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

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.

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Nephrotic Syndrome Clots Are Resistant to Fibrinolysis Despite Elevated Plasmin Generation

Blood ◽

10.1182/blood.v128.22.3767.3767 ◽

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.

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BβArg448Lys polymorphism is associated with altered fibrin clot structure and fibrinolysis in type 2 diabetes

Thrombosis and Haemostasis ◽

10.1160/th16-07-0554 ◽

2017 ◽

Vol 117 (02) ◽

pp. 295-302 ◽

Cited By ~ 1

Author(s):

Katie A. Greenhalgh ◽

Mark W. Strachan ◽

Saad Alzahrani ◽

Paul D. Baxter ◽

Kristina F. Standeven ◽

...

Keyword(s):

Type 2 Diabetes ◽

Fibrin Clot ◽

Clot Lysis ◽

Plasma Clot ◽

Lysis Time ◽

Increased Risk ◽

Fibrin Network ◽

Clot Lysis Time ◽

Fibrin Clots

SummaryBoth type 2 diabetes (T2DM) and Bß448Lys variant of fibrinogen are associated with dense fibrin clots, impaired fibrinolysis and increased cardiovascular risk. It was our objective to investigate whether BßArg448Lys adds to vascular risk by modulating fibrin network structure and/or fibrinolysis in diabetes. The primary aim was to study effects of BßArg448Lys on fibrin network characteristics in T2DM. Secondary aims investigated interactions between gender and BßArg448Lys substitution in relation to fibrin clot properties and vascular disease. Genotyping for BßArg448Lys and dynamic clot studies were carried out on 822 T2DM patients enrolled in the Edinburgh Type 2 Diabetes Study. Turbidimetric assays of individual plasma samples analysed fibrin clot characteristics with additional experiments conducted on clots made from purified fibrinogen, further examined by confocal and electron microscopy. Plasma clot lysis time in Bß448Lys was longer than Bß448Arg variant (mean ± SD; 763 ± 322 and 719 ± 351 seconds [s], respectively; p<0.05). Clots made from plasma-purified fibrinogen of individuals with Arg/Arg, Arg/Lys and Lys/Lys genotypes showed differences in fibre thickness (46.75 ± 8.07, 38.40 ± 6.04 and 25 ± 4.99 nm, respectively; p<0.001) and clot lysis time (419 ± 64, 442 ± 87 and 517 ± 65 s, respectively; p=0.02), directly implicating the polymorphism in the observed changes. Women with Bß448Lys genotype had increased risk of cerebrovascular events and were younger compared with Bß448Arg variant (67.2 ± 4.0 and 68.2 ± 4.4 years, respectively; p=0.035). In conclusion, fibrinogen Bβ448Lys variant is associated with thrombotic fibrin clots in diabetes independently of traditional risk factors. Prospective studies are warranted to fully understand the role of BβArg448Lys in predisposition to vascular ischaemia in T2DM with the potential to develop individualised antithrombotic management strategies.

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A Universal Heparin Antidote with Negligible Effect on Fibrin(ogen) and Plasma Coagulation

Blood ◽

10.1182/blood.v124.21.4231.4231 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4231-4231

Author(s):

Manu Thomas Kalathottukaren ◽

Rajesh A Shenoi ◽

Lai FL Benjamin ◽

Fred Rosell ◽

Jayachandran N Kizhakkedathu ◽

...

Keyword(s):

