Hypoalbuminemia Increases Fibrin Clot Density and Impairs Fibrinolysis

Hypoalbuminemia Increases Fibrin Clot Density and Impairs Fibrinolysis | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Hypoalbuminemia Increases Fibrin Clot Density and Impairs Fibrinolysis View ORCID Profile Amanda P. Waller , Katelyn J. Wolfgang , View ORCID Profile Zachary S. Stevenson , View ORCID Profile Lori A. Holle , View ORCID Profile Alisa S. Wolberg , View ORCID Profile Bryce A. Kerlin doi: https://doi.org/10.1101/2025.02.03.635731 Amanda P. Waller * Center for Clinical and Translational Research, Abigail Wexner Research Institute at Nationwide Children’s , Columbus, Ohio, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Amanda P. Waller Katelyn J. Wolfgang * Center for Clinical and Translational Research, Abigail Wexner Research Institute at Nationwide Children’s , Columbus, Ohio, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zachary S. Stevenson * Center for Clinical and Translational Research, Abigail Wexner Research Institute at Nationwide Children’s , Columbus, Ohio, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Zachary S. Stevenson Lori A. Holle † Department of Pathology Laboratory Medicine and UNC Blood Research Center, University of North Carolina , Chapel Hill, NC, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lori A. Holle Alisa S. Wolberg † Department of Pathology Laboratory Medicine and UNC Blood Research Center, University of North Carolina , Chapel Hill, NC, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alisa S. Wolberg Bryce A. Kerlin * Center for Clinical and Translational Research, Abigail Wexner Research Institute at Nationwide Children’s , Columbus, Ohio, USA ‡ Division of Hematology/Oncology/Blood & Marrow Transplantation, Nationwide Children’s Hospital , Columbus, Ohio, USA § Department of Pediatrics, The Ohio State University College of Medicine , Columbus, Ohio, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Bryce A. Kerlin For correspondence: Bryce.Kerlin{at}NationwideChildrens.org Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Background Hypoalbuminemia is a thrombotic disease risk biomarker. Albumin is a negative acute phase reactant and may thus be an indirect biomarker of thromboinflammation. However, nephrotic syndrome (resulting from non-inflammatory proteinuric glomerular disease) causes both hypoalbuminemia and elevated thrombotic risk. Hypofibrinolysis has been observed in nephrotic syndrome and published data suggest that albumin may directly enhance fibrinolysis. These observations suggest that albumin may directly influence thrombotic risk. Objective To test the hypothesis that hypoalbuminemia impairs fibrinolysis. Methods Hypoalbuminemic blood and plasma from nephrotic syndrome rat models and nephrotic patients were analyzed by thromboelastometry, clot lysis assays, fibrin clot turbidity, confocal microscopy, and immunoblotting. Some studies were conducted without vs. with albumin repletion. Plasma from an analbuminemic mutant rat model was used to confirm albumin-dependent observations. Results Hypoalbuminemia was associated with hypofibrinolysis in nephrotic whole blood. Albumin levels were positively associated with fibrinolysis in both nephrotic rat and patient plasma. Hypoalbuminemia accelerated fibrin clot formation in nephrotic rat plasma. Dense fibrin clots are known to be resistant to fibrinolysis and fibrin clot network density was increased in hypoalbuminemic plasma clots from both nephrotic rats and patients. Clots formed from hypoalbuminemic plasma contained less albumin than controls, and repletion with recombinant albumin to healthy control levels corrected both fibrin network density and fibrinolysis in nephrotic and analbuminemic rat plasma. Conclusion These data show that albumin directly increases fibrin network porosity and enhances fibrinolysis. Hypoalbuminemia may mechanistically contribute to nephrotic syndrome thrombotic complications and may similarly increase thrombotic risk in other hypoalbuminemic conditions. INTRODUCTION Hypoalbuminemia is a significant predictor of both thrombosis and cardiovascular disease [ 1 - 5 ]. Nephrotic syndrome (NS) is a glomerular disorder characterized by hypoalbuminemia due to massive proteinuria that is associated with a high-risk for life-threatening thrombotic complications [ 6 - 9 ]. Increased thrombotic risk in NS is generally attributed to its well-described acquired hypercoagulability that is caused by deranged protein homeostasis resulting from proteinuria [ 8 ]. In addition, however, hypofibrinolysis was described in patients with NS as early as 1974 and may play an underappreciated role in NS-associated thrombotic risk [ 8 , 10 - 15 ]. Importantly, decreased clot lysis has been associated with increased thrombotic risk in non-NS cohorts and recent evidence suggests that co-existent hypercoagulability and hypofibrinolysis may synergistically increase thrombosis risk [ 16 , 17 ]. We previously reported significant hypofibrinolysis in a rat NS model using rotational thromboelastometry and found that the hypofibrinolytic signal was directly correlated with urinary protein and indirectly correlated with plasma albumin [ 14 ]. Albumin was previously shown to partially correct hypofibrinolysis in clotted NS patient plasma [ 11 ]. The same study demonstrated that fibrin networks from NS patient plasma were more dense, with shorter fibrils and more branch points, a pattern that was partially corrected by albumin supplementation [ 11 ]. Other reports, not focused on NS, used plasma, purified fibrinogen, or fibrin monomers to demonstrate that albumin affects fibrin network structure and fibrinolysis [ 18 - 23 ]. However, many of these studies were performed with non-physiologic concentrations of albumin, fibrin(ogen), or both. Many were underpowered, sometimes demonstrating effects in only a single subject. It is thus unclear from these studies if albumin is a biologically important regulator of fibrinolysis. As a negative acute phase reactant, albumin is a potential thromboinflammation biomarker [ 3 ]. However, several studies suggest