant sex differences in endogenous PAI-1 concentrations, thus not allowing direct comparisons between sexes67. Treatment with siPAI-1 achieved significant knockdown of circulating PAI-1 concentrations in both male and female mice, however, female mice exhibited a greater inter-animal variability. Females have much greater variability of circuiting PAI-1 than males53, 54. To minimize experimental variability, all subsequent studies were performed in male mice.

Furthermore, in this study we assessed bleeding phenotype associated with decreased PAI-1 concentrations in three mouse bleeding injury models. Although there were no detectable differences in bleeding time or blood loss between mice treated with siPAI-1 or siLuc, future studies will investigate the safety and tolerability of siPAI-1 in large animal models, such as canine or swine bleeding models that have more similarities to the fibrinolysis system in humans38, 68. While humans with complete PAI-1 deficiency exhibit excessive bleeding, we do not expect siPAI-1 to cause a bleeding phenotype since ~1 ng/mL is considered sufficient to maintain hemostasis18, 69. Since siPAI-1 does not modulate PAI-1 protein levels in the heart or fat of mice, circulating concentrations are unlikely to drop below 1 ng/mL. Furthermore, we evaluated long-term toxicity by administering weekly injections of siPAI-1 or PBS to mice for 6 months. No pathological abnormalities were detected in the liver or heart and no signs of inflammation were observed in the cytokine analysis. Future studies will have to be performed to evaluate long-term tolerability in larger animal models.

siPAI-1 represents a mechanistically distinct approach compared to existing small-molecule and antibody-based PAI-1 inhibitors, such as TM5614 and MDI-2517. Whereas these agents transiently modulate PAI-1 activity for approximately six hours, siPAI-1 acts at the mRNA level, enabling a sustained and long-term reduction in PAI-1 expression for up to 10 days. Advances in siRNA delivery may enable the duration of knockdown to be extended even further in the future70. Current clinical PAI-1 inhibitors may reduce PAI-1 levels by approximately 60–80%, which is comparable to the reduction observed with siPAI-1. Furthermore, the thrombosis outcomes we observed with siPAI-1 are consistent with existing PAI-1 inhibitors16, 71, 72. In conclusion, PAI-1 concentration increases in various age-associated diseases, and siPAI-1 can be administered to decrease this elevation, normalizing the concentration. Decreasing PAI-1 levels could potentially have significant benefits for elderly, both by decreasing thrombosis and enhancing healthy aging. Furthermore, siPAI-1 can be applied as a research tool to address knowledge gaps of the role of PAI-1 in pathophysiological processes. Overall, siPAI-1 has potential as both a research tool and a therapeutic for a wide range of diseases.

Supplementary Material

Supplementary Material

Key points:

  • siRNA targeting PAI-1 encapsulated in lipid nanoparticles lowers PAI-1 levels in age-associated disease models with no overt toxicity

  • A reduction in circulating PAI-1 levels reduced thrombosis in young and aged mice and prolonged lifespan in a fast-aging mouse model.

ACKNOWLEDGMENTS

This work was supported by the American Heart Association (AHA) (Collaborative Science Award, 952422), by the National Institutes of Health (NIH) (R01HL166382) and by the Canadian Institutes of Health (CIHR) ((Doctoral award 187577 to F.F., Postdoctoral fellowship MFE181897 to W.S.H. and MFE414357 to C.L.). This research was also supported by grants from the National Institutes of Health (NIH) to JPL (R01 DK135649) and to QS (R01 HL102035), support from the USDA National Institute of Food and Agriculture, and the Albert C. and Lois E. Dehn Endowment to Michigan State University for Veterinary Medicine (Pathobiology and Diagnostic Investigation) to J.P.L. This research was also supported by the Shared Resource Labs of the Versiti Blood Research Institute, WI, USA, by the UBC Centre for Blood Research, BC, Canada, and by the Histology Core at Children’s Hospital of Wisconsin, USA. Schematics were made with BioRender.com.

