Efficacy of a Combination Therapy with Laronidase and Genistein in Treating Mucopolysaccharidosis Type I in a Mouse Model.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder caused by α-L-iduronidase deficiency. The standard treatment, enzyme replacement therapy with laronidase, has limited effectiveness in treating neurological symptoms due to poor blood-brain barrier penetration. An alternative is substrate reduction therapy using molecules, such as genistein, which crosses this barrier. This study evaluated the effectiveness of a combination of laronidase and genistein in a mouse model of MPS I. Over 12 weeks, MPS I and wild-type mice received laronidase, genistein, or both. Glycosaminoglycan (GAG) storage in visceral organs and the brain, its excretion in urine, and the serum level of the heparin cofactor II-thrombin (HCII-T) complex, along with behavior, were assessed. The combination therapy resulted in reduced GAG storage in the heart and liver, whereas genistein alone reduced the brain GAG storage. Laronidase and combination therapy decreased liver and spleen weights and significantly reduced GAG excretion in the urine. However, this therapy negated some laronidase benefits in the HCII-T levels. Importantly, the combination therapy improved the behavior of female mice with MPS I. These findings offer valuable insights for future research to optimize MPS I treatments.

Iatrogenic Pseudoaneurysm with Acute Arterial Thrombosis in Multiple Branches of the Lower Limbs Treated by Ultrasound-guided Thrombin Injection: A Case Report.

INTRODUCTION: With the development of vascular intervention, pseudoaneurysm complications are increasing. Ultrasound-guided thrombin injection (UGTI) is currently the treatment of choice for pseudoaneurysm, but the pharmacological properties of thrombin may trigger acute thrombosis within the vessel lumen. Despite a very low incidence, this type of primary arterial thrombosis is a serious complication of UGTI, and cases involving multiple branches of the lower limb arteries are particularly rare. CASE PRESENTATION: Here, we report a case of a 65-year-old male who underwent UGTI for the treatment of an iatrogenic pseudoaneurysm of the femoral artery complicated by acute thrombosis of multiple arteries in the lower limbs, and the patient ultimately underwent a successful thrombectomy. CONCLUSION: We reviewed the case and analyzed the possible etiologic causes, providing a reference for future clinical work.

Pharmacokinetic-Pharmacodynamic Modelling in Hemophilia A: Relating Thrombin and Plasmin Generation to Factor VIII Activity After Administration of a VWF/FVIII Concentrate.

BACKGROUND: Hemophilia A patients are treated with factor (F) VIII prophylactically to prevent bleeding. In general, dosage and frequency are based on pharmacokinetic measurements. Ideally, an alternative dose adjustment can be based on the hemostatic potential, measured with a thrombin generation assay (TGA), like the Nijmegen hemostasis assay. OBJECTIVE: The objective of this study was to investigate the predicted performance of a previously developed pharmacokinetic-pharmacodynamic model for FVIII replacement therapy, relating FVIII dose and FVIII activity levels with thrombin and plasmin generation parameters. METHODS: Pharmacokinetic and pharmacodynamic measurements were obtained from 29 severe hemophilia A patients treated with pdVWF/FVIII concentrate (Haemate P®). The predictive performance of the previously developed pharmacokinetic-pharmacodynamic model was evaluated using nonlinear mixed-effects modeling (NONMEM). When predictions of FVIII activity or TGA parameters were inadequate [median prediction error (MPE) > 20%], a new model was developed. RESULTS: The original pharmacokinetic model underestimated clearance and was refined based on a two-compartment model. The pharmacodynamic model displays no bias in the observed normalized thrombin peak height and normalized thrombin potential (MPE of 6.83% and 7.46%). After re-estimating pharmacodynamic parameters, EC50 and Emax values were relatively comparable between the original model and this group. Prediction of normalized plasmin peak height was inaccurate (MPE 58.9%). CONCLUSION: Our predictive performance displayed adequate thrombin pharmacodynamic predictions of the original model, but a new pharmacokinetic model was required. The pharmacodynamic model is not factor specific and applicable to multiple factor concentrates. A prospective study is needed to validate the impact of the FVIII dosing pharmacodynamic model on bleeding reduction in patients.