that albumin may also contribute mechanistically to fibrinolysis [ 11 , 18 , 19 , 21 - 23 ]. Thus, defining the role of albumin in fibrinolytic regulation may increase understanding of thrombotic pathogenesis not only in kidney disease, but also in other diseases associated with hypoalbuminemia [ 24 ]. Importantly, hypoalbuminemia is highly prevalent amongst hospitalized patients who are a group that is well-known to be at high risk for thrombosis [ 24 , 25 ]. We therefore tested the hypothesis that hypoalbuminemia impairs fibrinolysis using both NS and mutant analbuminemic rat models as well as NS patient samples [ 26 ]. MATERIALS AND METHODS Hypoalbuminemic Rat Models All procedures were approved by the Abigail Wexner Research Institute at Nationwide Children’s Hospital Institutional Animal Care and Use Committee (AR13-00027), in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Three hypoalbuminemic rat models were utilized in these studies ( Table S1 ). Two of these models are well-established NS models wherein toxin-mediated podocyte injury leads to proteinuric glomerular disease [ 27 , 28 ]. Hypoalbuminemia develops in both models due to massive albuminuria. We also examined genetically analbuminemic ( Alb -/- ) rats that were maintained on an inbred Fischer 344 background [ 29 - 32 ]. These rats have a naturally occurring 7 bp intron deletion that disrupts Alb mRNA splicing [ 29 , 30 ]. As a result, homozygous rats have no detectable plasma or tissue albumin [ 31 , 32 ]. Blood was collected from wild-type ( Alb +/+ ), heterozygous ( Alb +/- ), and homozygous (Alb -/- ) littermate rats using the above methods (∼150 g rats, age ∼6 weeks, n =5 animals/group). Both male and female rats were studied in the Alb -/- model whereas only male rats were considered in the NS models because they had less phenotypic variability in preliminary studies. Administration of puromycin aminonucleoside (PAN), a podocyte-specific toxin, is a well-established dose-dependent rat NS model (PAN-NS) [ 14 , 27 , 33 , 34 ]. We previously constructed a repository of plasma samples from Wistar rats with a range of plasma albumin levels by varying the PAN dose [ 14 ]. These 5 groups of rats (body weight ∼150 g, age ∼45-50 d) received a single dose of intravenous PAN (0 (sham carrier solution only), 25, 50, 100, or 150 mg/kg; n =6 animals/group) on Day 0 [ 14 ]. Sham treated rats in this group represent healthy controls and the PAN treated rats were divided into albumin quartiles. We also studied the podocin-promoter driven human Diphtheria Toxin Receptor ( pDTR ) transgene rat NS model ( pDTR -NS) on an inbred Fischer 344 background [ 28 , 33 ]. Expression of the pDTR transgene in this model is restricted to podocytes. Four groups of homozygous pDTR rats (∼150 g, age ∼6 weeks) were treated with intraperitoneal diphtheria toxin (DT) as follows: 0 (sham carrier solution only), 25, 50, or 75 ng/kg on day 0 and compared to a matched group of wild-type Fischer 344 rats treated with 50 ng/kg DT (WT+DT; n =5-8 animals/group). This strategy incorporates two types of healthy control: (1) Sham treated pDTR rats are presented as the 4 th albumin quartile whereas (2) The WT+DT rats are labeled as Controls, since WT rats do not express a DT receptor. Nadir plasma albumin occurs by day 10 in both models [ 27 , 28 , 35 ]. Thus, blood was collected on day 10 from the inferior vena cava into final concentration 0.32% NaCitrate / 1.45 µM Corn Trypsin Inhibitor (CTI; Haematologic Technologies Inc., Vermont, VT, USA), an aliquot of whole blood was used for rotational thromboelastometry (ROTEM) where indicated and the remainder was processed to Platelet Poor Plasma (PPP), within one hour, aliquoted, and frozen at -80°C until analyzed, as described previously [ 14 ]. Columbus Nephrotic Syndrome Cohort To translate the rat model observations to human hypoalbuminemia, assays were also performed on plasma samples from a previously described cohort of incident pediatric and adult NS patients from whom plasma was collected prior to initiation of NS-specific treatment [ 36 , 37 ]. This portion of the study was approved by the Nationwide Children’s Hospital Institutional Review Board (IRB12–00290) in reciprocity with The Ohio State University Wexner Medical Center IRB. After written informed consent (and assent when applicable) was obtained from each participant or parent/guardian, blood was collected into final concentration 0.32% sodium citrate / 1.45 μM CTI and processed to PPP as above. Plasma Albumin Quantitation Plasma albumin was quantified in all rat and human samples using the bromocresol purple (BCP) assay (QuantiChrom BCP; BioAssay Systems, Hayward, CA), according to the manufacturer’s instructions, as previously described [ 34 , 36 - 38 ]. Global Hemostasis Assays Whole blood ROTEM assays were performed within 20 min of specimen collection, using the INTEM (intrinsic pathway activation) assay in mini cups and pins without and with tranexamic acid (TXA; 1 mg per 100 µL of whole blood [ 39 ]), on a ROTEM delta instrument (Werfen, Barcelona, Spain). ROTEM reports maximal clot firmness (MCF) as the maximal waveform amplitude (in mm). Lysis at 60 minutes (LY60) is the percent decrease in amplitude from MCF at 60 minutes: [ 40 , 41 ]. Thrombin generation assays (TGA) were performed using the Technothrombin TGA kit (Technoclone, Vienna, Austria) on rat PPP diluted 1:1 with TGA buffer and TGA RC Low Reagent, and read on a Spectramax M2 fluorescent plate reader (Molecular Devices, Sunnyvale, California), as previously described [ 14 , 34 , 36 ]. Relative Fluorescence Units (RFU) were converted to thrombin (nM) concentrations using a standard curve and evaluated with Technoclone Evaluation Software. Fibrinolysis Assays Plasma Clot Lysis Assays (CLA) were adapted from a previously reported colorimetric method based upon release of Coomassie brilliant blue R-250 dye from fibrin clots [ 42 ]. Briefly, fibrin clot formation was initiated by adding thrombin (20 nM) and calcium chloride (10 mM) to PPP (diluted 1:4 with PBS) in the presence of R-250 dye (1 mg/ml). The plasma was allowed to clot at room temperature