Footnotes

DISCLOSURE OF CONFLICTS OF INTEREST

C.J.K., P.R.C., A.W.S., F.F. and L.J.J. are directors, shareholders and/or co-founders of companies developing RNA-therapies, including SeraGene Therapeutics, Inc. (C.J.K., P.R.C., A.W.S., L.J.J.), NanoVation Therapeutics, Inc. (C.J.K., P.R.C.), and Acuitas Therapeutics, Inc. (P.R.C.). Z.Z., W.D., H.L. are shareholders and co-founders of Milukee Therapeutics Inc. C.J.K., P.R.C., F.F., Z.Z., W.D., H.L., A.W.S. and L.J.J. have filed intellectual property on RNA-based therapies with the intention of commercializing these inventions. C.W.S., M.S., W.S.H., L.M.K., T.C., G.G.R., R.K., Y.Z., M.R.D., M.C., J.L., Q.S., H.Y., A.L.G., M.P. and Z.W. declare no conflict of interest.

Data Sharing Statement:

Requests for data sharing may be submitted to Christian J. Kastrup (ckastrup@versiti.org).

REFERENCES

  • (1).Lutsey PL; Zakai NA Epidemiology and prevention of venous thromboembolism. Nat Rev Cardiol 2023, 20 (4), 248–262. DOI: 10.1038/s41569-022-00787-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (2).Engbers MJ; van Hylckama Vlieg A; Rosendaal FR Venous thrombosis in the elderly: incidence, risk factors and risk groups. J Thromb Haemost 2010, 8 (10), 2105–2112. DOI: 10.1111/j.1538-7836.2010.03986.x. [DOI] [PubMed] [Google Scholar]
  • (3).North BJ; Sinclair DA The intersection between aging and cardiovascular disease. Circ Res 2012, 110 (8), 1097–1108. DOI: 10.1161/CIRCRESAHA.111.246876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (4).Sillen M; Declerck PJ Targeting PAI-1 in Cardiovascular Disease: Structural Insights Into PAI-1 Functionality and Inhibition. Front Cardiovasc Med 2020, 7, 622473. DOI: 10.3389/fcvm.2020.622473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (5).Sillen M; Declerck PJ A Narrative Review on Plasminogen Activator Inhibitor-1 and Its (Patho)Physiological Role: To Target or Not to Target? Int J Mol Sci 2021, 22 (5). DOI: 10.3390/ijms22052721. [DOI] [Google Scholar]
  • (6).Tjärnlund-Wolf A; Brogren H; Lo EH; Wang X Plasminogen activator inhibitor-1 and thrombotic cerebrovascular diseases. Stroke 2012, 43 (10), 2833–2839. DOI: 10.1161/STROKEAHA.111.622217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (7).Yatsenko T; Rios R; Nogueira T; Salama Y; Takahashi S; Tabe Y; Naito T; Takahashi K; Hattori K; Heissig B Corrigendum: Urokinase-type plasminogen activator and plasminogen activator inhibitor-1 complex as a serum biomarker for COVID-19. Front Immunol 2024, 15, 1390698. DOI: 10.3389/fimmu.2024.1390698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (8).Zuo Y; Warnock M; Harbaugh A; Yalavarthi S; Gockman K; Zuo M; Madison JA; Knight JS; Kanthi Y; Lawrence DA Plasma tissue plasminogen activator and plasminogen activator inhibitor-1 in hospitalized COVID-19 patients. medRxiv 2020. DOI: 10.1101/2020.08.29.20184358. [DOI] [Google Scholar]
  • (9).Baycan OF; Barman HA; Bolen F; Atici A; Erman H; Korkmaz R; Calim M; Atalay B; Aciksari G; Cekmen MB; et al. Plasminogen activator inhibitor-1 levels as an indicator of severity and mortality for COVID-19. North Clin Istanb 2023, 10 (1), 1–9. DOI: 10.14744/nci.2022.09076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (10).Yamamoto K; Takeshita K; Kojima T; Takamatsu J; Saito H Aging and plasminogen activator inhibitor-1 (PAI-1) regulation: implication in the pathogenesis of thrombotic disorders in the elderly. Cardiovasc Res 2005, 66 (2), 276–285. DOI: 10.1016/j.cardiores.2004.11.013. [DOI] [PubMed] [Google Scholar]