Effectiveness of a thrombin-gelatin flowable for treating severe liver bleeding: an experimental study.

BACKGROUND: Current scientific evidence has pointed out the relevance of hemostatic products for improving clinical outcomes in liver trauma, including increased survival rates and reductions in bleeding-related complications. The purpose of this study was to compare the use of the gelatin-thrombin flowable (Flowable) versus the standard technique of Packing in a new experimental liver injury model. METHODS: Twenty-four swine were prospectively randomized to receive either Flowable or standard packing technique. We used a novel severe liver injury model, in which the middle and left suprahepatic veins were selectively injured, causing an exsanguinating hemorrhage. The main outcome measure was the percentage of lost blood volume. RESULTS: The median total percentage of total blood volume per animal lost, from injury to minute 120, was significantly lower in the Flowable group (15.2%; interquartile range: 10.7-46.7%) than in the Packing group (64.9%; Interquartile range: 53.4-73.0%) (Hodges-Lehmann median difference: 41.1%; 95% CI: 18.9-58.0%, p = 0.0034). The 24-hour survival rate was significantly higher in the Flowable group (87.0%) than in the Packing group (0.0%) (Hazard ratio (HR) 0.08; 95% confidence interval 0.102 to 0.27; p < 0.0001). Mean-arterial pressure was significantly lower at minute 60 and 120 in the Flowable group than in the packing group (p = 0.0258 and p = 0.0272, respectively). At minute 120, hematocrit was higher in the Flowable than in the packing group (Hodges-Lehmann median difference: 5.5%; 95%CI: 1.0 to11.0, p = 0.0267). Finally, the overall-surgical-procedure was significantly shorter with Flowable than with Packing (Hodges-Lehmann median difference: 39.5 s, 95% CI: 25.0 to 54.0 s, p = 0.0004). CONCLUSIONS: The use of the Flowable was more effective in achieving hemostasis, reducing blood loss, and improving survival rates than standard packing in a severe porcine-liver bleeding model.

6-Gingerol ameliorates alveolar hypercoagulation and fibrinolytic inhibition in LPS-provoked ARDS via RUNX1/NF-κB signaling pathway.

BACKGROUND: Alveolar hypercoagulation and fibrinolytic inhibition play a central role in refractory hypoxemia in acute respiratory distress syndrome (ARDS), but it lacks effective drugs for prevention and treatment of this pathophysiology. Our previous experiment confirmed that RUNX1 promoted alveolar hypercoagulation and fibrinolytic inhibition through NF-κB pathway. Other studies demonstrated that 6-gingerol regulated inflammation and metabolism by inhibiting the NF-κB signaling pathway. We assume that 6-gingerol would ameliorate alveolar hypercoagulation and fibrinolytic inhibition via RUNX1/ NF-κB pathway in LPS-induced ARDS. METHODS: Rat ARDS model was replicated through LPS inhalation. Before LPS inhalation, the rats were intraperitoneally treated with different doses of 6-gingerol or the same volume of normal saline (NS) for 12 h, and then intratracheal inhalation of LPS for 24 h. In cell experiment, alveolar epithelial cell type II (AECII) was treated with 6-gingerol for 6 h and then with LPS for another 24 h. RUNX1 gene was down-regulated both in pulmonary tissue and in cells. Tissue factor (TF), plasminogen Activator Inhibitor 1(PAI-1) and thrombin were determined by Wester-blot (WB), qPCR or by enzyme-linked immunosorbent (ELISA). Lung injury score, pulmonary edema and pulmonary collagen III in rat were assessed. NF-κB pathway were also observed in vivo and in vitro. The direct binding capability of 6-gingerol to RUNX1 was confirmed by using Drug Affinity Responsive Target Stability test (DARTS). RESULTS: 6-gingerol dose-dependently attenuated LPS-induced lung injury and pulmonary edema. LPS administration caused excessive TF and PAI-1 expression both in pulmonary tissue and in AECII cell and a large amount of TF, PAI-1 and thrombin in bronchial alveolar lavage fluid (BALF), which all were effectively decreased by 6-gingerol treatment in a dose-dependent manner. The high collagen Ⅲ level in lung tissue provoked by LPS was significantly abated by 6-gingerol. 6-gingerol was seen to dramatically inhibit the LPS-stimulated activation of NF-κB pathway, indicated by decreases of p-p65/total p65, p-IKKß/total IKKß, and also to suppress the RUNX1 expression. RUNX1 gene knock down or RUNX1 inhibitor Ro5-3335 significantly enhanced the efficacies of 6-gingerol in vivo and in vitro, but RUNX1 over expression remarkably impaired the effects of 6-gingerol on TF, PAI-1 and on NF-κB pathway. DARTS result showed that 6-gingerol directly bond to RUNX1 molecules. CONCLUSIONS: Our experimental data demonstrated that 6-gingerol ameliorates alveolar hypercoagulation and fibrinolytic inhibition via RUNX1/NF-κB pathway in LPS-induced ARDS. 6-gingerol is expected to be an effective drug in ARDS.

