Proteinuria Severity Is Directly Correlated with Markers of Hypofibrinolysis in a Podocyte Specific Experimental Model of Nephrotic Syndrome

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1106-1106 ◽  
Author(s):  
Amanda P Waller ◽  
Ruchika Sharma ◽  
Shipra Agrawal ◽  
Roger C Wiggins ◽  
William E Smoyer ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Animal Models ◽  
Experimental Model ◽  
Diphtheria Toxin ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Clot Lysis ◽  
Plasma Clot ◽  
Podocyte Injury ◽  
Thrombotic Risk

Abstract Introduction Nephrotic syndrome (NS) is characterized by massive proteinuria (secondary to podocyte injury or dysfunction), hypoalbuminemia, and edema, and is associated with a complex acquired hypercoagulopathy and a high prevalence (~25%) of life-threatening thrombotic complications. However, anticoagulation is associated with a substantial risk for adverse bleeding events. Recently published epidemiology studies suggest that proteinuria severity is directly correlated with thrombotic risk. However, further validation of this candidate biomarker for thrombotic risk requires appropriate validation and adequate pathophysiologic explanation. We have recently demonstrated that proteinuria severity is directly proportional to hypercoagulability (as assessed by ex vivo and in vivo markers of thrombotic capacity),in two well-established animal models of NS (puromycin aminonucleoside (PAN) and Adriamycin (ADR) rats). Thromboelastometry studies also suggested a resistance to fibrinolysis during rat NS. Thus, the aim of the present study was to further delineate the relationship between proteinuria severity and hypofibrinolysis using a podocyte-specific rat model of NS. We hypothesized that hypofibrinolysis is directly proportional to severity of proteinuria. Methods Using a transgenic rat which expresses the human diphtheria toxin receptor (hDTR) on a podocyte specific promoter (podocin), we compared markers of global hemostasis (ROTEM) and fibrinolysis to proteinuria severity. A range of proteinuria severity was induced by a single I.P. injection of diphtheria toxin (0, 25, 50 & 75 ng/kg; n= 7-8/group). On Day 10 post-injection, morning spot urines were collected and analyzed for protein:creatinine ratio. Rats were then anesthetized and venous blood (IVC) was collected into 0.32% NaCitrate/1.45 µM Corn Trypsin Inhibitor [final concentrations] and immediately analyzed with ROTEM (whole blood; intem) before being spun down to platelet poor plasma (PPP). Plasma clot lysis assay (CLA) was performed using urokinase (50 IU). Thrombin and plasmin generation assays are currently being performed. Results There were significant differences (P<0.005) between the highest proteinuria hDTR rat group and controls, in both hypercoagulopathic (clot formation time, maximum clot firmness, and clot size (amplitude at 10 & 20 min)), and hypofibrinolytic (amount of lysis at 60 min; LI60) ROTEM parameters. Importantly, there was a significant negative correlation between proteinuria severity and LI60 (R2 =0.362, P=0.02), suggesting that hypofibrinolysis is directly proportional to podocyte injury and therefore disease severity during NS. Preliminary results from the CLA (n=2 control & 2 high proteinuria) also suggest a marked impairment (~50% difference) in plasma clot lysis time in proteinuria rats. Conclusions These results demonstrate that proteinuria severity is directly proportional to both hypercoagulability and hypofibrinolytic capacity in a podocyte-specific rodent model of NS, thus confirming our recent findings in two other well-established animal models of NS in a third, more specific, experimental model of glomerular disease. Importantly, these data also strongly suggest a marked impairment in fibrinolysis during NS, which is directly correlated with proteinuria severity. Therefore it appears that severe proteinuria is associated with both hypercoagulopathic and hypofibrinolytic defects in the coagulation system. Future studies will delve into the molecular mechanisms involved in these defects. Disclosures No relevant conflicts of interest to declare.

Nephrotic Syndrome Clots Are Resistant to Fibrinolysis Despite Elevated Plasmin Generation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3767-3767
Author(s):  
Amanda P Waller ◽  
Katelyn J Wolfgang ◽  
Roger C Wiggins ◽  
William E Smoyer ◽  
Bryce A. Kerlin
Keyword(s):  
Nephrotic Syndrome ◽  
Disease Severity ◽  
Diphtheria Toxin ◽  
Conflicts Of Interest ◽  
Venous Blood ◽  
Clot Formation ◽  
Clot Lysis ◽  
Transgenic Rat ◽  
Plasma Clot ◽  
The Relationship

