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.

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.

Abstract W P366: Hypothermia Does Not Affect Tissue Plasminogen Activator Activity in Acute Ischemic Stroke Patients as Measured by Thromboelastography

Stroke ◽  
2014 ◽  
Vol 45 (suppl_1) ◽  
Author(s):  
Joancy M Archeval-Lao ◽  
Hope Moser ◽  
Stephen A Riney ◽  
Jorge Kawano-Castillo ◽  
Stephanie A Parker ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Acute Ischemic Stroke ◽  
Venous Blood ◽  
Clot Formation ◽  
Clot Lysis ◽  
Stroke Patients ◽  
Blood Samples ◽  
Lytic Effect ◽  
Tissue Plasminogen ◽  
Coagulation Status

Background: Currently, t-PA is the only FDA approved treatment for acute ischemic stroke (AIS). Supplementing t-PA with therapeutic hypothermia is being evaluated, but cooler temperatures may affect the enzymatic activity of t-PA. Thromboelastography (TEG) determines coagulation status in whole blood, and has detected hypercoagulability in AIS patients compared to healthy controls. Using TEG, we evaluated the effect of variable degrees of hypothermia on clot formation and lysis in AIS patients receiving t-PA. Methods: Between June 2012 -July 2013, venous blood from 18 AIS patients receiving t-PA within 4.5 hours of symptom onset was collected prior to and 10 minutes after t-PA bolus. Blood samples were analyzed by TEG at 30°C, 33°C, and 37°C. The variables of interest were R (initiation of clot formation), K (speed of clot strengthening), Angle α (rate of clot formation), and LY30 (percentage of clot lysis over 30 minutes) (see figure). All statistical analyses were performed using SAS 9.3. Results: Baseline R averaged 6.0, 5.6, and 4.6 minutes at 30°C, 33°C, and 37°C (p=0.02),K averaged 2.5, 2.3, and 1.4 (p=0.01),and Angle averaged 59.1, 62.4, and 69.3(p=0.01), indicating slower clotting at lower temperatures. Post t-PA LY30 averaged 93.9, 93.9, 89.8 (p= 0.61, N=18) indicating no effect on t-PA lytic activity at lower temperatures. Conclusions: Our data suggest that hypothermia progressively slows clot formation in AIS patients but has no effect on the lytic effect of t-PA as measured by TEG.

scholarly journals Thrombin‐generating potential, plasma clot formation, and clot lysis are impaired in patients with bleeding of unknown cause

2019 ◽  
Vol 17 (9) ◽  
pp. 1478-1488 ◽  
Author(s):  
Stefanie Hofer ◽  
Cihan Ay ◽  
Judit Rejtö ◽  
Alisa S. Wolberg ◽  
Helmuth Haslacher ◽  
...  
Keyword(s):  
Clot Formation ◽  
Clot Lysis ◽  
Plasma Clot

scholarly journals Plasma clot formation and clot lysis to compare effects of different anticoagulation treatments on hemostasis in patients with atrial fibrillation

2018 ◽  
Vol 18 (3) ◽  
pp. 325-336 ◽  
Author(s):  
Oliver Königsbrügge ◽  
Günter Weigel ◽  
Peter Quehenberger ◽  
Ingrid Pabinger ◽  
Cihan Ay
Keyword(s):  
Atrial Fibrillation ◽  
Clot Formation ◽  
Clot Lysis ◽  
Plasma Clot

scholarly journals Correlation of Plasma MMP-1 and TIMP-1 Levels and the Colonic Mucosa Expressions in Patients with Ulcerative Colitis

2009 ◽  
Vol 2009 ◽  
pp. 1-5 ◽  
Author(s):  
Ying-De Wang ◽  
Xiao-Yan Tan ◽  
Ke Zhang
Keyword(s):  
Ulcerative Colitis ◽  
Disease Severity ◽  
Plasma Levels ◽  
Colonic Mucosa ◽  
Venous Blood ◽  
Normal Subjects ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Reverse Transcription Pcr ◽  
Matrix Metalloproteinase 1 ◽  
The Relationship

Background. Both plasma and mucosal levels of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) have been shown to be independently correlated with ulcerative colitis (UC), but their relationship with each other and to disease severity remains unclear. This study aims to evaluate the relationship between colonic mucosal and plasma levels of MMP-1 and TIMP-1 with each other and with the severity of ulcerative colitis (UC).Methods. Colonic mucosal lesions and venous blood samples were collected from 30 patients with UC and 15 normal subjects. Real-time reverse transcription-PCR and immunohistochemistry were used to determine colonic mucosal MMP-1 and TIMP-1 expression; ELISA was used to measure plasma levels of MMP-1 and TIMP-1.Results. Expression of colonic mucosal and plasma MMP-1 and TIMP-1 in patients with UC was significantly higher than that of controls (P<.05), and was positively correlated with disease severity (P<.05). Plasma MMP-1 and TIMP-1 levels were well correlated with their corresponding expression in colonic mucosa (P<.05,r=0.805 and 0.908).Conclusion. Plasma MMP-1 and TIMP-1 levels reflect their colonic mucosal expression to some extent in patients with UC. Plasma MMP-1 and TIMP-1, in particular, demonstrate the potential to become biomarkers to clinically diagnose UC, predict its severity, and guide further therapy.