for 45 minutes, the resulting clots were washed with 1 mL PBS, and resuspended in 1 mL of a urokinase-type plasminogen activator (uPA ) solution (200 IU/mL in PBS). The suspended clot was then gently rotated at room temperature and lysis was determined at 60 minutes by transferring 100 uL of the soluble supernatant fraction into a 96-well microtiter plate wherein absorbance was measured at 540 nm to quantitate R-250 dye released from the clot. Percent total clot lysis was calculated as a percentage of pooled normal plasma (PNP) clot lysis after blank subtraction. In some experiments, the assay conditions were modified to explore potential underlying mechanisms as follows: (a) thrombin (20 nM) without uPA, (b) low thrombin (5 nM) + uPA (200 IU/mL), (c) thrombin (20 nM) + uPA (200 IU/mL) + plasmin (1.2 µM), and (d) thrombin (20 nM) + high uPA (800 IU/mL) with incubation at 37°C on a rotator with endpoint measurement at 24 hrs. In other experiments, CLA was performed with tissue-type plasminogen activator (tPA; 20 nM) or plasmin (1.2 µM) in lieu of uPA or after immunodepleting or supplementing specific proteins, as indicated – most notably, recombinant human albumin (expressed in P. pastoris ; Millipore Sigma). Immunodepletion experiments were performed by incubating PPP with Dynabeads coated with indicated antibodies ( Table S2 ) according to the manufacturer’s instructions. Immunodepletion to ≤10% of unmodified PPP was confirmed by immunoblot prior to further testing. Spiking experiments were performed by supplementing PPP with indicated proteins ( Table S2 ) in amounts adequate to replete plasma to 100-200% of rat PNP values prior to clot formation. FXIII activity was inhibited by the addition of 20 µM 1,3,4,5-Tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride (T101, Zedira BmbH, Germany) prior to clot formation. Fibrin Clot Formation Kinetics and Network Density Plasma clot formation kinetics were determined in a turbidimetric assay at absorbance 405 nm, as described previously [ 43 - 45 ]. Basal turbidity varied between samples, likely due to varying lipid levels, so baseline turbidity was normalized to zero for all samples. Fibrin network density was assessed by laser scanning confocal microscopy using fluorescently-labeled fibrinogen as a tracer, as described previously [ 43 , 46 ]. In brief, clots were formed in vitro using PPP diluted 1:1 in HBS, in the presence of AlexaFluor488-conjugated fibrinogen (160 µM), phospholipids and tissue factor (final concentrations ≈ 1uM and 0.5pM, respectively; RC Low reagent), and calcium chloride (10 mM). In some experiments, recombinant human albumin was added to the PPP prior to clot formation. Clots were then assessed by laser scanning confocal microscopy followed by ImageJ analysis of the following variables: (1) Fibrin Area: AlexaFluor488-positive integrated optical density (IOD), (2) Fibrin Density: percentage of randomly placed grid crosshairs with intersecting fibrin fibers, as previously reported [ 44 ], and (3) Fibrin Porosity: AlexaFluor488-negative IOD. Enzyme-Linked Immunosorbent Assays, Immunoblots, Antibodies, and Other Reagents ELISA assays were used to determine rat PPP concentrations of fibrinolysis pathway components as specified in Table S2 , all ELISA kits were validated for use with the respective rat proteins and performed according to the manufacturer’s instructions. Immunoblot and magnetic bead pulldown assays were performed using rat-specific antibodies against proteins of interest ( Table S2 ). For fibrin clot immunoblot experiments, rat PPP was clotted by addition of 20 nM thrombin and 10 mM calcium chloride at RT for 60 min. The resulting clot was then washed once with PBS and solubilized in M-PER (mammalian-protein extraction reagent) containing protease and phosphatase inhibitor cocktails ( Table S2 ). Equal amounts (10 µg) of total protein (mixed with Laemmli buffer with β-mercaptoethanol) were loaded in each lane, resolved by 12% SDS-PAGE, and transferred to a PVDF membrane. The membranes were blocked with 5% nonfat dry milk solution, incubated overnight with respective primary antibodies (see Table S2 for concentrations) followed by incubation with their corresponding secondary HRP-conjugated antibodies. Semi-quantitative determination of each protein was performed by autoradiography after revealing the antibody-bound protein by enhanced chemiluminescence reaction. The density of the bands on scanned autoradiographs was quantified using ImageJ. Where indicated, the relative quantity of each protein of interest was expressed as a ratio to total fibrin(ogen) within the same clot. See Table S2 for all other reagents. Statistics One- or two-way ANOVA (analysis of variance) was used for multiple group comparisons. When a significant difference was identified by ANOVA, post hoc tests were performed using the Student–Newman–Keuls technique. GraphPad Prism (Boston, MA) software was used for all statistical analyses and for preparation of figures. Statistical significance was defined as P <0.05. Data are presented as mean ± SE. RESULTS Rotational Thromboelastometry Revealed a Hypofibrinolytic Defect in Hypoalbuminemic Whole Blood Using a ROTEM assay (INTEM activator reagent with added uPA), we previously observed less degradation of clot firmness over time in PAN-NS whole blood, strongly suggestive of a hypofibrinolytic defect [ 14 ]. Here we show that this signal was reproducible in a standard ROTEM assay (INTEM without uPA) in which LY60 was directly proportional to plasma albumin ( Fig. 1A-C ). To confirm that this signal was fibrinolysis-dependent, we performed additional ROTEM analyses without and with the addition of tranexamic acid, a potent fibrinolysis inhibitor. These experiments demonstrated that LY60 was significantly reduced by tranexamic acid independently of albumin concentration ( Fig. 1D-E ). We next sought to validate these observations in the pDTR -NS rat model in which hypoalbuminemia, proteinuria, and hypercoagulopathy are proportional to DT dose ( Fig. S1 ), consistent with prior observations in PAN-NS [ 14 , 34 ]. ROTEM revealed decreased LY60 in