  • (11).Vaughan DE; Rai R; Khan SS; Eren M; Ghosh AK Plasminogen Activator Inhibitor-1 Is a Marker and a Mediator of Senescence. Arterioscler Thromb Vasc Biol 2017, 37 (8), 1446–1452. DOI: 10.1161/ATVBAHA.117.309451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (12).Kortlever RM; Higgins PJ; Bernards R Plasminogen activator inhibitor-1 is a critical downstream target of p53 in the induction of replicative senescence. Nat Cell Biol 2006, 8 (8), 877–884. DOI: 10.1038/ncb1448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (13).Juhan-Vague I; Alessi MC; Mavri A; Morange PE Plasminogen activator inhibitor-1, inflammation, obesity, insulin resistance and vascular risk. J Thromb Haemost 2003, 1 (7), 1575–1579. DOI: 10.1046/j.1538-7836.2003.00279.x. [DOI] [PubMed] [Google Scholar]
  • (14).McDonald AP; Meier TR; Hawley AE; Thibert JN; Farris DM; Wrobleski SK; Henke PK; Wakefield TW; Myers DD Aging is associated with impaired thrombus resolution in a mouse model of stasis induced thrombosis. Thromb Res 2010, 125 (1), 72–78. DOI: 10.1016/j.thromres.2009.06.005. [DOI] [PubMed] [Google Scholar]
  • (15).Booth NA; Simpson AJ; Croll A; Bennett B; MacGregor IR Plasminogen activator inhibitor (PAI-1) in plasma and platelets. Br J Haematol 1988, 70 (3), 327–333. DOI: 10.1111/j.1365-2141.1988.tb02490.x. [DOI] [PubMed] [Google Scholar]
  • (16).Cesari M; Pahor M; Incalzi RA Plasminogen activator inhibitor-1 (PAI-1): a key factor linking fibrinolysis and age-related subclinical and clinical conditions. Cardiovasc Ther 2010, 28 (5), e72–91. DOI: 10.1111/j.1755-5922.2010.00171.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (17).Adam MP; Feldman J; Mirzaa GM; Pagon RA; Wallace SE; Amemiya A GeneReviews. 1993.
  • (18).Iwaki T; Tanaka A; Miyawaki Y; Suzuki A; Kobayashi T; Takamatsu J; Matsushita T; Umemura K; Urano T; Kojima T; et al. Life-threatening hemorrhage and prolonged wound healing are remarkable phenotypes manifested by complete plasminogen activator inhibitor-1 deficiency in humans. J Thromb Haemost 2011, 9 (6), 1200–1206. DOI: 10.1111/j.1538-7836.2011.04288.x. [DOI] [PubMed] [Google Scholar]
  • (19).Mehta R; Shapiro AD Plasminogen activator inhibitor type 1 deficiency. Haemophilia 2008, 14 (6), 1255–1260. DOI: 10.1111/j.1365-2516.2008.01834.x. [DOI] [PubMed] [Google Scholar]
  • (20).Fay WP; Parker AC; Condrey LR; Shapiro AD Human plasminogen activator inhibitor-1 (PAI-1) deficiency: characterization of a large kindred with a null mutation in the PAI-1 gene. Blood 1997, 90 (1), 204–208. [PubMed] [Google Scholar]
  • (21).Khan SS; Shah SJ; Klyachko E; Baldridge AS; Eren M; Place AT; Aviv A; Puterman E; Lloyd-Jones DM; Heiman M; et al. A null mutation in. Sci Adv 2017, 3 (11), eaao1617. DOI: 10.1126/sciadv.aao1617. [DOI] [Google Scholar]
  • (22).Gene Variant in Amish a Clue to Better Aging. Genetic Engineering & Biotechnology News, Feb 1, 2018; Vol. 38. [Google Scholar]
  • (23).Reinke AA; Li SH; Warnock M; Shaydakov ME; Guntaka NS; Su EJ; Diaz JA; Emal CD; Lawrence DA Dual-reporter high-throughput screen for small-molecule. J Biol Chem 2019, 294 (5), 1464–1477. DOI: 10.1074/jbc.RA118.004885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (24).Hennan JK; Elokdah H; Leal M; Ji A; Friedrichs GS; Morgan GA; Swillo RE; Antrilli TM; Hreha A; Crandall DL Evaluation of PAI-039 [{1-benzyl-5-[4-(trifluoromethoxy)phenyl]-1H-indol-3-yl}(oxo)acetic acid], a novel plasminogen activator inhibitor-1 inhibitor, in a canine model of coronary artery thrombosis. J Pharmacol Exp Ther 2005, 314 (2), 710–716. DOI: 10.1124/jpet.105.084129. [DOI] [PubMed] [Google Scholar]