Expert panel recommendations for topical hemostatic agent use in varied bleeding sites and situations during neuro-spine surgeries.

Intraoperative bleeding poses a substantial challenge, particularly in neuro-spine surgeries leading to complications such as hematomas, infections, and hemodynamic instability. Despite their proven efficacy, use of topical hemostatic agents (THAs) lacks comprehensive published literature and guidelines particularly in the Indian setting. The present study provides the first-ever Indian expert panel recommendations for effective adjunct THA use in different intraoperative bleeding sites and situations in neuro-spine surgeries. A comprehensive approach, encompassing a literature review, followed by experience sharing in a meeting using a survey helped integrate expert opinions in the form of practical algorithms to guide THA selection. Our survey results revealed a strong inclination towards specific THAs, flowable gelatin + thrombin being choice of THA for difficult to access and problematic bleeding situations during tumor removal/resection, transsphenoidal hypophysectomy and skull-based procedures. Both oxidized regenerated cellulose (ORC)/Fibrillar and flowable gelatin + thrombin were recommended for continuous oozing. ORC/Fibrillar was preferred for arteriovenous and cavernous malformations. This expert-panel guidance on THA use aims to optimize hemostat use practices and improve surgical outcomes in neuro-spine surgery.

Effectiveness of gelatin matrix with human thrombin for reducing blood loss in palliative decompressive surgery with posterior spinal fusion for metastatic spinal tumors.

BACKGROUND: This study aimed to investigate the effect of gelatin matrix with human thrombin (GMHT) on blood loss and survival time in patients with metastatic spinal tumors treated with palliative decompression surgery with posterior spinal fusion. METHODS: We retrospectively reviewed 67 consecutive patients with metastatic spinal tumors who underwent palliative decompression surgery with posterior spinal fusion. We compared patients in whom GMHT was not used during surgery with those in whom GMHT was used. The following baseline characteristics were evaluated: age, height, weight, sex, metastatic tumor diagnosis, medical history, use of antiplatelet drug, use of anticoagulant drug, use of NSAIDs, smoking, preoperative PLT value, preoperative APTT, preoperative PT-INR, Karnofsky Performance Status score, Charlson comorbidities index score, the percentage of patients who received perioperative chemotherapy, main tumor level, Frankel category, revised Tokuhashi score, spinal instability neoplastic score (SINS), number of fusion segments, operation time, intraoperative blood loss, drainage blood loss, red blood cell transfusion, hemoglobin level, total protein (TP), albumin values, total blood loss (TBL), hidden blood loss, postoperative bed rest and postoperative survival time. Perioperative complications were assessed. RESULTS: Age, height, weight, sex, metastatic tumor diagnosis, medical history, use of antiplatelet drug, use of anticoagulant drug, use of NSAIDs, smoking, preoperative PLT value, preoperative APTT, preoperative PT-INR, CCI score, main level of tumors, SINS score, preoperative Tokuhashi score and number of fusion segments did not differ significantly between the two groups. Operation time, intraoperative blood loss, postoperative drainage blood loss, and TBL were significantly decreased in the group with GMHT than in the group without GMHT. The total number of perioperative complications was significantly lesser in the group with GMHT than in the group without GMHT. The median postoperative survival time was significantly longer in the GMHT group than in the group without GMHT. CONCLUSION: GMHT should be considered a valid option for the treatment of patients with metastatic spinal tumors with a short life expectancy.