hypoalbuminemic pDTR -NS whole blood ( Fig. 1F-H ). Tranexamic acid significantly reduced LY60 independently of albumin concentration in this model as well ( Fig. 1I-J ). These data confirm that ROTEM LY60 is a fibrinolysis-specific variable and suggest that nephrotic hypoalbuminemia is associated with reduced clot lysis. Download figure Open in new tab Figure 1: Rotational Thromboelastometry Revealed a Hypofibrinolytic Defect in Hypoalbuminemic Whole Blood. ( A , F ) Representative ROTEM tracings from 1 st and 4 th albumin IQR PAN-( A ) and pDTR -( F ) nephrotic rats. ( B , G ) %Lysis (LY60) determined by ROTEM was directly correlated with plasma albumin concentration in PAN-( B ) and pDTR -( G ) nephrotic rats. ( C , H ) %Lysis by albumin IQR in PAN-( C ) and pDTR -( H ) nephrotic rats with healthy control rats for both models. ( D - E, I - J ) Additional groups of PAN- and pDTR -nephrotic rats with and without hypoalbuminemia ( D , I ) demonstrated that TXA impaired lysis independently of albumin levels ( E , J ). ROTEM: rotational thromboelastometry; IQR: interquartile range; PAN: puromycin aminonucleoside; pDTR : podocin-promoter driven diphtheria toxin receptor transgene; LY60: Lysis at 60 minutes; TXA: tranexamic acid; Hypofibrinolysis was Proportional to Hypoalbuminemia We next assessed the effect of albumin on clot lysis in PPP. In rats with PAN-NS ( Fig. 2A-B ) or pDTR -NS ( Fig. 2C-D ), as well as in NS patient plasma clots ( Fig. 2E-F ), uPA-mediated lysis decreased proportionally to reduced albumin concentrations. A modified CLA performed on pDTR -NS PPP clots revealed minimal lysis when uPA was omitted ( Fig. S2A ). Albumin concentration-dependent fibrinolysis persisted when a lower concentration of thrombin (5 nM instead of 20 nM) was used to form the clot ( Fig. S2B ), suggesting that hypofibrinolysis due to hypoalbuminemia is not dependent upon thrombin concentration [ 14 , 34 , 36 , 45 , 47 ]. In this assay, fibrinolysis is dependent upon uPA-mediated conversion of endogenous plasminogen to plasmin. However, initiation of lysis with both uPA and exogenous plasmin did not meaningfully alter the results ( Fig. S2C ), suggesting that the defect is not due to increased activity of plasminogen activator inhibitors. Quadrupling the uPA concentration did not enable complete lysis of hypoalbuminemic clots despite concomitantly increasing the assay duration to 24 hours ( Fig. S2D ). Performing the assay with tPA ( Fig. S2E ) or plasmin ( Fig. S2F ) in lieu of uPA gave similar results. Collectively, these data suggest that clots formed from hypoalbuminemic plasma are inherently resistant to fibrinolysis. Download figure Open in new tab Figure 2: Hypofibrinolysis was Proportional to Hypoalbuminemia. ( A , C , E ) Representative residual clots at the clot lysis assay endpoint (60 minutes) from PAN- ( A ) or pDTR - ( C ) nephrotic rat plasmas and from nephrotic patient plasmas ( E ; annotated with albumin IQR group ( A, C ) or level ( E )). ( B , D , F ) Lysis, expressed as a percentage of rat ( B , D ) or human ( E ) PNP, in PAN- ( B ) and pDTR - ( D ) nephrotic rat plasma clots (and healthy control rat clots) and in clots from nephrotic patients with hypoalbuminemia (<3.5 g/dL) vs. normal albumin levels (≥3.5 g/dL). PAN: puromycin aminonucleoside; pDTR : podocin-promoter driven diphtheria toxin receptor transgene; PNP: pooled normal plasma; IQR: interquartile range; WT: wild-type; DT: diphtheria toxin; n =6 rats/group ( A-B ); n =5-8 rats/group ( C-D ); n =7-16 patients/group ( E-F ); * P <0.05, *** P <0.001. Fibrin Clot Formation Kinetics were Enhanced in Hypoalbuminemic Plasma Accelerated fibrin clot formation and peak turbidity are associated with fibrinolytic resistance [ 45 , 48 - 55 ]. Thus, we sought to determine if plasma fibrin clot formation kinetics were altered in hypoalbuminemic pDTR -NS rat plasma. Interestingly, both fibrin clot formation rate (Vmax) and peak turbidity increased in proportion to hypoalbuminemia severity ( Fig. 3 ). These data suggest that albumin may regulate fibrin clot formation kinetics. Download figure Open in new tab Figure 3: Fibrin Clot Formation Kinetics were Enhanced in Hypoalbuminemic Plasma. ( A ) Representative fibrin clot formation tracings from pDTR -nephrotic and a healthy control rat (annotated with plasma albumin levels and albumin IQR group). ( B ) The rate of fibrin formation (Vmax) was inversely proportional to plasma albumin level. ( C ) Vmax by albumin IQR group. ( D ) Peak turbidity was inversely proportional to plasma albumin level. ( E ) Peak turbidity by albumin IQR group. WT: wild-type; DT: diphtheria toxin; pDTR : podocin-promoter driven diphtheria toxin receptor transgene; IQR: interquartile range; Vmax: clotting rate; n =5-8 rats/group; * P <0.05. Fibrin Clot Network Density was Inversely Related to Plasma Albumin Increased thrombin generation, hyperfibrinogenemia, and enhanced plasma clot formation kinetics are co-existent in NS and have all been associated with greater fibrin network density wherein the fibrin fibers are thinner and more tightly packed [ 14 , 44 , 45 , 56 ]. Here we found that fibrin area and density increased as albumin concentration decreased in both the pDTR -NS rat model and in NS patient plasma ( Fig. 4 ) Fibrin network density was significantly and positively correlated with ETP (R=0.498; P< 0.01), plasma fibrinogen (R=0.146; P <0.05), and peak turbidity (R=0.364; P <0.05) in the pDTR -NS rat model. These data show that fibrin network density is associated with thrombin generation, fibrinogen levels, and fibrin clot formation kinetics during nephrotic syndrome and further demonstrate that density increases during hypoalbuminemia. Download figure Open in new tab Figure 4: Fibrin Clot Network Density was Inversely Related to Plasma Albumin. Fibrin clots were formed under glass coverslips in the presence of AlexaFlour488-labeled fibrinogen and imaged by laser scanning confocal microscopy. ( A , E ) Representative photomicrographs of clots formed from pDTR -rat ( A ) and nephrotic patient ( E ) plasma (annotated with albumin IQR group ( A ) or level ( E )). ( B , F ) Fibrin