  • (25).Vousden KA; Lundqvist T; Popovic B; Naiman B; Carruthers AM; Newton P; Johnson DJD; Pomowski A; Wilkinson T; Dufner P; et al. Discovery and characterisation of an antibody that selectively modulates the inhibitory activity of plasminogen activator inhibitor-1. Sci Rep 2019, 9 (1), 1605. DOI: 10.1038/s41598-019-38842-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (26).Sillen M; Weeks SD; Strelkov SV; Declerck PJ Structural Insights into the Mechanism of a Nanobody That Stabilizes PAI-1 and Modulates Its Activity. Int J Mol Sci 2020, 21 (16). DOI: 10.3390/ijms21165859. [DOI] [Google Scholar]
  • (27).Peng S; Xue G; Gong L; Fang C; Chen J; Yuan C; Chen Z; Yao L; Furie B; Huang M A long-acting PAI-1 inhibitor reduces thrombus formation. Thromb Haemost 2017, 117 (7), 1338–1347. DOI: 10.1160/TH16-11-0891. [DOI] [PubMed] [Google Scholar]
  • (28).Xi X; Liu N; Wang Q; Chu Y; Yin Z; Ding Y; Lu Y ACT001, a novel PAI-1 inhibitor, exerts synergistic effects in combination with cisplatin by inhibiting PI3K/AKT pathway in glioma. Cell Death Dis 2019, 10 (10), 757. DOI: 10.1038/s41419-019-1986-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (29).Sasaki K; Fujiwara T; Ochi T; Ono K; Kato H; Onodera K; Ichikawa S; Fukuhara N; Onishi Y; Yokoyama H; et al. TM5614, an Inhibitor of Plasminogen Activator Inhibitor-1, Exerts an Antitumor Effect on Chronic Myeloid Leukemia. Tohoku J Exp Med 2022, 257 (3), 211–224. DOI: 10.1620/tjem.2022.J036. [DOI] [PubMed] [Google Scholar]
  • (30).Hirai T; Asano K; Ito I; Miyazaki Y; Sugiura H; Agirbasli M; Kobayashi S; Kobayashi M; Shimada D; Natsume I; et al. A randomized double-blind placebo-controlled trial of an inhibitor of plasminogen activator inhibitor-1 (TM5614) in mild to moderate COVID-19. Sci Rep 2024, 14 (1), 165. DOI: 10.1038/s41598-023-50445-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (31).Study To Antagonize Plasminogen Activator Inhibitor - 1 in Severe COVID-19. NIH Clinical Trials [Google Scholar]
  • (32).Fujimura T; Yoshino K; Kato H; Fukushima S; Ishizuki S; Otsuka A; Matsushita S; Amagai R; Muto Y; Yamazaki E; et al. A phase II multicentre study of plasminogen activator inhibitor-1 inhibitor (TM5614) plus nivolumab for treating anti-programmed cell death 1 antibody-refractory malignant melanoma: TM5614-MM trial. Br J Dermatol 2024, 191 (5), 691–697. DOI: 10.1093/bjd/ljae231. [DOI] [PubMed] [Google Scholar]
  • (33).Fujimura T; Yoshino K; Nakamura M; Kato H; Ito T; Maekawa T; Fujisawa Y; Matsushita S; Amagai R; Yamazaki E; et al. Efficacy and safety of TM5614 in combination with paclitaxel in the treatment of paclitaxel-resistant cutaneous angiosarcoma: Phase II study protocol. Exp Dermatol 2024, 33 (1), e14976. DOI: 10.1111/exd.14976. [DOI] [PubMed] [Google Scholar]
  • (34).Kim YK RNA therapy: rich history, various applications and unlimited future prospects. Exp Mol Med 2022, 54 (4), 455–465. DOI: 10.1038/s12276-022-00757-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (35).Sparmann A; Vogel J RNA-based medicine: from molecular mechanisms to therapy. EMBO J 2023, 42 (21), e114760. DOI: 10.15252/embj.2023114760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (36).Leung AK; Tam YY; Cullis PR Lipid nanoparticles for short interfering RNA delivery. Adv Genet 2014, 88, 71–110. DOI: 10.1016/B978-0-12-800148-6.00004-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (37).Strilchuk AW; Meixner SC; Leung J; Safikhan NS; Kulkarni JA; Russell HM; van