Patients with moderate hemophilia A and B with a severe bleeding phenotype have an increased burden of disease.

BACKGROUND: Patients with moderate hemophilia express varying bleeding phenotypes. OBJECTIVES: To assess the burden of disease in patients with moderate hemophilia and a mild or severe phenotype incorporating the thrombin generation profile. METHODS: This sub-study of the 6th Hemophilia in the Netherlands study, analyzed data of adults with moderate hemophilia A or B. Patient characteristics and information on bleeding tendency, joint status, and quality of life were obtained from electronic patient files and self-reported questionnaires. A severe bleeding phenotype was defined as an annual bleeding rate ≥5, an annual joint bleeding rate ≥3, and/or the use of secondary/tertiary prophylaxis, and a mild phenotype vice versa. TG was measured with the Nijmegen Hemostasis Assay. RESULTS: This study included 116 patients: 21% had a severe phenotype of whom 46% used prophylaxis. Patients with a severe phenotype treated on demand reported a higher median annual bleeding rate (7), annual joint bleeding rate (3), and more frequently an impaired joint (77%) than patients with a severe phenotype on prophylaxis (2; 0; 70%) or patients with a mild phenotype (0; 0; 47%). Furthermore, patients with a severe phenotype treated on demand experienced a more decreased quality of life. Despite similar factor activity levels, patients with a severe phenotype had a lower thrombin peak height and thrombin potential (0.7%; 0.06%) than patients with a mild phenotype (21.3%; 46.8%). CONCLUSION: Patients with moderate hemophilia and a severe phenotype treated on demand displayed a high burden of disease as well as a low thrombin generation profile advocating them toward more intensive prophylactic treatment.

Low molecular weight heparin decreases pro-coagulant activity in clinical MSC products.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are multipotent adult cells that can be isolated from tissues including bone marrow [MSC(BM)], adipose [MSC(AT)] and umbilical cord [MSC(CT)]. Previous studies have linked expression of tissue factor (TF) on MSC surfaces to a procoagulant effect. Venous thromboembolism (VTE), immediate blood-mediated inflammatory reaction (IBMIR) and microvascular thrombosis remain a risk with intravascular MSC therapy. We examined the effect of low molecular weight heparin (LMWH) on clinical-grade MSCs using calibrated automated thrombography (CAT). METHODS: Clinical grade MSC(BM)s, MSC(AT)s and MSC(CT)s harvested at passage 4 were added to normal pooled plasma (NPP) to a final concentration of either 400 000 or 50 000 cells/mL. LMWH was added to plasma in increments of 0.1 U/mL. Thrombin generation (TG) was measured using CAT. Flow cytometry was conducted on the cells to measure MSC phenotype and TF load. RESULTS: Presence of MSCs decreased lag time and increased peak TG. All cell lines demonstrated a dose response to LMWH, with MSC(AT) demonstrating the least thrombogenicity and most sensitivity to LMWH. TG was significantly reduced in all cell lines at doses of 0.2 U/mL LMWH and higher. DISCUSSION: All MSC types and concentrations had a decrease in peak thrombin and TG with increasing amounts of LMWH. While this in vitro study cannot determine optimal dosing, it suggests that LMWH can be effectively used to lower the risk of VTE associated with intravascular administration of MSCs. Future in vivo work can be done to determine optimal dosing and effect on IBMIR and VTE.

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.

Ruggedized Self-Propelling Hemostatic Gauze Delivers Low Dose of Thrombin and Systemic Tranexamic Acid and Achieves High Survival in Swine With Junctional Hemorrhage.