Abstract Introduction Nephrotic syndrome (NS) is characterized by massive proteinuria (secondary to podocyte injury), hypoalbuminemia, and edema. Importantly, NS is associated with a complex acquired hypercoagulopathy and a high prevalence (~25%) of life-threatening thrombotic complications. We have recently demonstrated that NS disease severity is directly proportional to hypercoagulability,in two well-established animal models of NS (puromycin aminonucleoside (PAN) and Adriamycin (ADR) rats). Furthermore, thromboelastometry studies by our group and others have suggested a resistance to fibrinolysis in both rats and humans with NS. Increasing evidence suggests that myriad abnormalities of the hemostatic system may be implicated in the pathogenesis of altered fibrin clot formation during NS; however the relationship between hypofibrinolysis and NS disease severity requires further validation and the mechanisms underlying the hypofibrinolytic defect remain unknown. Thus, the aim of the present study was to delineate the relationship between disease severity (proteinuria and hypoalbuminemia) and hypofibrinolysis using two rodent models of NS: a transgenic rat expressing the human diphtheria toxin receptor (hDTR) in a podocyte-specific manner and the well-established PAN rat model. We hypothesized that hypofibrinolysis is directly proportional to NS disease severity. Methods Using the hDTR rat, we compared markers of global hemostasis (ROTEM) and fibrinolysis to disease severity. A range of severity was induced by a single I.P. injection of diphtheria toxin (0, 25, 50 & 75 ng/kg; n= 7-8/group). On day 10 post-injection, morning spot urines were collected and analyzed for protein:creatinine ratio. Rats were then anesthetized and venous blood (IVC) was collected into 0.32% NaCitrate/1.45 µM Corn Trypsin Inhibitor [final concentrations] and immediately analyzed with ROTEM (whole blood; intem) before being spun down to platelet poor plasma (PPP). Plasma clot lysis assay (CLA) was performed using urokinase (50 IU). Plasmin generation was measured using a fluorogenic substrate in plasma with addition of 10 nM tissue plasminogen activator (tPA). Results Hypercoagulopathy in the hDTR NS model: Disease severity (proteinuria and hypoalbuminemia) was significantly correlated with endogenous thrombin potential, peak thrombin, and hypercoagulopathic ROTEM parameters (clot formation time, intermediate firmness (amplitude at 10 & 20 min), and maximum clot firmness). Hypofibrinolysis: The amount of lysis at 60 min (LI60) on ROTEM was significantly correlated with proteinuria in both the DTR and PAN models (R2=0.167; P=0.015 & R2=0.740; P<0.001, respectively), suggesting that NS thrombi are resistant to fibrinolysis in a manner that is proportional to disease severity. These findings were confirmed with the CLA, such that plasma clot lysis was significantly negatively correlated with proteinuria (R2=0.196; P=0.007 & R2=0.214; P=0.010) and significantly positively correlated with hypoalbuminemia (R2=0.310; P<0.001 & R2=0.240; P=0.006), in the DTR & PAN models, respectively. Plasmin Generation Assay: Plasmin generation increased with disease severity, such that it was highly correlated with proteinuria and hypoalbuminemia in both models (P<0.001; Figure). Interestingly, there was no correlation between plasmin generation and plasma clot lysis. (P=0.101 & P=0.126 in DTR & PAN rats, respectively). Conclusions NS disease severity is directly proportional to hypercoagulability in a podocyte-specific rodent model of NS; thus confirming our previously published findings in a podocyte-specific experimental model of NS. Moreover, this hypercoagulopathy leads to formation of a clot that is resistant to fibrinolysis. Paradoxically, the increased plasmin generation implies that the fibrinolytic system is also hyperactive in NS. Thus, it must be concluded that the clot is resistant, even to this increased plasmin that is generated. Future studies will explore the mechanisms underlying and in vivo significance of this fibrinolytic resistance. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Hypofibrinolytic Defect of Nephrotic Syndrome Is Directly Proportional to Fibrin Network Density

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1218-1218
Author(s):  
Amanda P. Waller ◽  
Katelyn J Wolfgang ◽  
Bryce A. Kerlin
Keyword(s):  
Nephrotic Syndrome ◽  
Disease Severity ◽  
Diphtheria Toxin ◽  
Thrombin Generation ◽  
Fibrin Clot ◽  
Network Density ◽  
Clot Lysis ◽  
Plasma Clot ◽  
Fibrin Network ◽  
Clot Structure