der Meel R; Sutherland MR; Owens AP; Palumbo JS; et al. Sustained depletion of FXIII-A by inducing acquired FXIII-B deficiency. Blood 2020, 136 (25), 2946–2954. DOI: 10.1182/blood.2020004976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (38).Strilchuk AW; Hur WS; Batty P; Sang Y; Abrahams SR; Yong ASM; Leung J; Silva LM; Schroeder JA; Nesbitt K; et al. Lipid nanoparticles and siRNA targeting plasminogen provide lasting inhibition of fibrinolysis in mouse and dog models of hemophilia A. Sci Transl Med 2024, 16 (735), eadh0027. DOI: 10.1126/scitranslmed.adh0027. [DOI] [Google Scholar]
  • (39).Juang LJ; Hur WS; Silva LM; Strilchuk AW; Francisco B; Leung J; Robertson MK; Groeneveld DJ; La Prairie B; Chun EM; et al. Suppression of fibrin(ogen)-driven pathologies in disease models through controlled knockdown by lipid nanoparticle delivery of siRNA. Blood 2022, 139 (9), 1302–1311. DOI: 10.1182/blood.2021014559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (40).Akinc A; Maier MA; Manoharan M; Fitzgerald K; Jayaraman M; Barros S; Ansell S; Du X; Hope MJ; Madden TD; et al. The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat Nanotechnol 2019, 14 (12), 1084–1087. DOI: 10.1038/s41565-019-0591-y. [DOI] [PubMed] [Google Scholar]
  • (41).Zhang L; More KR; Ojha A; Jackson CB; Quinlan BD; Li H; He W; Farzan M; Pardi N; Choe H Effect of mRNA-LNP components of two globally-marketed COVID-19 vaccines on efficacy and stability. NPJ Vaccines 2023, 8 (1), 156. DOI: 10.1038/s41541-023-00751-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (42).Cullis PR; Felgner PL The 60-year evolution of lipid nanoparticles for nucleic acid delivery. Nat Rev Drug Discov 2024, 23 (9), 709–722. DOI: 10.1038/s41573-024-00977-6. [DOI] [PubMed] [Google Scholar]
  • (43).Ferraresso F; Strilchuk AW; Juang LJ; Poole LG; Luyendyk JP; Kastrup CJ Comparison of DLin-MC3-DMA and ALC-0315 for siRNA Delivery to Hepatocytes and Hepatic Stellate Cells. Mol Pharm 2022, 19 (7), 2175–2182. DOI: 10.1021/acs.molpharmaceut.2c00033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (44).Ferraresso F; Leung J; Kastrup CJ RNA therapeutics to control fibrinolysis: review on applications in biology and medicine. J Thromb Haemost 2024, 22 (8), 2103–2114. DOI: 10.1016/j.jtha.2024.04.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (45).Brodsky J Alnylam Presents New Long-Term Results from Global Open-Label Extension Study of ONPATTRO® (patisiran) at the European Academy of Neurology Virtual Congress 2020. Lindenboom CR, Ed.; Alnylam Therapeutics: 2020; Vol. https://investors.alnylam.com/press-release?id=24856. [Google Scholar]
  • (46).Kularatne RN; Crist RM; Stern ST The Future of Tissue-Targeted Lipid Nanoparticle-Mediated Nucleic Acid Delivery. Pharmaceuticals (Basel) 2022, 15 (7). DOI: 10.3390/ph15070897. [DOI] [Google Scholar]
  • (47).Alessi MC; Poggi M; Juhan-Vague I Plasminogen activator inhibitor-1, adipose tissue and insulin resistance. Curr Opin Lipidol 2007, 18 (3), 240–245. DOI: 10.1097/MOL.0b013e32814e6d29. [DOI] [PubMed] [Google Scholar]
  • (48).Correia ML; Haynes WG A role for plasminogen activator inhibitor-1 in obesity: from pie to PAI? Arterioscler Thromb Vasc Biol 2006, 26 (10), 2183–2185. DOI: 10.1161/01.ATV.0000244018.24120.70. [DOI] [PubMed] [Google Scholar]