Abstract 354: Clots Are Potent Triggers of Inflammatory Cell Gene Expression: Indications for Timely Fibrinolysis

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Robert A Campbell ◽  
Adriana Vieira-de-Abreu ◽  
Jesse W Rowley ◽  
Zechariah G Franks ◽  
Matthew T Rondina ◽  
...  
Keyword(s):  
Gene Expression ◽  
Whole Blood ◽  
Inflammatory Responses ◽  
Vascular Diseases ◽  
Clot Formation ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Plasma Clot ◽  
Monocyte Chemoattractant Protein 1

Objective: Blood vessel wall damage often results in the formation of a fibrin clot that traps inflammatory cells, including monocytes. The effect of clot formation and subsequent lysis on the expression of monocyte-derived genes involved in the development and progression of ischemic stroke and other vascular diseases, however, is unknown. Determine if clot formation and lysis regulates the expression of human monocyte-derived genes that modulate vascular diseases. Approach and Results: We performed Next Generation RNA sequencing on monocytes extracted from whole blood clots. Thousands of mRNAs were differentially expressed by monocytes from clotted versus unclotted whole blood, including upregulation of interleukin 8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1). Clotted plasma also increased expression of IL-8 and MCP-1, which far exceeded responses observed in LPS-stimulated monocytes. Upregulation of IL-8 and MCP-1 occurred in a thrombin-independent, but fibrin-dependent manner. Fibrinolysis initiated shortly after plasma clot formation (i.e., 1-2 hours) reduced the synthesis of IL-8 and MCP-1, while delayed fibrinolysis was far less effective. Consistent with these in vitro models, monocytes embedded in unresolved thrombi from patients undergoing thrombectomy stained positively for IL-8 and MCP-1. Conclusions: These findings demonstrate that clots are potent inducers of monocyte gene expression, and that timely fibrinolysis attenuates inflammatory responses. Dampening of inflammatory gene expression by timely clot lysis may contribute to the clinically-proven efficacy of fibrinolytic drug treatment within hours of stroke onset.

Abstract T P57: TEG Does Not Correlate with Clot Subtype or Clinical Response to tPA

Stroke ◽  
2015 ◽  
Vol 46 (suppl_1) ◽  
Author(s):  
Mark M McDonald ◽  
Jeremy Wetzel ◽  
Andrea Elliott ◽  
Ritvij Bowry ◽  
Jorge F Kawano-Castillo ◽  
...  
Keyword(s):  
Clinical Response ◽  
Venous Blood ◽  
Interactive Effects ◽  
Clot Formation ◽  
Clot Lysis ◽  
Future Research ◽  
Number Of Patients ◽  
Coagulation Status ◽  
Multiple Variables

Background: Thromboelastography (TEG) measures coagulation status in venous blood. tPA thrombolysis is affected by multiple variables including whether clots are erythrocyte or platelet-rich. We hypothesized that TEG would correlate with clot subtype and response to tPA including rapid clinical improvement (RCI), recanalization, and hemorrhagic transformation (HT). Methods: 176 acute ischemic stroke patients between 11/09 and 06/14 treated with tPA were prospectively enrolled. Venous blood for TEG was drawn before and 10 minutes after tPA bolus. Pre-tPA measures of speed and strength of clot formation (R, Delta, K, Angle, MA, and G) and post-tPA measure of clot lysis (LY30) were analyzed. Hyperdense artery (HDA) on CT was a biomarker for erythrocyte-rich clot. RCI was defined as 8 point improvement on NIHSS or total NIHSS of 0,1 at 36 hours. HT was defined as any blood on follow up imaging within 36 hours. Recanalization was defined as resolution of baseline vascular occlusion on follow up CT or MR angiogram within 36 hours. Multivariable linear regression models compared TEG parameters after adjusting for potential confounding and interactive effects. Results: No differences in pre- or post-tPA TEG were found between patients with (n=32) or without (n=102) RCI. Also, there was no correlation between TEG and HDA on CT. Clot strength was decreased in patients with recanalization (lower MA and G, p = 0.02 and p = 0.03). Clotting was slightly prolonged (longer delta, p = 0.046) in patients with HT. Discussion: Our data do not show a robust association between TEG and clot subtype or clinical response to tPA. It is likely that arterial clot lysis is determined by factors unrelated to coagulation status as measured by TEG in the venous circulation. Though we found a correlation between TEG and recanalization, the number of patients with recanalization data was too small to detect an effect on clinical outcome. Similar to our previous findings, speed of clot formation may be related to risk of bleeding. Conclusion: It is unlikely that TEG will be useful in guiding tPA therapy. Future research should focus on local arterial influences on clot lysis. Further study of TEG in hemorrhage is indicated.