Area (AlexaFlour488-positive integrated optical density (IOD)). ( C , G ) Fibrin network density (percentage of randomly placed grid crosshairs with intersecting fibers). ( D , H ) Fibrin clot porosity (AlexaFlour488-positive IOD). FITC: fluorescein isothiocyanate; WT: wild-type; DT: diphtheria toxin; pDTR : podocin-promoter driven diphtheria toxin receptor transgene; IQR: interquartile range; n =5-8 rats/group ( A-E ); n =7-16 patients/group ( F-J ); * P <0.05, ** P <0.01, *** P <0.001. Hypoalbuminemic Plasma Clots Contained Less Albumin To determine the mechanisms mediating altered clot lysis, we quantified the levels of fibrinolysis pathway proteins in PPP. Although both plasminogen deficiency and plasminogen urinary loss have been reported in NS patients [ 57 ], plasminogen concentrations were not significantly correlated with albumin in pDTR -NS plasma ( Fig. S3A ). Consistent with previous reports, hyperfibrinogenemia was observed in hypoalbuminemic plasma ( Fig. S3B ) [ 8 , 14 ]. Plasminogen activator inhibitor-1 (PAI-1), α2-macroglobulin, and uPA levels were also significantly increased in hypoalbuminemic plasma ( Fig. S3C-S3E ). There were no significant changes in the concentrations of tPA, TAFI, or α2-AP ( Fig. S3F-S3H ). We next used immunoblotting to determine intra-clot quantities of albumin and fibrinolytic system components in pDTR -NS plasma clots relative to plasma albumin levels ( Figs. 5 and S4 ). Unsurprisingly, intra-clot albumin was decreased in concert with decreasing plasma albumin concentration ( Fig. 5A ). We expected to find higher quantities of fibrin(ogen) in the densest clots (from the most hypoalbuminemic plasmas). To our surprise, clot fibrin(ogen) content was decreased in hypoalbuminemic plasma ( Fig. 5B ), such that the least dense clots (those with the highest albumin levels) had the highest fibrin(ogen) content. Moreover, the lowest albumin quartile plasma clots had a lower albumin:fibrin(ogen) content ratio than the highest albumin quartile clots ( Fig. 5C ). Interestingly, although the levels of the fibrinolysis inhibitors PAI-1, α2AP, α2M, and TAFI increased with hypoalbuminemia (relative to fibrin(ogen), Fig. S5 ), immunodepleting these inhibitors from nephrotic, hypoalbuminemic plasma did not correct the hypofibrinolytic defect in CLA experiments ( Fig. S6 ). Likewise, although intra-clot levels of tPA, uPA, PAI-2, factor XII, factor XI, and prekallikrein decreased with hypoalbuminemia (relative to fibrin(ogen), Fig. S7 ), supplementing plasma to achieve increased levels of these proteins (100-200% of rat PNP) did not correct the lysis in CLA experiments ( Fig. S8 ). We also used a transglutaminase inhibitor, T101, to determine if albumin inhibits factor XIII-dependent fibrin cross-linking and thereby enhances fibrinolysis; however, factor XIII inhibition did not significantly alter CLA results ( Fig. S9 ). These data show that plasma albumin concentrations may influence the incorporation of various fibrinolytic components into fibrin clots, but that these changes do not directly influence fibrinolytic susceptibility. Download figure Open in new tab Figure 5: Hypoalbuminemic Plasma Clots Contained Less Albumin. ( A , B ) Fibrin clots were formed from pDTR -rat plasma, washed, solubilized in M-PER, then immunoblotted for albumin ( A ) and fibrin(ogen) β-chain ( B ); representative immunoblots are shown above each graph. Data are expressed relative to fibrin clots formed from rat PNP (rPNP). ( C ) Intra-clot albumin content (relative to fibrin(ogen)). ( D-F ) Clot content of albumin ( D ) and fibrin(ogen) β-chain ( E ), as well as albumin:fibrin(ogen) ratio ( F ) were directly correlated with plasma albumin. pDTR : podocin-promoter driven diphtheria toxin receptor transgene; M-PER: mammalian protein extraction reagent; IQR: interquartile range; n=5-8 rats/group; ** P <0.01, *** P <0.001. Albumin Repletion Corrected Hypofibrinolysis and Fibrin Network Density The negative findings with manipulation of fibrinolytic system components prompted us to directly test albumin repletion. Interestingly, dissolving lyophilized recombinant albumin into pDTR -NS plasma samples to achieve the upper limit of the healthy rat reference range (4.5 g/dL; 677 µM) enhanced fibrinolysis to values that were no longer significantly different from controls ( Fig. 6 ) [ 58 ]. In contrast, supplementation with an equimolar amount of a control protein from the serpin protein family (ovalbumin [OvA]) had no effect on fibrinolysis [ 59 , 60 ]. Moreover, albumin repletion decreased fibrin network density to values no different than control clots following albumin repletion whereas OvA addition had no significant effect ( Fig. 7 ). Taken together with the intra-clot albumin data, these observations suggest that clot-incorporated albumin reduces fibrin network density and increases fibrinolytic susceptibility. Download figure Open in new tab Figure 6: Albumin Repletion Corrected Hypofibrinolysis. ( A , B ) Representative residual clots at the clot lysis assay endpoint (60 minutes) from pDTR -nephrotic rat plasmas supplemented with recombinant Albumin ( A ) or Ovalbumin ( B ) ( Figure 2C shows representative residual clots of unmanipulated samples). ( C ) Lysis, expressed as a percentage of rat PNP values, is corrected by rAlbumin, but not Ovalbumin, supplementation. WT: wild-type; DT: diphtheria toxin; pDTR : podocin-promoter driven diphtheria toxin receptor transgene; rAlbumin: recombinant albumin; OvA: ovalbumin (note: OvA is a serpin-family, not an albumin-family, protein); n=5-8 rats/group; * P <0.05, ** P <0.01, *** P <0.001. Download figure Open in new tab Figure 7: Albumin Repletion Corrected Fibrin Network Density. ( A ) Representative photomicrographs of clots formed from 1 st and 4 th albumin IQR pDTR -rat plasma (as in Figure 4A ) with Ovalbumin or rAlbumin supplementation. ( B ) Fibrin area. ( C ) Fibrin network density. ( D ) Fibrin clot porosity. IQR: interquartile range; WT: wild-type; DT: diphtheria toxin; pDTR : podocin-promoter driven diphtheria toxin receptor transgene; rAlbumin: recombinant human albumin; OvA: ovalbumin (note: OvA is a serpin-family, not an albumin-family, protein); n=8 rats/group; * P <0.05, ** P <0.01, *** P <0.001. Albumin Dictated Fibrinolytic Susceptibility and Fibrin Network Density in Analbuminemic Rat Plasma To determine if albumin modulates fibrinolysis in settings beyond nephrotic syndrome, we examined plasma samples from a mutant analbuminemic rat model [ 29 - 32 ]. Consistent with the nephrotic-hypoalbuminemic data presented above, Alb -/- plasma clots were resistant to fibrinolysis and this hypofibrinolytic defect was normalized with the addition of albumin ( Fig. 8A ). Alb -/- rat blood was also hypofibrinolytic by ROTEM and albumin repletion significantly corrected the LY60 values to that of wild-type ( Alb +/+ ) controls ( Fig. S10 ). Albumin supplementation supported fibrinolysis with similar half-maximal effective concentration (EC 50 ) values in both nephrotic and Alb -/- plasma ( Fig. S11 ). Moreover, fibrin network density was increased in clotted Alb -/- plasma and reverted to normal following albumin supplementation ( Fig. 8B ). Collectively, these data confirm that albumin levels are an independent determinant of fibrinolytic susceptibility and fibrin clot density. Download figure Open in new tab Figure 8: Albumin Dictated Fibrinolytic Susceptibility and Fibrin Network Density in Albumin Knockout Plasma. ( A ) Representative residual clots at the clot lysis assay endpoint (60 minutes) from albumin knockout rat plasmas (as in Figure 4A ) without and with recombinant Albumin (rAlbumin) supplementation. ( B ) Lysis, expressed as a percentage of rat PNP values, was diminished in homozygous and heterozygous albumin knockout rat clots and was corrected by rAlbumin supplementation. ( C ) Representative photomicrographs of clots formed from albumin knockout rat plasmas without and with rAlbumin supplementation. ( D ) Fibrin area. ( E ) Fibrin network density. ( F ) Fibrin clot porosity. rAlbumin: recombinant albumin; n=5 rats/group; *** P <0.001. DISCUSSION In this study, hypoalbuminemia resulted in dense fibrin clots that were resistant to fibrinolysis and both effects were corrected by restoration of albumin concentrations to healthy control levels. These effects were observed in 2 hypoalbuminemic rat nephrotic syndrome (NS) models, hypoalbuminemic NS patient plasma, and in mutant analbuminemic rats. Importantly, systemic inflammation is not a feature of these rat models, enabling us to discern the effects of hypoalbuminemia independently of the negative acute phase response behavior of albumin that may be related to thromboinflammation [ 3 , 27 - 32 ]. Subjecting the clots to various profibrinolytic conditions and immunodepleting fibrinolysis inhibitors did not substantially improve fibrinolysis, suggesting that hypoalbuminemic plasma clots are inherently resistant to fibrinolysis. Multiple studies have demonstrated that dense fibrin clots are less susceptible to fibrinolysis [ 53 , 61 - 68 ]. In the present study, hypoalbuminemic clots displayed increased density that was correctable with recombinant albumin supplementation. Together, these data strongly suggest that fibrin clot density and fibrinolytic susceptibility are, at least partly, dictated by plasma albumin levels. Thus, the loss of albumin-dependent effects on fibrin clot structure may help explain why hypoalbuminemic patients are at higher risk for thrombosis and cardiovascular disease [ 1 - 4 ]. We and others have demonstrated hypofibrinolysis by a variety of methods in NS patients and animal models [ 10 - 15 ]. In a previous study, in vivo thrombosis modeling in analbuminemic ( Alb -/- ) rats revealed larger thrombi and less frequent thrombus embolization compared to albumin sufficient control rats [ 69 ]. The authors of that paper speculated that decreased thrombus embolization resulted from diminished in vivo fibrinolytic activity. In the present study, we observed diminished fibrinolysis in our ex vivo assays with both hypoalbuminemic NS and Alb -/- plasmas. The present data strongly suggest that hypoalbuminemia is associated with denser fibrin clots that are resistant to fibrinolysis. Previous studies have demonstrated that albumin supplementation enhances fibrinolysis of clotted nephrotic syndrome plasma [ 11 , 19 ]. Our fibrinolysis assay results confirm these observations and further demonstrate the same effect in Alb -/- plasma. The latter data provide the most conclusive evidence to date that albumin directly enhances fibrinolysis and are consistent with the data demonstrating decreased thrombus embolization in Alb -/- rats [ 69 ]. Significantly lower tPA and higher α2-AP activities were observed in Alb -/- plasma and, in a purified biochemistry system, both rat and human albumin enhanced tPA activation of plasminogen, leading the authors to conclude that albumin contributes to fibrinolytic activity [ 69 ]. Meanwhile, we were unable to demonstrate albumin-dependent enhancement of tPA activity against its chromogenic substrate (data not shown). Nonetheless, the present and prior data together strongly suggest that albumin enhances fibrinolytic capacity. While the molecular mechanism underlying this behavior remains unresolved, the present data suggest that albumin directly influences fibrin clot structure which, in turn, dictates its fibrinolytic susceptibility. Our results demonstrate that albumin regulates fibrin clot structure resulting in decreased fibrin network density. These data are consistent with previous reports that used a variety of approaches to show that albumin concentrations alter fibrin structure [ 11 , 20 , 22 , 23 ]. Under scanning confocal laser microscopy, both nephrotic and Alb -/- samples demonstrated increased fibrin network density that was correctable with albumin supplementation, resulting in thicker fibers and increased porosity [ 11 ]. In purified systems, at very low concentrations (≤5 μM) albumin decreased turbidity but as the levels increased (6-100 μM) turbidity increased, suggesting that albumin influences fibrin fiber density in a concentration-dependent manner [ 20 ]. Unfortunately, those experiments were conducted at non-physiologic