Abstract Introduction Nephrotic syndrome (NS) is characterized by massive proteinuria (secondary to podocyte injury), hypoalbuminemia, and edema. Importantly, NS is associated with a complex acquired hypercoagulopathy and a high incidence (~25%) of life-threatening thrombotic complications. Both hypercoagulopathy and hypofibrinolysis are described contributors to NS-related VTE risk. However, the mechanisms underlying the latter are poorly understood. We previously showed NS disease severity is directly proportional to both hypercoagulopathy and fibrinolytic resistance There is evidence that fibrin clot structural density contributes to clot stability and has been observed in the presence of both increased plasma thrombin generation and fibrinogen levels, both of which we have previously demonstrated in NS. Thus the aim of the present study was to investigate the mechanistic relationship between fibrin clot structure and fibrinolysis using two rodent models of NS and a cohort of human NS patients. We hypothesized that hypofibrinolysis arises from increased fibrin network density in a manner directly proportional to NS disease severity. Methods Using two well-established rat models of NS, transgenic diphtheria toxin receptor (DTR) and puromycin aminonucleoside (PAN), we compared fibrinolytic markers to disease severity. A range of severity was induced by a single injection of diphtheria toxin (0-75 ng/kg IP) or PAN (0-150 mg/kg IV). On day 10 post-injection, morning spot urines were collected and analyzed for protein:creatinine ratio (uPr:Cr). Rats were then anesthetized and venous blood (IVC) was collected into 0.32% NaCitrate/1.45 µM Corn Trypsin Inhibitor and spun down to platelet poor plasma (PPP). Samples were also collected from a local cohort of pediatric and adult NS patients (n=23), along with the corresponding clinical lab data for each patient. Plasma clot lysis assay (CLA) was performed using urokinase (50 IU) +/- plasminogen (2.4 uM), on clots initiated with high (20 nM) or low (5 nM) thrombin. Clot fibrin network structure was visualized/assessed by laser scanning confocal microscopy using fluorescently-labeled fibrinogen as a tracer. Fibrinolytic markers in plasma were measured by ELISA. Results Hypofibrinolysis: Previous findings of a hypofibrinolytic defect was confirmed with the CLA, such that plasma clot lysis at 60 min was significantly negatively correlated with proteinuria (R2=0.196; P=0.007 & R2=0.214; P=0.010) and significantly positively correlated with hypoalbuminemia (R2=0.310; P<0.001 & R2=0.240; P=0.006), in the DTR & PAN models, respectively. Additionally, plasma clot lysis by CLA was decreased in NS patients with uPrCr ≥2 (n=16) vs. <2 mg/mg (n=7) (96.1 vs 55.2 %, respectively; P=0.041). Similar results were found when the assay was repeated using high or low thrombin concentrations or increased UK (200 IU), with and without the addition of physiologic amounts of plasminogen. When the assay was performed in the absence of UK (0 IU), lysis at 60 min was drastically reduced (~17%) with no difference between groups. Mechanisms of Hypofibrinolysis: Fibrin network density increased with disease severity such that it was positively correlated with proteinuria (P=0.022) and negatively correlated with hypoalbuminemia (P=0.01) in our DTR rat model, with similar results seen in our human samples (Figure). As expected, fibrin network density was negatively correlated with plasma clot lysis (P=0.04), while plasma fibrinogen concentration (P=0.017), and thrombin generation (P=0.047) were positively correlated with fibrin density. There was no correlation with plasma uPA, PAI-1, a2AP, tPA, TAFI, or plasminogen. Conclusions These data suggest that nephrotic plasma forms thrombi with a denser fibrin network that is resistant to fibrinolysis, in a manner that is proportional to disease severity. The significant correlation between thrombin generation and fibrin network density suggest that plasma thrombotic potential may be a key mechanism contributing to the altered clot structure and impaired clot lysis of NS. Current studies are exploring the mechanisms underlying and in vivo significance of fibrinolytic resistance in our rat NS models. Disclosures No relevant conflicts of interest to declare.

Thrombin Generation Is Directly Correlated To Proteinuria Severity In An Experimental Model Of Nephrotic Syndrome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3615-3615
Author(s):  
Amanda P. Waller ◽  
William E. Smoyer ◽  
Melinda A. Chanley ◽  
Marvin T. Nieman ◽  
Bryce A. Kerlin
Keyword(s):  
Nephrotic Syndrome ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Linear Regression Analysis ◽  
Vena Cava ◽  
Surrogate Marker ◽  
Urinary Protein ◽  
Thrombotic Risk ◽  
Α Angle ◽  
Dose Dependent