INTRODUCTION: Hemorrhage is responsible for 91% of preventable prehospital deaths in combat. Bleeding from anatomic junctions such as the groin, neck, and axillae make up 19% of these deaths, and reports estimate that effective control of junctional hemorrhage could have prevented 5% of fatalities in Afghanistan. Hemostatic dressings are effective but are time-consuming to apply and are limited when proper packing and manual pressure are not feasible, such as during care under fire. CounterFlow-Gauze is a hemostatic dressing that is effective without compression and delivers thrombin and tranexamic acid into wounds. Here, an advanced prototype of CounterFlow-Gauze, containing a range of low thrombin doses, was tested in a lethal swine model of junctional hemorrhage. Outcomes were compared with those of Combat Gauze, the current dressing recommended by Tactical Combat Casualty Care. MATERIALS AND METHODS: CounterFlow-Gauze containing thrombin doses of 0, 20, 200, and 500 IU was prepared. Swine received femoral arteriotomies, and CounterFlow-Gauze was packed into wounds without additional manual compression. In a separate study using a similar model of junctional hemorrhage without additional compression, CounterFlow-Gauze containing 500 IU thrombin was tested and compared with Combat Gauze. In both studies, the primary outcomes were survival to 3 h and volume of blood loss. RESULTS: CounterFlow-Gauze with 200 and 500 IU had the highest 3-h survival, achieving 70 and 75% survival, respectively. CounterFlow-Gauze resulted in mean peak plasma tranexamic acid concentrations of 9.6 ± 1.0 µg/mL (mean ± SEM) within 3 h. In a separate study with smaller injury, CounterFlow-Gauze with 500 IU achieved 100% survival to 3 h compared with 92% in Combat Gauze animals. CONCLUSIONS: An advanced preclinical prototype of CounterFlow-Gauze formulated with a minimized thrombin dose is highly effective at managing junctional hemorrhage without compression. These results demonstrate that CounterFlow-Gauze could be developed into a feasible alternative to Combat Gauze for hemorrhage control on the battlefield.

Effective Prevention of Arterial Thrombosis with Albumin-Thrombin Inhibitor Conjugates.

Thromboprophylaxis is indicated in patients at an elevated risk of developing thrombotic disorders, typically using direct oral anticoagulants or low-molecular-weight heparins. We postulated that transient thromboprophylaxis (days-weeks) could be provided by a single dose of an anticoagulant engineered for prolonged pharmacokinetics. In the present work, d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone (PPACK) was used as a model anticoagulant to test the hypothesis that conjugation of thrombin inhibitors to the surface of albumin would provide durable protection against thrombotic insults. Covalent conjugates were formed between albumin and PPACK using click chemistry, and they were tested in vitro using a thrombin activity assay and a clot formation assay. Thromboprophylactic efficacy was tested in mouse models of arterial thrombosis, both chemically induced (FeCl3) and following ischemia-reperfusion (transient middle cerebral artery occlusion; tMCAO). Albumin-PPACK conjugates were shown to have nanomolar potency in both in vitro assays, and following intravenous injection had prolonged circulation. Conjugates did not impact hemostasis (tail clipping) or systemic coagulation parameters in normal mice. Intravenous injection of conjugates prior to FeCl3-induced thrombosis provided significant protection against occlusion of the middle cerebral and common carotid arteries, and injection immediately following ischemia-reperfusion reduced stroke volume measured 3 days after injury by ∼40% in the tMCAO model. The data presented here provide support for the use of albumin-linked anticoagulants as an injectable, long-circulating, safe thromboprophylactic agent. In particular, albumin-PPACK provides significant protection against thrombosis induced by multiple mechanisms, without adversely affecting hemostasis.

Thrombin Generation Decrease After LMWH Administration in an Antithrombin-Deficient Pregnant Woman With a Homozygous HBS II Mutation.