Binding Affinity ◽

Lag Time ◽

Fibrin Clot ◽

Coagulation System ◽

Clot Lysis ◽

Plasma Clot ◽

Fibrin Polymerization ◽

Maximum Absorbance ◽

Fiber Size ◽

Clot Structure

Abstract Background and Objective Anticoagulants play a pivotal role in the treatment of thromboembolic disorders. Haemorrhage in surgical patients receiving anticoagulants is a major concern. Antidotes are administered to counteract anticoagulation and to restore normal hemostasis. To date, protamine sulphate (PS), a cationic polypeptide is the only clinically approved antidote for unfractionated heparin. PS has toxic side effects and limitations. Inability of PS to completely reverse low molecular weight heparins and fondaparinux is due to its low binding affinity to these drugs. However, PS interacts with coagulation proteins such as fibrinogen to form aggregates which leads to cardiovascular adverse effects. Recently, we developed a synthetic universal heparin reversal agent (UHRA) with high binding affinity to heparins. In vivo studies revealed that UHRA completely reverse the activity of all clinical available parenteral anticoagulants and is nontoxic. This study aims to demonstrate the nontoxic nature of UHRA by assessing its influence on fibrinogen, fibrin clot architecture, plasma clotting and clot lysis. Methods UHRA was developed by incorporating tertiary amine based heparin binding groups on a dendritic hyperbranched polyglycerol scaffold and capping it with methoxy polyethylene glycol chains. Recalcification and tissue factor (TF) initiated turbidimetric plasma clotting assays was performed to understand the impact of UHRA on coagulation system. The interaction of UHRA on fibrinogen was investigated by fibrinogen aggregation assay, fibrin polymerization assay and by spectroscopic analysis (fluorescence and circular dichroism (CD)). The influence of UHRA on fibrin clot architecture was evaluated by scanning electron microscopy (SEM).The anticoagulant neutralization (heparins) by UHRA was studied by fluorogenic thrombin generation assay (TGA) in human platelet-rich plasma (PRP). The lysis of TF-induced plasma clot containing UHRA or PS exposed to exogenous tissue plasminogen activator (t-PA) was studied by turbidimetric assay. Results and discussion Results from the plasma clotting assays showed that UHRA did not alter the clotting parameters compared to PS (TF initiated lag time and maximum absorbance, control vs UHRA 200 mcg/mL, p=0.21 and 0.16, respectively; lag time and maximum absorbance in recalcification, control vs UHRA 200mcg/mL, p=0.08 and 0.13, respectively) suggesting that UHRA has no effect on coagulation system at the concentration studied (Figure 1). Unlike protamine, the fibrinogen aggregation and fibrin polymerization assay was not influenced by UHRA over a broad range of concentrations from 0.05mg/mL to 1mg/mL. Together with tryptophan fluorescence quenching measurements (Figure 2) and fibrinogen secondary structure measurements corroborates that UHRA is not interacting with fibrinogen. The results are quite different from PS and other synthetic cationic polymers which interact with fibrinogen eliciting aggregation and conformational changes. Fibrin clots generated in presence of UHRA (even at 0.5 mg/mL) showed similar structure and fiber size remains same as normal fibrin clot (control vs UHRA 0.5 mg/mL clot, p= 0.12) (Figure 3). On the other hand, fibrin clots formed in the presence of 0.05mg/mL PS (clinical dose) increased the fiber size and changed the clot structure dramatically (control vs PS 0.05mg/mL clot, p< 0.0001). Our plasma clot lysis studies in the presence of exogenous t-PA demonstrate that UHRA did not enhance clot degradation unlike protamine. UHRA restored thrombin levels in anticoagulated PRP (heparinized) demonstrating the efficacy. Conclusion and significance Our studies demonstrate that universal heparin antidote, UHRA, has negligible impact on fibrinogen, fibrin polymerization, clot structure, clot degradation and the coagulation system revealing their excellent hemocompatibility compared to protamine. Our results support the fact that UHRA could be an ideal antidote to restore hemostasis following invasive surgical procedures and to address bleeding complications by heparin based anticoagulants. Figure 1 Figure 1. Figure 3 Figure 3. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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Thrombin generation and fibrin clot structure after vitamin D supplementation

Endocrine Connections ◽

10.1530/ec-19-0429 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1447-1454 ◽

Cited By ~ 1

Author(s):

Marc Blondon ◽

Emmanuel Biver ◽

Olivia Braillard ◽

Marc Righini ◽

Pierre Fontana ◽

...