Abstract Introduction Thromboembolism is a common complication of nephrotic syndrome (NS). The massive urinary protein loss (proteinuria) of NS results in an acquired, complex hypercoagulopathy. Recent epidemiologic studies have demonstrated that severity of proteinuria in patients with NS is independently predictive for thrombotic risk. Nephrotic-range proteinuria is known to result in acquired deficiencies of antithrombin and free protein S as well as accumulation of procoagulants such as factors V and VIII and von Willebrand factor. However, published cohort studies reveal that the severity of these derangements are quite variable, thus the net effect of proteinuria on thrombotic potential remains unknown. Therefore, we hypothesized that proteinuria severity directly correlates with extent of hypercoagulopathy. Methods PAN (puromycin aminonucleoside)-induced rat NS is known to induce glomerular injury with peak proteinuria at day ∼9 following intravenous injection. Severity of proteinuria and global hemostasis were compared in five groups of male Wistar rats (body weight ∼150 g) receiving a single i.v. injection of PAN at 0 (saline), 25, 50, 100, or 150 mg/kg (n=4/group). Morning spot urines collected on days 0 (before PAN injection) & 9 were analyzed for urinary [protein]:[creatinine] ratio (uPr:Cr). After urine collection on day 9 the rats were anesthetized with isoflurane and blood was collected from the inferior vena cava into 0.32% NaCitrate/1.45 µM Corn Trypsin Inhibitor [final concentrations]. Rotational thromboelastometry (ROTEM) was performed on whole blood within 20 min of collection, using the INTEM (activated intrinsic pathway) assay with and without urokinase (35 ng). Platelet poor plasma (PPP) was prepared from the remaining blood sample. Thrombin generation assays (Technothrombin TGA) were performed on PPP diluted 1:1 with buffer. Results As expected, PAN-treated rats displayed escalating dose-dependent increases in proteinuria at 9 days post-injection (Fig A). The highest dose groups exhibited differential derangements in ROTEM parameters, such that clot formation time (CFT) was decreased and α-angle was increased in rats receiving 100 & 150 mg/kg PAN vs. the sham group (P&lt;0.05; Table). Maximum clot firmness (MCF), amplitude at 10 min (A10) & 20 min (A20), and lysis index at 60 min (LI60) were also significantly higher with the 100 & 150 mg/kg doses, compared to the 0, 25, & 50 groups (P&lt;0.05). Linear regression analysis of uPr:Cr and all measured ROTEM parameters demonstrated that proteinuria was negatively correlated with CFT (R2=0.612, P&lt;0.001), and positively correlated with MCF, A10, A20, & α-angle (respective R2=0.662, R2=0.496, R2=0.536, & R2=0.674; P&lt;0.001 for each parameter). The amount of urokinase-induced fibrinolysis at 60 min was inversely related to proteinuria (LI60; R2=0.674, P&lt;0.001). TGA parameters also exhibited a dose-dependent effect (Table & Fig B). Peak thrombin and endogenous thrombin potential (ETP) were higher in the 100 & 150 mg/kg dose groups vs. 0, 25 & 50 mg/kg groups (P&lt;0.05). There was a positive correlation between uPr:Cr and parameters of thrombin generation (peak thrombin R2=0.751; ETP R2=0.854; P&lt;0.001; Fig C & D). Conclusion These experiments demonstrate that proteinuria severity in the PAN-induced rat NS model is directly proportional to hypercoagulability as assessed by ROTEM and TGA. This suggests that proteinuria may have biologic relevance as an easily measured surrogate marker for hypercoagulopathy severity in NS. Analysis of inducible thrombus formation in PAN-induced rat NS is currently underway to determine the physiologic relevance of these findings. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Direct Oral Anticoagulants Reduce Hypercoagulopathy and Preserve Podocyte Function in an Experimental Model of Glomerular Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 26-26
Author(s):  
Amanda P. Waller ◽  
Katelyn J Wolfgang ◽  
Tasha K Wilkie ◽  
Sagar Bhayana ◽  
Bryce A. Kerlin
Keyword(s):  
Diphtheria Toxin ◽  
Glomerular Disease ◽  
Rodent Model ◽  
Ckd Progression ◽  
Glomerular Diseases ◽  
Podocyte Injury ◽  
Thrombotic Risk ◽  
Randomized Controlled ◽  
Glomerular Proteinuria