The cases of antithrombin (AT)-deficient pregnant women with a homozygous HBS II mutation are relatively rare and are accompanied by an increased thrombophilic risk, which is manifested by increased thrombin generation (TG). It is very difficult to ensure their prophylactic treatment during pregnancy. We aimed to determine the utility of the thrombin generation assay (TGA) and anti-factor Xa (anti-FXa) test to monitor the effects of a prophylactic dose of low-molecular-weight heparin (LMWH) in a 28-year-old woman with homozygous AT deficiency caused by mutation c.391C > T#, (p.Leu131Phe†) in the SERPINC1 gene and to compare the findings with those from a group of pregnant and non-pregnant women also treated with LMWH. TG monitoring was chosen due to severe AT deficiency that was manifested by low levels of anti-FXa activity when monitoring the efficacy of LMWH treatment. A significant decrease in TG was detected in all monitored groups (P < .05). There were no thrombotic complications during the whole pregnancy of the woman with AT deficiency. Consistent monitoring of TG with LMWH anticoagulant therapy administration during pregnancy together with AT administration before and after delivery may improve the overall condition of pregnant women and the quality of their care.

Efficacy of gelatin-thrombin matrix sealants for blood loss in single-level transforaminal lumbar interbody fusion.

Although gelatin-thrombin matrix sealants have been used successfully in other surgery types, their effect on reducing blood loss during single-level transforaminal lumbar interbody fusion is unclear. We thus examined the efficacy of gelatin-thrombin matrix sealants for reducing blood loss during such surgery. We analyzed 102 patients who underwent single-level transforaminal lumbar interbody fusion for lumbar degenerative disease. We compared body mass index, surgical time, intraoperative blood loss, postoperative blood loss, true total blood loss, hidden blood loss, the proportion of blood transfusion, blood pressure pre- and post-surgery (systolic and diastolic), and pre-and post-surgery laboratory data (hemoglobin, hematocrit, platelets, prothrombin time, activated partial thromboplastin time, and D-dimer) between patients in whom gelatin-thrombin matrix sealants were (GTMS group) or were not (control group) used during surgery. One-week postoperative epidural hematoma size was measured using magnetic resonance imaging. The GTMS and control groups included 54 (24 males and 30 females) and 48 patients (19 males and 29 females). Intraoperative, true total, and hidden blood loss; epidural hematoma size; and hospitalization duration were significantly lower in the GTMS than in the control group. Intraoperative blood loss correlated with surgical time (R = 0.523, P = .001), body mass index (R = 0.221, P = .036), and the amount of gelatin-thrombin matrix sealant used (r = -0.313, P = .002). In multivariate linear regression analysis using intraoperative blood loss as the dependent variable, surgical time (standardization coefficient 0.516, P = .001) and amount of gelatin-thrombin matrix sealant used (standardization coefficient -0.220, P = .032) were independently related factors. In our study, the GTMS group had significantly less intraoperative true total and hidden blood loss than did the control group. Thus, use of gelatin-thrombin matrix sealants reduce perioperative blood loss in transforaminal lumbar interbody fusion.

Protease-Activated Receptor 1-Mediated Damage of Podocytes in Diabetic Nephropathy.

There is clinical evidence that increased urinary serine proteases are associated with the disease severity in the setting of diabetic nephropathy (DN). Elevation of serine proteases may mediate [Ca2+]i dynamics in podocytes through the protease-activated receptors (PARs) pathway, including associated activation of nonspecific cation channels. Cultured human podocytes and freshly isolated glomeruli were used for fluorescence and immunohistochemistry stainings, calcium imaging, Western blot analysis, scanning ion conductance microscopy, and patch clamp analysis. Goto-Kakizaki, Wistar, type 2 DN (T2DN), and a novel PAR1 knockout on T2DN rat background rats were used to test the importance of PAR1-mediated signaling in DN settings. We found that PAR1 activation increases [Ca2+]i via TRPC6 channels. Both human cultured podocytes exposed to high glucose and podocytes from freshly isolated glomeruli of T2DN rats had increased PAR1-mediated [Ca2+]i compared with controls. Imaging experiments revealed that PAR1 activation plays a role in podocyte morphological changes. T2DN rats exhibited a significantly higher response to thrombin and urokinase. Moreover, the plasma concentration of thrombin in T2DN rats was significantly elevated compared with Wistar rats. T2DNPar1-/- rats were embryonically lethal. T2DNPar1+/- rats had a significant decrease in glomerular damage associated with DN lesions. Overall, these data provide evidence that, during the development of DN, elevated levels of serine proteases promote an excessive [Ca2+]i influx in podocytes through PAR1-TRPC6 signaling, ultimately leading to podocyte apoptosis, the development of albuminuria, and glomeruli damage. ARTICLE HIGHLIGHTS: Increased urinary serine proteases are associated with diabetic nephropathy. During the development of diabetic nephropathy in type 2 diabetes, the elevation of serine proteases could overstimulate protease-activated receptor 1 (PAR1). PAR1 signaling is involved in the development of DN via TRPC6-mediated intracellular calcium signaling. This study provides fundamental knowledge that can be used to develop efficient therapeutic approaches targeting serine proteases or corresponding PAR pathways to prevent or slow the progression of diabetes-associated kidney diseases.