  • (49).Ferraresso F; Badior K; Seadler M; Zhang Y; Wietrzny A; Cau MF; Haugen A; Rodriguez GG; Dyer MR; Cullis PR; et al. Protein is expressed in all major organs after intravenous infusion of mRNA-lipid nanoparticles in swine. Mol Ther Methods Clin Dev 2024, 32 (3), 101314. DOI: 10.1016/j.omtm.2024.101314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (50).Shi Q; Fahs SA; Mattson JG; Yu H; Perry CL; Morateck PA; Schroeder JA; Rapten J; Weiler H; Montgomery RR A novel mouse model of type 2N VWD was developed by CRISPR/Cas9 gene editing and recapitulates human type 2N VWD. Blood Adv 2022, 6 (9), 2778–2790. DOI: 10.1182/bloodadvances.2021006353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (51).Chen Y; Schroeder JA; Kuether EL; Zhang G; Shi Q Platelet gene therapy by lentiviral gene delivery to hematopoietic stem cells restores hemostasis and induces humoral immune tolerance in FIX(null) mice. Mol Ther 2014, 22 (1), 169–177. DOI: 10.1038/mt.2013.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (52).Ferraresso FKC. Pharmacological depletion of plasminogen activator inhibitor – 1.
  • (53).Asselbergs FW; Williams SM; Hebert PR; Coffey CS; Hillege HL; Navis G; Vaughan DE; van Gilst WH; Moore JH Gender-specific correlations of plasminogen activator inhibitor-1 and tissue plasminogen activator levels with cardiovascular disease-related traits. J Thromb Haemost 2007, 5 (2), 313–320. DOI: 10.1111/j.1538-7836.2007.02311.x. [DOI] [PubMed] [Google Scholar]
  • (54).Schoenhard JA; Asselbergs FW; Poku KA; Stocki SA; Gordon S; Vaughan DE; Brown NJ; Moore JH; Williams SM Male-female differences in the genetic regulation of t-PA and PAI-1 levels in a Ghanaian population. Hum Genet 2008, 124 (5), 479–488. DOI: 10.1007/s00439-008-0573-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (55).Wadhera RK; Steen DL; Khan I; Giugliano RP; Foody JM A review of low-density lipoprotein cholesterol, treatment strategies, and its impact on cardiovascular disease morbidity and mortality. J Clin Lipidol 2016, 10 (3), 472–489. DOI: 10.1016/j.jacl.2015.11.010. [DOI] [PubMed] [Google Scholar]
  • (56).Declerck PJ; Gils A; De Taeye B Use of mouse models to study plasminogen activator inhibitor-1. Methods Enzymol 2011, 499, 77–104. DOI: 10.1016/B978-0-12-386471-0.00005-5. [DOI] [PubMed] [Google Scholar]
  • (57).Kurosu H; Yamamoto M; Clark JD; Pastor JV; Nandi A; Gurnani P; McGuinness OP; Chikuda H; Yamaguchi M; Kawaguchi H; et al. Suppression of aging in mice by the hormone Klotho. Science 2005, 309 (5742), 1829–1833. DOI: 10.1126/science.1112766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (58).Otto GP; Rathkolb B; Oestereicher MA; Lengger CJ; Moerth C; Micklich K; Fuchs H; Gailus-Durner V; Wolf E; Hrabě de Angelis M Clinical Chemistry Reference Intervals for C57BL/6J, C57BL/6N, and C3HeB/FeJ Mice (Mus musculus). J Am Assoc Lab Anim Sci 2016, 55 (4), 375–386. [PMC free article] [PubMed] [Google Scholar]
  • (59).Chander N; Basha G; Yan Cheng MH; Witzigmann D; Cullis PR Lipid nanoparticle mRNA systems containing high levels of sphingomyelin engender higher protein expression in hepatic and extra-hepatic tissues. Mol Ther Methods Clin Dev 2023, 30, 235–245. DOI: 10.1016/j.omtm.2023.06.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (60).Cubellis MV; Wun TC; Blasi F Receptor-mediated internalization and degradation of urokinase is caused by its specific inhibitor PAI-1. EMBO J 1990, 9 (4), 1079–1085. DOI: 10.1002/j.1460-2075.1990.tb08213.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (61).Suzuki Y; Ishihara H Difference in the lipid nanoparticle technology employed in three approved siRNA (Patisiran) and mRNA (COVID-19 vaccine) drugs. Drug Metab Pharmacokinet 2021, 41, 100424. DOI: 10.1016/j.dmpk.2021.100424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (62).Baluta MM; Vintila MM PAI-1 Inhibition - Another Therapeutic Option for Cardiovascular Protection. Maedica (Bucur) 2015, 10 (2), 147–152. [PMC free article] [PubMed] [Google Scholar]