Proteinuric glomerular diseases are a leading cause of chronic kidney disease (CKD). Both pre-CKD glomerular disease and established CKD are major risk factors for thrombosis. Glomerular capillary podocyte injury is a key determinant of CKD progression and results in massive proteinuria accompanied by an acquired hypercoagulopathy that drives thrombotic risk. Unfortunately, the routine use of anticoagulant prophylaxis during glomerular proteinuria (GP) remains controversial due to both a lack of agreement regarding indications and no randomized controlled trial data demonstrating both safety and efficacy. We have recently used rat glomerular disease models to reveal that: (1) Proteinuria is directly correlated with hypercoagulopathy and in vivo thrombosis and (2) Thrombin, the key effector enzyme of the coagulation system, directly injures podocytes during proteinuria. What is not yet known is the ability of direct oral anticoagulant (DOAC) therapy to improve these important CKD and thrombosis outcomes. Thus, the aim of the present study is to determine if DOACs simultaneously reduce podocytopathy and enable effective thromboprophylaxis during GP. We hypothesized that DOACs would simultaneously preserve podocyte function and reduce hypercoagulopathy, in a podocyte-specific rodent model of glomerular disease. We utilized the podocin promotor-human diphtheria toxin receptor (pDTR) transgenic rat model to induce highly specific podocyte injury following a single I.P. injection of 50 ng/kg diphtheria toxin (DT). DT-induced proteinuria was subsequently treated daily by oral gavage with 1) Dabigatran (20 mg/kg; Dabi), 2) Rivaroxaban (3 mg/kg; Riva), or 3) Sham (saline) and compared to healthy controls (n=3-6/group). Morning spot urine and citrated plasma samples were collected from each group at day 10 post-DT. Endogenous Thrombin Potential (ETP) was measured by Technothrombin TGA assay, without and with DOAC-Stop reagent. Glomeruli were isolated from the kidney, dissociated into single-cell suspensions and analyzed by flow cytometry following immunofluorescent antibody and TUNEL staining. Both Dabi and Riva significantly reduced proteinuria (Fig A) and podocytopathy (TUNEL positive podocyte fraction; Fig B), while concomitantly correcting elevated ETP levels (Fig C open symbols). Addition of DOAC-Stop (Fig C closed symbols) revealed an insignificant (P=0.18) trend toward partial ETP reduction, consistent with DOAC-mediated reduction of the underlying GP-mediated hypercoagulopathy (via indirect, antiproteinuric effects). In conclusion, dabigatran and rivaroxaban reduce proteinuria and enhance podocyte health in concert with alleviation of the acquired hypercoagulopathy in a podocyte-specific rodent model of glomerular disease. Overall these data suggest DOAC treatment as a novel approach to simultaneously reduce both podocytopathy and thrombotic co-morbidities during glomerular disease. Additional experiments using this model to determine DOAC efficacy on in vivo thrombosis are in progress. Results from these preclinical studies should inform subsequent randomized controlled DOAC trials that may transform care for patients with glomerular disease by mitigating their risk of both CKD progression and thrombosis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Endogenous Thrombin Potential is Directly Correlated with Proteinuria Severity in Both Nephrotic Syndrome Patients and an Animal Model of Nephrotic Syndrome

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4243-4243
Author(s):  
Amanda P Waller ◽  
Marvin T. Nieman ◽  
William E. Smoyer ◽  
Bryce A. Kerlin
Keyword(s):  
Animal Model ◽  
Nephrotic Syndrome ◽  
Animal Models ◽  
Serum Albumin ◽  
Thrombin Generation ◽  
Significant Positive Correlation ◽  
Newly Diagnosed ◽  
Thrombotic Risk ◽  
Positive Correlation ◽  
Endogenous Thrombin Potential

Abstract Introduction Nephrotic syndrome (NS) is characterized by massive proteinuria, hypoalbuminemia, and edema, and is associated with a complex acquired hypercoagulopathy and a high prevalence (~25%) of life-threatening thrombotic complications. However, anticoagulation is associated with a substantial risk for adverse bleeding events. Recently published epidemiology studies suggest that proteinuria severity is directly correlated with thrombotic risk. However, further validation of this candidate biomarker for thrombotic risk requires appropriate validation and adequate pathophysiologic explanation. Results from recent experiments in our laboratory provide further evidence suggesting that proteinuria severity is directly proportional to hypercoagulability (as assessed by ex vivo and in vivo markers of thrombotic capacity) in two well-established animal models of NS (puromycin aminonucleoside (PAN) and Adriamycin (ADR) rats). Thus the aim of the present study was to further determine the relationship between proteinuria severity and endogenous thrombin potential (ETP), using a local cohort of prevalent and newly-diagnosed NS patients. We hypothesized that ETP is directly proportional to severity of proteinuria in human NS. Methods In order to fully examine proteinuria as a potential biomarker of thrombotic risk in NS, and to evaluate the usefulness of rodent NS models to study the NS-associated hypercoagulopathy, we first compared established markers of thrombin generation and global hemostasis in blood collected from the inferior vena cava (IVC) of male Wistar rats with PAN- or ADR- induced nephrosis (see above) exhibiting a range of proteinuria levels (morning spot urines analyzed for [protein:creatinine], uPr:Cr). Pediatric and adult patients from Nationwide Children's Hospital and the Ohio State University's Wexner Medical Center who were not currently taking anticoagulant or antiplatelet agents and had no prior history of VTE were enrolled at the time of diagnosis with NS or during follow-up. Peripheral venous blood from these patients (6 children and 11 adults) was collected into 0.32% NaCitrate/1.45 µM Corn Trypsin Inhibitor [final concentrations] and immediately spun down to platelet poor plasma (PPP). Thrombin generation assays (TGA) were then performed and ETP correlated to corresponding spot urines analyzed for uPr:Cr, as obtained from clinical lab records. This study was approved by the Nationwide Children's Hospital IRB, which has a reciprocity agreement with the OSUWMC IRB. Results When the final results from our experimental animal model studies were merged, the combined TGA data from both PAN and ADR models of NS revealed a significant positive correlation between proteinuria severity and thrombin generation (ETP; R2=0.837, P<0.001; Fig A&B). Thus, we sought to determine if this relationship was also present in human patients with NS. As expected, our local cohort of prevalent and newly-diagnosed NS patients exhibited a range of proteinuria values (median (range): 3.93 mg/mg (0.11-20.2)). Importantly, analysis of thrombin generation in these NS patients demonstrated that proteinuria severity was directly proportional to hypercoagulability (Fig C), such that there was a significant positive correlation between Ur Pr:Cr and ETP (R2=0.251, P=0.041; Fig D). In contrast, although plasma albumin levels were significantly negatively correlated with ETP in our rodent models (R2=0.852, P<0.001), we found no correlation between serum albumin and ETP in our human patient cohort (R2=0.062, P=0.314). Conclusions These data confirm that proteinuria severity in both rodents and humans with NS is directly proportional to ETP, an established marker of thrombin generation that is known to correlate with thrombotic risk. Taken together, these data strongly suggest that proteinuria severity is a promising biomarker for thrombotic risk in NS, and that it may be superior to serum albumin. Animal models of NS may provide outstanding opportunities to further define the molecular pathophysiology by which proteinuria enhances thrombin generation and clinical risk for thrombosis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Limited Role of Antithrombin Deficiency in Hypercoagulopathy Associated with Nephrotic Syndrome