Keyword(s):

Vitamin D ◽

Vitamin D Deficiency ◽

Thrombin Generation ◽

Fibrin Clot ◽

Clot Lysis ◽

High Dose ◽

Severe Vitamin ◽

Lysis Time ◽

Clot Structure ◽

Clot Lysis Time

Objective Vitamin D deficiency is associated with increased risks of arterial and venous cardiovascular events. Hypothetically, supplementation with vitamin D may lead to a less prothrombotic phenotype, as measured by global coagulation assays and fibrin clot structure. Methods In this prospective cohort study, we enrolled adult outpatients attending the Primary Care Division of the Geneva University Hospitals with a severe vitamin D deficiency (25-hydroxyvitamin-D3 (25-OHD) <25 nmol/L), excluding obese patients or with a recent acute medical event. We evaluated changes in coagulation times, thrombin generation assay, clot formation and clot lysis time, 25-OHD and parathormone before and 1–3 months after cholecalciferol oral supplementation with one-time 300,000 IU then 800 IU daily. Paired t-tests with a two-sided alpha of 0.05 compared absolute mean differences. Results The 48 participants had a mean age of 43.8 ± 13.8 years. After supplementation, 25-OHD levels increased from 17.9 ± 4.6 nmol/L to 62.5 ± 20.7 nmol/L 6.4 ± 3.0 weeks after inclusion. Endogenous thrombin potential and thrombin generation peak values both decreased significantly (−95.4 nM × min (95%CI −127.9 to −62.8), P < 0.001; −15.1 nM (−23.3 to −6.8), P < 0.001). The maximum absorbance by turbidimetry decreased significantly (P = 0.001) after supplementation. There was no change in clot lysis time, coagulation times or plasminogen activator inhibitor-1 and homocysteine levels. Conclusions In severe vitamin D deficiency, a high-dose cholecalciferol supplementation was associated with a reduction in thrombin generation and an average decreased number of fibrin protofibrils per fibers and fibrin fiber size measured by turbidimetry. This suggests that severe vitamin D deficiency may be associated with a potentially reversible prothrombotic profile.

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

Blood ◽

10.1182/blood.v126.23.1106.1106 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1106-1106 ◽

Cited By ~ 1

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.

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Effect of Tranexamic Acid on Coagulation and Fibrin Clot Properties in Children Undergoing Craniofacial Surgery

Thrombosis and Haemostasis ◽

10.1055/s-0039-3402762 ◽

2020 ◽

Vol 120 (03) ◽

pp. 392-399 ◽

Cited By ~ 1

Author(s):

Christian Fenger-Eriksen ◽

Alexander D'Amore Lindholm ◽

Lisbeth Krogh ◽

Tobias Hell ◽

Martin Berger ◽

...

Keyword(s):

Data Analysis ◽

Blood Loss ◽

Tranexamic Acid ◽

Thrombin Generation ◽

Blood Clot ◽

Fibrin Clot ◽

Clot Formation ◽

Clot Lysis ◽

Clot Strength ◽

Clot Structure

Abstract Objective Craniosynostosis surgery in small children is very often associated with a high blood loss. Tranexamic acid (TXA) reduces blood loss during this procedure, although the potential underlying coagulopathy in these children is not known in detail. Objective was to determine the nature of any coagulopathy found during and after craniosynostosis surgery and to characterize the effect of TXA on fibrin clot formation, clot strength, and fibrinolysis. Materials and Methods Thirty children received either TXA (bolus dose of 10 mg/kg followed by 8 hours continuous infusion of 3 mg/kg/h) or placebo. Dynamic whole blood clot formation assessed by thromboelastometry, platelet count, dynamic thrombin generation/thrombin-antithrombin, clot lysis assay, and fibrinogen/factor XIII (FXIII) levels were measured. Additionally, clot structure was investigated by real-time live confocal microscopy and topical data analysis. Results Increased ability of thrombin generation was observed together with a tendency toward shortened activated partial thromboplastin time and clotting time. Postoperative maximum clot firmness was higher among children receiving TXA. FXIII decreased significantly during surgery in both groups.Resistance toward tissue plasminogen activator-induced fibrinolysis was higher in children that received TXA, as evidenced by topical data analysis and by a significant longer lysis time. Fibrinogen levels were higher in the TXA group at 24 hours. Conclusion A significant coagulopathy mainly characterized by changes in clot stability and not parameters of thrombin generation was reported. Tranexamic acid improved clot strength and reduced fibrinolysis, thereby avoiding reduction in fibrinogen levels.