fibrin(ogen)-to-albumin molar ratios making the results difficult to put into physiologic context. Interestingly, when fibrin fiber density is estimated by turbidity, albumin has been reported to decrease fiber thickness in purified fibrinogen clots but to increase thickness in clotted plasma samples [ 22 ]. Similarly, in magnetic birefringence assays of clotted plasma, albumin appears to enhance fibrin fiber thickness [ 23 ]. These latter data suggest that interactions with other plasma components may be involved in the mechanism by which albumin regulates fibrin structure. Collectively, the present and previously published data strongly suggest that, at physiologically relevant concentrations, albumin increases fibrin fiber thickness, decreases network density, and increases porosity. Some authors have proposed volume exclusion as a possible explanation for the latter observations [ 23 ]. However, both our data demonstrating that a control protein does not alter fibrin clot structure and the purified fibrinogen reports (which also included control proteins) argue against this possibility [ 22 ]. Thus, the molecular mechanism underlying the effect of albumin on fibrin network density remains unexplained. Whereas the previously published Alb -/- rat thrombosis model study is consistent with our ex vivo observations, future studies should explore these phenomena using in vivo models to more fully understand their physiologic relevance [ 69 ]. With the exception of the ROTEM experiments, the present data was predominantly generated in platelet poor plasma, negating potential interactions with cellular blood components and endothelium, emphasizing the need for in vivo translation of these observations [ 70 ]. Hyperfibrinogenemia is a feature of both NS and the Alb -/- rat and hyperfibrinogenemia is associated with both increase clot density and fibrinolytic resistance [ 8 , 14 , 44 , 69 ]. While it is likely that hyperfibrinogenemia contributed to the hypofibrinolytic phenotypes shown in these data, albumin was able to modify lysis of these samples, strongly suggesting that albumin has a fibrinogen-independent effect on fibrinolysis. Interestingly, fibrinogen-to-albumin ratio determination has recently emerged as a novel biomarker for thromboembolic risk and outcomes [ 71 , 72 ]. It remains unclear from the presently available data if albumin regulates clot structure via direct mechanisms (e.g. intramolecular interactions with fibrin(ogen)) or if these effects are indirect (perhaps via interactions with other components of the fibrin clot or coagulation system) [ 73 ]. Despite these limitations, the available data strongly suggest that albumin is an important regulator of fibrin clot structure and fibrinolytic susceptibility. Hypoalbuminemia is associated with both venous and atherothrombotic cardiovascular disease [ 1 - 4 ]. In this study we found that hypofibrinolysis is a feature of hypoalbuminemia. In turn, hypofibrinolysis is associated with increased risk for both venous and arterial thrombosis, suggesting a mechanistic link between these risk factors [ 16 , 17 ]. Future studies should thus focus on determining the molecular mechanisms by which albumin regulates fibrin clot structure and fibrinolytic susceptibility. AUTHOR CONTRIBUTIONS BAK directed and supervised all experimental design, data acquisition, and analysis, edited the manuscript and figures, and compiled the manuscript for submission. APW and BAK drafted the manuscript together. ASW critically revised the manuscript, provided important intellectual content, and supervised the fibrin clot formation kinetic studies which were performed in her lab by LAH. APW, KJW, and ZSS conducted all other experiments, analyzed data, and prepared figure components. All authors approved the submitted version of the manuscript. DECLARATION OF COMPETING INTERESTS There are no competing interests to disclose. ACKNOWLEDGEMENTS This work was supported by grants from the National Institute Health (NIH) grants R01 DK124549 to B. A. Kerlin and R01 HL126974 and R01 HL147894 to A. S. Wolberg as well as a research endowment from the George and Elizabeth Kelly Foundation (Lewis Center, OH) to B. A. Kerlin. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors are indebted to Samir V. Parikh, Brad H. Rovin, and William E. Smoyer for recruiting patients into the Columbus Nephrotic Syndrome Cohort. pDTR transgenic rat breeders were a kind gift from Roger C. Wiggins and Bruce A. Molitoris. REFERENCES ↵ Bhasin N , Roe DJ , Saboda K , Journeycake J , Moreno V , Lentz SR . Association of low serum albumin with venous thrombosis in pediatric patients . Thromb Res . 2022 ; 218 : 48 – 51 . doi: 10.1016/j.thromres.2022.08.008 . OpenUrl CrossRef PubMed Folsom AR , Lutsey PL , Heckbert SR , Cushman M . Serum albumin and risk of venous thromboembolism . Thromb Haemost . 2010 ; 104 : 100 – 4 . doi: 10.1160/TH09-12-0856 . OpenUrl CrossRef PubMed ↵ Ronit A , Kirkegaard-Klitbo DM , Dohlmann TL , Lundgren J , Sabin CA , Phillips AN , Nordestgaard BG , Afzal S . Plasma Albumin and Incident Cardiovascular Disease: Results From the CGPS and an Updated Meta-Analysis . Arterioscler Thromb Vasc Biol . 2020 ; 40 : 473 – 82 . doi: 10.1161/ATVBAHA.119.313681 . OpenUrl CrossRef PubMed ↵ Violi F , Novella A , Pignatelli P , Castellani V , Tettamanti M , Mannucci PM , Nobili A , Group RS . Low serum albumin is associated with mortality and arterial and venous ischemic events in acutely ill medical patients. Results of a retrospective observational study . Thromb Res . 2023 ; 225 : 1 – 10 . doi: 10.1016/j.thromres.2023.02.013 . OpenUrl CrossRef PubMed ↵ Valeriani E , Pannunzio A , Palumbo IM , Bartimoccia S , Cammisotto V , Castellani V , Porfidia A , Pignatelli P , Violi F . Risk of venous thromboembolism and arterial events in patients with hypoalbuminemia: a comprehensive meta-analysis of more than 2 million patients . J Thromb Haemost . 2024 ; 22 : 2823 – 33 . doi: 10.1016/j.jtha.2024.06.018 . OpenUrl CrossRef PubMed ↵ Mahalingasivam V , Booth J , Sheaff M , Yaqoob M . Nephrotic syndrome in adults . Acute Med . 