  • (63).Zheng Z; Nakamura K; Gershbaum S; Wang X; Thomas S; Bessler M; Schrope B; Krikhely A; Liu RM; Ozcan L; et al. Interacting hepatic PAI-1/tPA gene regulatory pathways influence impaired fibrinolysis severity in obesity. J Clin Invest 2020, 130 (8), 4348–4359. DOI: 10.1172/JCI135919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (64).Dai W; Zhang H; Lund H; Zhang Z; Castleberry M; Rodriguez M; Kuriakose G; Gupta S; Lewandowska M; Powers HR; et al. Intracellular tPA-PAI-1 interaction determines VLDL assembly in hepatocytes. Science 2023, 381 (6661), eadh5207. DOI: 10.1126/science.adh5207. [DOI] [Google Scholar]
  • (65).Zhu Y; Carmeliet P; Fay WP Plasminogen activator inhibitor-1 is a major determinant of arterial thrombolysis resistance. Circulation 1999, 99 (23), 3050–3055. DOI: 10.1161/01.cir.99.23.3050. [DOI] [PubMed] [Google Scholar]
  • (66).Siebert AE; Brake MA; Verbeek SC; Johnston AJ; Morgan AP; Cleuren AC; Jurek AM; Schneider CD; Germain DM; Battistuzzi FU; et al. Identification of genomic loci regulating platelet plasminogen activator inhibitor-1 in mice. J Thromb Haemost 2023, 21 (10), 2917–2928. DOI: 10.1016/j.jtha.2023.06.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (67).DiMusto PD; Lu G; Ghosh A; Roelofs KJ; Su G; Zhao Y; Lau CL; Sadiq O; McEvoy B; Laser A; et al. Increased PAI-1 in females compared with males is protective for abdominal aortic aneurysm formation in a rodent model. Am J Physiol Heart Circ Physiol 2012, 302 (7), H1378–1386. DOI: 10.1152/ajpheart.00620.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (68).Cau MF; Ferraresso F; Seadler M; Badior K; Zhang Y; Ketelboeter LM; Rodriguez GG; Chen T; Ferraresso M; Wietrzny A; et al. siRNA-mediated reduction of a circulating protein in swine using lipid nanoparticles. Mol Ther Methods Clin Dev 2024, 32 (2), 101258. DOI: 10.1016/j.omtm.2024.101258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (69).Chan JC; Duszczyszyn DA; Castellino FJ; Ploplis VA Accelerated skin wound healing in plasminogen activator inhibitor-1-deficient mice. Am J Pathol 2001, 159 (5), 1681–1688. DOI: 10.1016/S0002-9440(10)63015-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (70).Whitehead KA; Langer R; Anderson DG Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov 2009, 8 (2), 129–138. DOI: 10.1038/nrd2742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (71).Khoukaz HB; Ji Y; Braet DJ; Vadali M; Abdelhamid AA; Emal CD; Lawrence DA; Fay WP Drug Targeting of Plasminogen Activator Inhibitor-1 Inhibits Metabolic Dysfunction and Atherosclerosis in a Murine Model of Metabolic Syndrome. Arterioscler Thromb Vasc Biol 2020, 40 (6), 1479–1490. DOI: 10.1161/ATVBAHA.119.313775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (72).Gorlatova NV; Cale JM; Elokdah H; Li D; Fan K; Warnock M; Crandall DL; Lawrence DA Mechanism of inactivation of plasminogen activator inhibitor-1 by a small molecule inhibitor. J Biol Chem 2007, 282 (12), 9288–9296. DOI: 10.1074/jbc.M611642200. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material
NIHMS2150465-supplement-Supplementary_Material.pdf (263.4KB, pdf)

Data Availability Statement

Requests for data sharing may be submitted to Christian J. Kastrup (ckastrup@versiti.org).

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