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Aβ delays fibrin clot lysis by altering fibrin structure and attenuating plasminogen binding to fibrin

Blood ◽

10.1182/blood-2011-11-389668 ◽

2012 ◽

Vol 119 (14) ◽

pp. 3342-3351 ◽

Cited By ~ 58

Author(s):

Daria Zamolodchikov ◽

Sidney Strickland

Keyword(s):

Structural Changes ◽

Fibrin Clot ◽

Brain Parenchyma ◽

Amyloid Peptide ◽

Clot Lysis ◽

Fibrin Network ◽

Β Amyloid Peptide ◽

Clot Structure ◽

The Brain ◽

Fibrin Clots

Abstract Alzheimer disease is characterized by the presence of increased levels of the β-amyloid peptide (Aβ) in the brain parenchyma and cerebral blood vessels. This accumulated Aβ can bind to fibrin(ogen) and render fibrin clots more resistant to degradation. Here, we demonstrate that Aβ42 specifically binds to fibrin and induces a tighter fibrin network characterized by thinner fibers and increased resistance to lysis. However, Aβ42-induced structural changes cannot be the sole mechanism of delayed lysis because Aβ overlaid on normal preformed clots also binds to fibrin and delays lysis without altering clot structure. In this regard, we show that Aβ interferes with the binding of plasminogen to fibrin, which could impair plasmin generation and fibrin degradation. Indeed, plasmin generation by tissue plasminogen activator (tPA), but not streptokinase, is slowed in fibrin clots containing Aβ42, and clot lysis by plasmin, but not trypsin, is delayed. Notably, plasmin and tPA activities, as well as tPA-dependent generation of plasmin in solution, are not decreased in the presence of Aβ42. Our results indicate the existence of 2 mechanisms of Aβ42 involvement in delayed fibrinolysis: (1) through the induction of a tighter fibrin network composed of thinner fibers, and (2) through inhibition of plasmin(ogen)–fibrin binding.

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Effects of actin filaments on fibrin clot structure and lysis

Blood ◽

10.1182/blood.v80.4.928.928 ◽

1992 ◽

Vol 80 (4) ◽

pp. 928-936 ◽

Cited By ~ 23

Author(s):

PA Janmey ◽

JA Lamb ◽

RM Ezzell ◽

S Hvidt ◽

SE Lind

Keyword(s):

Actin Filaments ◽

Fibrin Clot ◽

Actin Binding ◽

Clot Lysis ◽

Plasma Gelsolin ◽

Fibrin Network ◽

Viscoelastic Effects ◽

Rheologic Properties ◽

Clot Structure ◽

Fibrin Clots

Abstract The muscle and cytoskeletal protein actin is released from cells as a consequence of cell death and interacts with components of the hemostatic and fibrinolytic systems, including platelets, plasmin, and fibrin. We report here that incorporation of actin filaments into fibrin clots changes their viscoelastic properties by increasing their shear modulus at low deforming stresses and by nearly eliminating their tendency to become more rigid with increasing deformation (ie, exhibit strain-hardening). The viscoelastic effects depended on the length of the actin filaments as shown by the effects of the plasma filament- severing protein, gelsolin. Binding of actin to fibrin clots also varied with actin filament length. The plasma actin-binding proteins gelsolin and vitamin D-binding protein reduced, but did not eliminate, the incorporation of actin in the clot. Fluorescence microscopy showed a direct association of rhodamine-labeled actin filaments with the fibrin network. Incubation of clots containing long actin filaments in solutions containing physiologic concentrations of gelsolin (2 mumol/L) released 60% of the actin trapped in the clot. Reduction of the actin content of a fibrin clot by incubation in a gelsolin-containing solution resulted in an increased rate of clot lysis. The ability of plasma gelsolin to shorten actin filaments may therefore be of physiologic and potentially of therapeutic importance insofar as gelsolin-mediated diffusion of actin from the clot may restore the clot's rheologic properties and render it more sensitive to the lytic action of plasmin.