Intrathecal autologous thrombin-activated condensed platelet cytokines in chronic neurodegenerative disease: A hypothesis for anti-inflammatory and regenerative response.

Choroid plexus insufficiency or glymphatic stasis are often classified as prequels to harmful accretion of toxic proteins in neurodegenerative disease. Cognitive decline and disordered neuronal signaling subsequently become cardinal features of Alzheimer's disease (AD), typically progressing with amyloid-ß and tau protein accumulation. For Parkinson's disease (PD), α-synuclein deposits and dopamine depletion are linked to impaired movement, resting tremor, and rigidity. Importantly, both diagnoses feature hyperinflammation and intrathecal cytokine changes. Thus far, numerous clinical trials have produced nothing effective for AD or PD, yet the anti-inflammatory and regenerative potential of autologous platelet-rich plasma (PRP) remains largely unexamined in this context. Our report explores a proposed Phase I study on intrathecal condensed plasma growth factors processed from thrombin-activated PRP as monotherapy for AD or PD. The concept gains support from related work where cytokines of platelet origin successfully lowered inflammation, corrected background fibrosis, deactivated abnormal cells, and recovered local tissue function-all desirable outcomes in AD and PD. While PRP-mediated effects on membrane potentials, cellular signaling, electrolyte balance, and water clearance are less well characterized, experimental data suggest these pathways could likewise influence glymphatic drainage to ameliorate proteinopathies. As a well-tolerated 'orthobiologic' with no hypersensitivity risk, intrathecal PRP and its derivatives bring advantages over synthetic pharmaceuticals. If age-associated neuroinflammation in AD and PD is an upstream event inciting or contributing to neural disruption, then dampening local oxidative stress by a patient's own platelet cytokines (successful in other contexts) could offer therapeutic relevance to these neurodegenerative conditions as well.

Technical and clinical outcomes following EUS-guided thrombin injection and coil implantation for parastomal varices.

BACKGROUND AND AIMS: Bleeding from parastomal varices causes significant morbidity and mortality. Treatment options are limited, particularly in high-risk patients with significant underlying liver disease and other comorbidities. The use of EUS-guided embolisation coils combined with thrombin injection in gastric varices has been shown to be safe and effective. Our institution has applied the same technique to the treatment of parastomal varices. METHODS: A retrospective review was performed of 37 procedures on 24 patients to assess efficacy and safety of EUS-guided injection of thrombin, with or without embolisation coils for treatment of bleeding parastomal varices. All patients had been discussed in a multidisciplinary team meeting, and correction of portal hypertension was deemed to be contraindicated. Rebleeding was defined as stomal bleeding that required hospital admission or transfusion. RESULTS: All patients had significant parastomal bleeding at the time of referral. 100% technical success rate was achieved. 70.8% of patients had no further significant bleeding in the follow-up period (median 26.2 months) following one procedure. 1-year rebleed-free survival was 80.8% following first procedure. 7 patients (29.1%) had repeat procedures. There was no significant difference in rebleed-free survival following repeat procedures. Higher age was associated with higher risk of rebleeding. No major procedure-related complications were identified. CONCLUSIONS: EUS-guided thrombin injection, with or without embolisation coils, is a safe and effective technique for the treatment of bleeding parastomal varices, particularly for patients for whom correction of portal venous hypertension is contraindicated.

Role of Nitric Oxide in the Altered Calcium Homeostasis of Platelets from Rats with Biliary Cirrhosis.