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 294-294
Author(s):  
Eman Abdelghani ◽  
Amanda P. Waller ◽  
Katelyn J Wolfgang ◽  
Bryce A. Kerlin
Keyword(s):  
Nephrotic Syndrome ◽  
Disease Severity ◽  
Thrombin Generation ◽  
Molecular Mechanisms ◽  
Pediatric Patients ◽  
Conflicts Of Interest ◽  
Plasma Albumin ◽  
Steroid Resistant ◽  
Steroid Sensitive ◽  
At Deficiency

Abstract Background: Nephrotic syndrome (NS) is associated with an acquired hypercoagulopathy that drives venous thromboembolic comorbidities. The molecular mechanisms underlying NS-associated hypercoagulopathy are incompletely understood, however previous studies proposed marked urinary loss of antithrombin (AT) as a primary etiology. We have previously demonstrated, in rodent NS models, that there is no correlation between AT antigen and thrombin generation, which we have previously used to characterize the hypercoagulopathy. The objective of this study is to examine the association between AT antigen, AT activity, and hypercoagulopathy along with NS disease severity markers (proteinuria and plasma albumin) in 2 cohorts that include both pediatric and adult NS patients. Methods: Plasma samples were obtained from both cohorts as follows: (1) Samples were obtained on presentation from 147 adult and pediatric patients participating in the NEPTUNE prospective observational cohort study (2) Samples were obtained on presentation and after 7 weeks of glucocorticoid therapy (GC) in steroid sensitive and steroid resistant pediatric patients participating in the PNRC study. AT antigen and activity assays were performed using ELISA and chromogenic assays, respectively. Thrombin generation assay, plasma albumin, and urine protein-to-creatinine ratio were determined as previously described. Results: There was no significant relationship between AT antigen or AT activity and thrombin generation parameters. AT antigen and activity also did not correlate with NS severity markers (plasma albumin and proteinuria) in the NEPTUNE cohort (Figure1). In PNRC pediatric cohort, no significant correlation was found between AT antigen and thrombin generation or other disease severity markers. However, AT activity was significantly associated with both plasma albumin and endogenous thrombin potential at presentation (Figure 2) and at follow-up. Interestingly, GC therapy significantly improved AT activity in steroid-sensitive but not steroid-resistant NS patients (Figure 3). A comprehensive literature review and meta-analysis was also performed which revealed that clinically significant AT deficiency (&lt;0.7 IU/mL) is uncommon in both pediatric and adult NS patients. Conclusion: AT antigen does not correlate with thrombin generation-defined hypercoagulopathy in adult or pediatric NS patients. AT activity does correlate with hypercoagulopathy in pediatric NS patients and improves significantly with successful remission inducing therapy. These data suggest that pediatric NS patients may have a qualitative, but not quantitative, AT deficiency that is responsive to therapy. The mechanism underlying this qualitative deficiency should be characterized in future studies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Thrombin Induces Protease Activated Receptor (PAR)-3/-4 Interaction in Human Podocytes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3488-3488
Author(s):  
Sharma Ruchika ◽  
Amanda P Waller ◽  
Shipra Agrawal ◽  
Berend H Isermann ◽  
William E Smoyer ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Molecular Mechanisms ◽  
Thrombin Inhibitor ◽  
Dependent Manner ◽  
Glomerular Injury ◽  
Podocyte Injury ◽  
Proximity Ligation ◽  
Control Peptide ◽  
Activation Peptide