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Effects of actin filaments on fibrin clot structure and lysis

Blood ◽

10.1182/blood.v80.4.928.bloodjournal804928 ◽

1992 ◽

Vol 80 (4) ◽

pp. 928-936

Author(s):

PA Janmey ◽

JA Lamb ◽

RM Ezzell ◽

S Hvidt ◽

SE Lind

Keyword(s):

Actin Filaments ◽

Fibrin Clot ◽

Actin Binding ◽

Clot Lysis ◽

Plasma Gelsolin ◽

Fibrin Network ◽

Viscoelastic Effects ◽

Rheologic Properties ◽

Clot Structure ◽

Fibrin Clots

The muscle and cytoskeletal protein actin is released from cells as a consequence of cell death and interacts with components of the hemostatic and fibrinolytic systems, including platelets, plasmin, and fibrin. We report here that incorporation of actin filaments into fibrin clots changes their viscoelastic properties by increasing their shear modulus at low deforming stresses and by nearly eliminating their tendency to become more rigid with increasing deformation (ie, exhibit strain-hardening). The viscoelastic effects depended on the length of the actin filaments as shown by the effects of the plasma filament- severing protein, gelsolin. Binding of actin to fibrin clots also varied with actin filament length. The plasma actin-binding proteins gelsolin and vitamin D-binding protein reduced, but did not eliminate, the incorporation of actin in the clot. Fluorescence microscopy showed a direct association of rhodamine-labeled actin filaments with the fibrin network. Incubation of clots containing long actin filaments in solutions containing physiologic concentrations of gelsolin (2 mumol/L) released 60% of the actin trapped in the clot. Reduction of the actin content of a fibrin clot by incubation in a gelsolin-containing solution resulted in an increased rate of clot lysis. The ability of plasma gelsolin to shorten actin filaments may therefore be of physiologic and potentially of therapeutic importance insofar as gelsolin-mediated diffusion of actin from the clot may restore the clot's rheologic properties and render it more sensitive to the lytic action of plasmin.

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Altered fibrin clot structure/function in patients with antiphospholipid syndrome: association with thrombotic manifestation

Thrombosis and Haemostasis ◽

10.1160/th13-11-0980 ◽

2014 ◽

Vol 112 (08) ◽

pp. 287-296 ◽

Cited By ~ 21

Author(s):

Magdalena Celińska-Löwenhoff ◽

Teresa Iwaniec ◽

Agnieszka Padjas ◽

Jacek Musiał ◽

Anetta Undas

Keyword(s):

Structure Function ◽

Antiphospholipid Syndrome ◽

Thrombotic Event ◽

Fibrin Clot ◽

Clot Lysis ◽

Lag Phase ◽

Lysis Time ◽

Clot Structure ◽

Clot Lysis Time ◽

D Dimer

SummaryWe tested the hypothesis that plasma fibrin clot structure/function is unfavourably altered in patients with antiphospholipid syndrome (APS). Ex vivo plasma clot permeability, turbidity and susceptibility to lysis were determined in 126 consecutive patients with APS enrolled five months or more since thrombotic event vs 105 controls. Patients with both primary and secondary APS were characterised by 11% lower clot permeability (p<0.001), 4.8% shorter lag phase (p<0.001), 10% longer clot lysis time (p<0.001), and 4.7% higher maximum level of D-dimer released from clots (p=0.02) as compared to the controls. Scanning electron microscopy images confirmed denser fibrin networks composed of thinner fibres in APS. Clots from patients with “triple-antibody positivity” were formed after shorter lag phase (p=0.019) and were lysed at a slower rate (p=0.004) than in the remainder. Clots from APS patients who experienced stroke and/or myocardial infarction were 8% less permeable (p=0.01) and susceptible to lysis (10.4% longer clot lysis time [p=0.006] and 4.5% slower release of D-dimer from clots [p=0.01]) compared with those following venous thromboembolism alone. Multivariate analysis adjusted for potential confounders showed that in APS patients, lupus anticoagulant and “triple-positivity” were the independent predictors of clot permeability, while “triple-positivity” predicted lysis time. We conclude that APS is associated with prothrombotic plasma fibrin clot phenotype, with more pronounced abnormalities in arterial thrombosis. Molecular background for this novel prothrombotic mechanism in APS remains to be established.

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