INTRODUCTION: Previously, we found that intracellular calcium (Ca2+) homeostasis is altered in platelets from an experimental model of liver cirrhosis, namely the bile-duct-ligated (BDL) rat. These alterations are compatible with the existence of a hypercoagulable state. OBJECTIVE: In the present study, we analyzed the role of nitric oxide in the abnormal calcium signaling responses of an experimental cirrhosis model, the bile duct-ligated rat. METHODS: Chronic treatment with L-NAME was used to inhibit NO production in a group of control and BDL animals, and the responses compared to those obtained in a control and BDL untreated group (n = 6 each). The experiments were conducted on isolated platelets loaded with fura-2, using fluorescence spectrometry. RESULTS: Chronic treatment with L-NAME increased thrombin-induced Ca2+ release from internal stores in both control and BDL rats. However, the effect was significantly greater in the BDL rats (p < 0.05). Thrombin-induced calcium entry from the extracellular space was also elevated but at lower doses and, similarly in both control and BDL platelets, treated with the NO synthesis inhibitor. Capacitative calcium entry was also enhanced in the control platelets but not in platelets from BDL rats treated with L-NAME. Total calcium in intracellular stores was elevated in untreated platelets from BDL rats, and L-NAME pretreatment significantly (p < 0.05) elevated these values both in controls and in BDL but significantly more in the BDL rats (p < 0.05). CONCLUSIONS: Our results suggest that nitric oxide plays a role in the abnormal calcium signaling responses observed in platelets from BDL rats by interfering with the mechanism that releases calcium from the internal stores.

Lower extremity pseudoaneurysms and their interventional radiological management: a pictorial review.

Lower extremity pseudoaneurysms (PsAs) are mostly developed after traumatic or iatrogenic injury to the arteries. Unless treated, they can be complicated by adjacent mass effects, distal embolism, secondary infection, and rupture. Imaging helps in the diagnosis and planning of therapeutic intervention. Ultrasonography (USG) is often diagnostic, while CT angiography aids in vascular mapping required for intervention. Image-guided therapy offers to manage these pseudoaneurysms in a minimally invasive approach, obviating the need for surgery. A smaller, superficial, and narrow-necked PsA can easily be managed with local USG-guided compression or thrombin injection. When the percutaneous approach is not a feasible option, PsA from expendable arteries can also be managed with coiling or glue injection. Wide-necked PsA from an unexpendable artery necessitates stent graft placement, although coiling of the neck may be a viable and cheaper alternative for a long- and narrow-necked PsA. Presently, vascular closure devices are also used to seal a small arterial rent through a direct percutaneous approach. This pictorial review entails various techniques to deal with lower extremity pseudoaneurysms. An idea about the various intervention radiological approaches will help in choosing appropriate methods to tackle lower extremity pseudoaneurysms.

An update on novel therapies for treating patients with arterial thrombosis.

INTRODUCTION: Antithrombotic therapy field is undergoing rapid and significant changes during the past decade. In addition to new therapeutic strategies with existing targets, investigators are exploring the potential use of new targets to address unmet needs to treat patients with arterial diseases. AREAS COVERED: We aim to provide an update on and a comprehensive review of the antithrombic agents that are being explored in patients with arterial diseases. We discuss latest developments with respect to upstream antiplatelet agents, and collagen and thrombin pathway inhibitors. We searched PubMed databases for English language articles using keywords: antiplatelet agents, thrombin pathway inhibitors, collagen receptors, arterial disease. EXPERT OPINION: Despite implementation of potent P2Y12 inhibitors, there are numerous unmet needs in the treatment of arterial diseases including ceiling effect of currently available antiplatelet agents along with and an elevated risk of bleeding. The latter observations encouraged investigators to explore new targets that can attenuate the generation of platelet-fibrin clot formation and subsequent ischemic event occurrences with minimal effect on bleeding. These targets include collagen receptors on platelets and thrombin generation including FXa, FXIa, and FXIIa. In addition, investigators are studying novel antiplatelet agents/strategies to facilitate upstream therapy in high-risk patients.