Abstract INTRODUCTION Nephrotic Syndrome, one of the most common forms of glomerular disease, is characterized by massive proteinuria with structural and functional injury of specialized glomerular cells called podocytes.We and others have shown that thrombin generation is enhanced in nephrotic syndrome. Recent in vitro studies have demonstrated that exposure to high concentrations of thrombin can injure podocytes, suggesting that thrombin may exacerbate glomerular injury. However, the molecular mechanisms by which thrombin induces podocyte injury are not yet known. Thrombin activates platelets, leukocytes, and other cells via the protease activated receptor (PAR) system. Thus, we hypothesized that thrombin exacerbates glomerular injury by enhancing podocyte apoptosis in a PAR-dependent manner. METHODS Experiments were performed with differentiated, conditionally immortalized human podocytes. After 36 hours of thrombin (20nM) exposure podocyte apoptosis was determined by TUNEL assay. Specific PAR antibodies and activating peptides were utilized to determine which PARs mediate thrombin-induced podocyte apoptosis. Specific PAR antibodies (ab) included hPAR1ab (ATAP2), hPAR2 ab (SAM11), hPAR3 ab (8E8), hPAR4 ab (H-120). Activation peptides (AP) included PAR1 AP (TFFLRNPNDKNH2), PAR2 AP (SLIGRLNH2); PAR3 AP (TFRGAPOH); PAR4 AP (AYPGKFNH2) and a control peptide (FSLLRNNH2). Phalloidin assays were used to evaluate structural changes in the actin-cytoskeleton as a marker of podocyte stress. Proximity ligation assays (PLAs) allow detection of protein-protein interactions at the molecular level. PLAs were performed on podocytes grown in an 8-well chamber slide system. PLA was carried out using the DUOLinkTM kit (Sigma-Aldrich) according to the manufacturer's instructions and as described previously. Oligonucleotide-conjugated secondary antibodies against mouse and rabbit primary antibodies were used and protein-protein associations were detected by microscopy as bright red dots. One-Way ANOVA and t-tests were used to determine statistical significance (SigmaPlotTM). RESULTS Thrombin exposure induced a significant increase in apoptosis of human podocytes from 1.8% to 42.87% (p <0.05). Blockade of PAR-3 or PAR-4 resulted in a significant decrease in apoptosis [9.2% with hPAR-3 ab and 11.7% with hPAR-4ab] (p <0.05). Inhibition of thrombin enzymatic activity with hirudin, a direct thrombin inhibitor, also resulted in a decrease in apoptosis to 2.1% (p <0.05). In comparison to a control peptide, PAR-4 activation peptide significantly increased apoptosis from 1.7% to 40.1% (p <0.05), while PAR-3 activation peptide did not. Analogous results were seen with the phalloidin assay. Thrombin caused actin cytoskeletal changes, while PAR-3 and PAR-4 blockade ameliorated these changes. In addition, only activation with PAR-4 activating peptide resulted in loss of actin stress fibers. These findings suggest that, in human podocytes, thrombin signaling is mediated via PAR-4, in a PAR-3-dependent manner. Thus to directly assess this hypothesis, we performed proximity ligation assays with PAR-3/PAR-4 antibodies in the presence and absence of thrombin which revealed the presence of PAR-3/PAR-4 interactions in thrombin exposed podocytes, but not in control podocytes. The quantification of the in situ PLA signals per cell was performed with a BZ900 microscope and software (Keyence, Osaka, Japan) equipped with automated 'dynamic cell count" feature (p =0.03). CONCLUSIONS Thrombin-induced injury is mediated in a PAR-dependent fashion in human podocytes. Specifically, in this in vitro model, thrombin-induced apoptosis appears to be mediated in a PAR-3/-4 dependent manner. Furthermore, these data suggest that thrombin induced podocyte injury may be mediated in a manner dependent on interactions between PAR-3 and -4. This is a novel finding, as PAR-3/-4 interactions are known to mediate mouse platelet signaling, but have not been previously described in human cells. Collectively, our findings suggest that interrupting thrombin-mediated podocyte injury may offer a novel therapeutic approach to reduce podocyte injury during nephrotic syndrome. Disclosures No relevant conflicts of interest to declare.