scholarly journals FC 015LACK OF PLASMINOGEN RELATES TO A HYPERCOAGULABLE STATE IN MICE WITH EXPERIMENTAL NEPHROTIC SYNDROME

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Mengyun Xiao ◽  
Stefanie Hammer ◽  
Wissam A Khalel ◽  
Lisann Pelzl ◽  
Bernhard N Bohnert ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Alpha Angle ◽  
Formation Time ◽  
Clot Formation ◽  
Hypercoagulable State ◽  
Clot Lysis ◽  
Activity Measurement ◽  
Clotting Time ◽  
Plasminogen Deficiency ◽  
Clot Formation Time

Abstract Background and Aims Urinary excretion of the fibrinolytic enzyme plasminogen has been identified as a characteristic feature of nephrotic syndrome (NS) in both human and experimental mouse models. Lack of plasminogen may lead to a hypercoagulable state and thrombosis, and mice with plasminogen deficiency have been shown to suffer from developing spontaneous thrombosis. However, the role of plasminogen in hypercoagulable state and thrombosis in an experimental nephrotic syndrome has not been investigated before. Method We investigated the relationship between plasminogen and a hypercoagulable state in an inducible nephrotic mouse model with conditional podocyte-specific podocin deletion (Nphs2Δipod * Plg+/+, n=12). The Nphs2Δipod mice with constitutive plasminogen knockout were used as negative plasminogen control (Nphs2Δipod * Plg-/-, n=15). All mice received a daily oral doxycycline administration for 2 weeks for NS induction. The last day of doxycycline treatment was set as day 0. Spot urine was collected daily for proteinuria and urinary plasmin activity measurement. Citrate blood was collected from each mouse before induction of NS, 7 days and 21 days after induction, respectively (Nphs2Δipod * Plg+/+ mice, n=4/timepoint; Nphs2Δipod * Plg-/- mice, n=5/timepoint). A global assessment of coagulation (extrinsic coagulation test, EX test) was examined by ClotPro® system. Besides, fibrinolysis was tested by adding tissue plasminogen activator (TPA test). Results According to the EX test, uninduced mice with plasminogen deficiency showed a significantly reduced clotting time (CT, Plg-/- vs. Plg+/+, 42 ± 1s vs. 54 ± 4s, p=0.0213), and decreased clot formation time (CFT, Plg-/- vs. Plg+/+, 82 ± 5s vs. 206 ± 28s p&lt;0.0001) with a larger alpha-angle (Plg-/- vs. Plg+/+, 75 ± 1° vs. 66 ± 2°, p=0.0041). The maximum clot firmness (MCF) was significantly increased in uninduced plasminogen knockout mice (Plg-/- vs. Plg+/+, 45 ± 0.5mm vs. 32 ± 2.5mm p&lt;0.0001). According to the TPA test, uninduced Nphs2Δipod *Plg-/-mice had a faster velocity of clot formation (α-angle, 75.6 ± 0.2° vs. 66.5 ± 1.6°, p=0.0254) and did not show any clot lysis in contrast to uninduced nphs2Δipod * plg+/+mice. After induction of NS, both Nphs2Δipod * Plg-/-mice and Nphs2Δipod * Plg+/+ mice developed massive proteinuria to a comparable extent (Plg-/- vs. Plg+/+on day 21, 218 ± 46mg/mg crea vs. 203 ± 28mg/mg crea), and plasminuria was detectable in nephrotic nphs2Δipod * plg+/+ mice. With the ongoing loss of plasminogen in the urine, CT and CFT was significantly reduced in nephrotic Nphs2Δipod * Plg+/+ mice. MCF was significantly increased with a faster velocity of clot formation measured by both the EX and TPA test. Moreover, clot lysis was significantly reduced. In nephrotic nphs2Δipod *plg-/-mice at day 21, there was also a tendency towards a decrease in CT, CFT and an increased velocity of clot formation. According to both EX and TPA test, there were no significant differences between the genotypes in nephrotic mice any more. Conclusion The results highlight that loss of plasminogen in the nephrotic state contributes to a hypercoagulable state with shortened clotting time, clot formation time, increased clot firmness, and most strikingly, loss of clot lysis. Changes in nephrotic wild-type mice were similar to mice with constitutive plasminogen deficiency, indicating that loss of plasminogen plays a role in the hypercoagulable state of nephrotic syndrome.

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