Recombinant Variants of Tissue-Type Plasminogen Activator Containing Amino Acid Substitutions in the Finger Domain

1992 ◽  
Vol 68 (06) ◽  
pp. 672-677 ◽  
Author(s):  
Hitoshi Yahara ◽  
Keiji Matsumoto ◽  
Hiroyuki Maruyama ◽  
Tetsuya Nagaoka ◽  
Yasuhiro Ikenaka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Half Life ◽  
Tissue Type ◽  
Clot Lysis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.

A Plasma Clot Lysis Assay Based on the Release of Fibrin Degradation Products: Application to the Diagnosis of Hypofibrinolytic States

1990 ◽  
Vol 63 (01) ◽  
pp. 076-081 ◽  
Author(s):  
Pascale Gaussem ◽  
Sophie Gandrille ◽  
Pascale Molho-Sabatier ◽  
Loïc Capron ◽  
Jean-Noël Fiessinger ◽  
...  
Keyword(s):  
Degradation Products ◽  
Venous Occlusion ◽  
Lower Limbs ◽  
Clot Lysis ◽  
Plasma Clot ◽  
Vein Thrombosis ◽  
Fibrin Degradation Products ◽  
Before And After ◽  
Euglobulin Clot Lysis Time ◽  
Pai 1

SummaryUsing a monoclonal antibody-based assay, we measured the fibrin degradation product release in the supernatant of plasma clots obtained before and after venous occlusion (VO) in 30 patients with definite or suspected vascular thrombosis (19 definite and 2 suspected deep vein thrombosis, 6 recurrent superficial thrombophlebitis, 3 arterial occlusions of lower limbs). tPA and PAI-1 concentrations were determined using ELISA assays; the post-occlusion values were corrected for haemoconcentration. The increase in tPA during VO was correlated with haemoconcentration (r = 0.74), but 3 patients had ineffective VO (<2% increase in proteins). The fibrinolytic response to VO was evaluated using the shortening of the time necessary for the release of 200 μg of fibrin degradation products per mg of fibrinogen (Δ T 200). Two among the 27 patients with effective VO were bad responders with a Δ T 200 <3 h (whereas all the others had Δ T 200 >10 h). These patients had respectively a deficient tPA release (Δ tPA = 1 ng/ml) and an elevated PAI-1 level at rest (33 ng/ml). Several other patients were bad responders in terms of tPA release or of shortening of the euglobulin clot lysis time but they had a normal Δ T 200. This plasma clot test reflects the ability of free tPA to bind to fibrin (the amount of which depends on the level of tPA and PAI-1), and may be useful in the diagnosis of a hypofibrinolytic state.

Functional Properties of p-Anisoylated Plasmin-Staphylokinase Complex

1993 ◽  
Vol 70 (02) ◽  
pp. 326-331 ◽  
Author(s):  
H R Lijnen ◽  
B Van Hoef ◽  
R A G Smith ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Rate Constant ◽  
Time Course ◽  
Bolus Injection ◽  
Similar Rate ◽  
Clot Lysis ◽  
Lag Phase ◽  
Plasma Clot ◽  
Rapid Clearance ◽  
Stoichiometric Complex

SummaryThe kinetic and fibrinolytic properties of a reversibly acylated stoichiometric complex between human plasmin and recombinant staphylokinase (plasmin-STAR complex) were evaluated. The acylation rate constant of plasmin-STAR by p-amidinophenyl-p’-anisate-HCI was 52 M-1 s-1 and its deacylation rate constant 1.2 × 10-4 s-1 (t½ of 95 min) which are respectively 50-fold and around 3-fold lower than for the plasmin-streptokinase complex. The acylated complex was stable as evidenced by binding to lysine-Sepharose. However, following an initial short lag phase, the acylated plasmin-STAR complex activated plasminogen at a similar rate as the unblocked complex, whereas the acylated plasmin-streptokinase complex did not activate plasminogen. These findings indicate that STAR, unlike streptokinase, dissociates from its acylated complex with plasmin in the presence of excess plasminogen. In agreement with this hypothesis, the time course of the lysis of a 125I-fibrin labeled plasma clot submerged in citrated human plasma, is similar for acylated plasmin-STAR, unblocked plasmin-STAR and free STAR (50% clot lysis in 2 h requires 12 nM of each agent). The plasma clearances of STAR-related antigen following bolus injection in hamsters were 1.0 to 1.5 ml/min for acylated plasmin-STAR, unblocked plasmin-STAR and free STAR, as a result of short initial half-lives of 2.0 to 2.5 min.The dissociation of the anisoylated plasmin-STAR complex and its consequent rapid clearance suggest that it has no apparent advantages as compared to free STAR for clinical thrombolysis.

Sign in / Sign up

Export Citation Format

Share Document