2018 ; 17 : 36 – 43 . OpenUrl PubMed Wang CS , Greenbaum LA . Nephrotic Syndrome . Pediatr Clin North Am . 2019 ; 66 : 73 – 85 . doi: 10.1016/j.pcl.2018.08.006 . OpenUrl CrossRef PubMed ↵ Kerlin BA , Ayoob R , Smoyer WE . Epidemiology and pathophysiology of nephrotic syndrome-associated thromboembolic disease . Clin J Am Soc Nephrol . 2012 ; 7 : 513 – 20 . doi: 10.2215/CJN.10131011 . OpenUrl Abstract / FREE Full Text ↵ Mahmoodi BK , ten Kate MK , Waanders F , Veeger NJ , Brouwer JL , Vogt L , Navis G , van der Meer J . High absolute risks and predictors of venous and arterial thromboembolic events in patients with nephrotic syndrome: results from a large retrospective cohort study. Circulation . 2008 ; 117 : 224-30 . doi: 10.1161/CIRCULATIONAHA.107.716951 . OpenUrl CrossRef PubMed ↵ Thomson C , Forbes CD , Prentice CR , Kennedy AC . Changes in blood coagulation and fibrinolysis in the nephrotic syndrome . Q J Med . 1974 ; 43 : 399 – 407 . OpenUrl PubMed ↵ Collet JP , Mishal Z , Lesty C , Mirshahi M , Peyne J , Baumelou A , Bensman A , Soria J , Soria C . Abnormal fibrin clot architecture in nephrotic patients is related to hypofibrinolysis: influence of plasma biochemical modifications: a possible mechanism for the high thrombotic tendency? Thromb Haemost . 1999 ; 82 : 1482 – 9 . OpenUrl CrossRef PubMed Web of Science Gandrille S , Jouvin MH , Toulon P , Remy P , Fiessinger JN , Roncato M , Moatti N , Aiach M . A study of fibrinogen and fibrinolysis in 10 adults with nephrotic syndrome . Thromb Haemost . 1988 ; 59 : 445 – 50 . OpenUrl CrossRef PubMed Ishikawa T , Nakajima Y , Omae T , Ogiwara K , Nogami K . Comprehensive coagulation and fibrinolytic potential in the acute phase of pediatric patients with idiopathic nephrotic syndrome evaluated by whole blood-based rotational thromboelastometry . Pediatr Nephrol . 2022 ; 37 : 1605 – 14 . doi: 10.1007/s00467-021-05366-4 . OpenUrl CrossRef PubMed ↵ Kerlin BA , Waller AP , Sharma R , Chanley MA , Nieman MT , Smoyer WE . Disease Severity Correlates with Thrombotic Capacity in Experimental Nephrotic Syndrome . J Am Soc Nephrol . 2015 ; 26 : 3009 – 19 . doi: 10.1681/ASN.2014111097 . OpenUrl Abstract / FREE Full Text ↵ Shimizu K . Thromboelastographic analysis of coagulation and fibrinolysis in rats with nephrotic syndrome induced by daunomycin . Nihon Jinzo Gakkai Shi . 1991 ; 33 : 491 – 6 . OpenUrl PubMed ↵ Lisman T . Decreased Plasma Fibrinolytic Potential As a Risk for Venous and Arterial Thrombosis . Seminars in thrombosis and hemostasis . 2017 ; 43 : 178 – 84 . doi: 10.1055/s-0036-1585081 . OpenUrl CrossRef PubMed ↵ Lisman T , de Groot PG , Meijers JC , Rosendaal FR . Reduced plasma fibrinolytic potential is a risk factor for venous thrombosis . Blood . 2005 ; 105 : 1102 – 5 . doi: 10.1182/blood-2004-08-3253 . OpenUrl Abstract / FREE Full Text ↵ Carr ME , Jr . Turbidimetric evaluation of the impact of albumin on the structure of thrombin-mediated fibrin gelation . Haemostasis . 1987 ; 17 : 189 – 94 . doi: 10.1159/000215742 . OpenUrl CrossRef PubMed Web of Science ↵ Gandrille S , Aiach M . Albumin concentration influences fibrinolytic activity in plasma and purified systems . Fibrinolysis . 1990 ; 4 : 225 – 32 . doi: 10.1016/0268-9499(90)90019-g . OpenUrl CrossRef ↵ Galanakis DK , Lane BP , Simon SR . Albumin modulates lateral assembly of fibrin polymers: evidence of enhanced fine fibril formation and of unique synergism with fibrinogen . Biochemistry . 1987 ; 26 : 2389 – 400 . doi: 10.1021/bi00382a046 . OpenUrl CrossRef PubMed ↵ de Sain-van der Velden MGM , Smolders HC , van Rijn HJM , Voorbij HAM . Does albumin play a role in fibrinolysis by its inhibition of plasminogen activation? Fibrinolysis and Proteolysis . 2000 ; 14 : 242 – 6 . doi: 10.1054/fipr.2000.0067 . OpenUrl CrossRef ↵ Nair CH , Azhar A , Dhall DP . The effects of some plasma proteins on fibrin network structure . Blood Coagul Fibrinolysis . 1990 ; 1 : 469 – 73 . doi: 10.1097/00001721-199010000-00019 . OpenUrl CrossRef PubMed ↵ Torbet J . Fibrin assembly in human plasma and fibrinogen/albumin mixtures . Biochemistry . 1986 ; 25 : 5309 – 14 . doi: 10.1021/bi00366a048 . OpenUrl CrossRef PubMed ↵ Gounden V , Vashisht R , Jialal I. Hypoalbuminemia . StatPearls: StatPearls Publishing Copyright © 2023, StatPearls Publishing LLC ., 2023 . ↵ Cushman M . Epidemiology and risk factors for venous thrombosis . Semin Hematol . 2007 ; 44 : 62 – 9 . doi: 10.1053/j.seminhematol.2007.02.004 . OpenUrl CrossRef PubMed Web of Science ↵ Chen Z , He Y , Shi B , Yang D . Human serum albumin from recombinant DNA technology: challenges and strategies . Biochimica et biophysica acta . 2013 ; 1830 : 5515 – 25 . doi: 10.1016/j.bbagen.2013.04.037 . OpenUrl CrossRef ↵ Pippin JW , Brinkkoetter PT , Cormack-Aboud FC , Durvasula RV , Hauser PV , Kowalewska J , Krofft RD , Logar CM , Marshall CB , Ohse T , Shankland SJ . Inducible rodent models of acquired podocyte diseases . American journal of physiology Renal physiology . 2009 ; 296 : F213 – 29 . doi: 10.1152/ajprenal.90421.2008 . OpenUrl CrossRef PubMed Web of Science ↵ Wharram BL , Goyal M , Wiggins JE , Sanden SK , Hussain S , Filipiak WE , Saunders TL , Dysko RC , Kohno K , Holzman LB , Wiggins RC . Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene . J Am Soc Nephrol . 2005 ; 16 : 2941 – 52 . doi: 10.1681/ASN.2005010055 . OpenUrl Abstract / FREE Full Text ↵ Esumi H , Okui M , Sato S , Sugimura T , Nagase S . Absence of albumin mRNA in the liver of analbuminemic rats . Proc Natl Acad Sci U S A . 1980 ; 77 : 3215 – 9 . doi: 10.1073/pnas.77.6.3215 . OpenUrl Abstract / FREE Full Text ↵ Esumi H , Takahashi Y , Sato S , Nagase S , Sugimura T . A seven-base-pair deletion in an intron of the albumin gene of analbuminemic rats . Proc Natl Acad Sci U S A . 1983 ; 80 : 95 – 9 . doi: 10.1073/pnas.80.1.95 . OpenUrl Abstract / FREE Full Text ↵ Nagase S , Shimamune K , Shumiya S . Albumin-deficient rat mutant. Science . 1979 ; 205 : 590-1 . doi: 10.1126/science.451621 . OpenUrl CrossRef PubMed ↵ Sugiyama K , Emori T , Nagase S . Absence of albumin in tissues of analbuminemic rats . J Biochem . 1980 ; 88 : 1413 – 7 . doi: 10.1093/oxfordjournals.jbchem.a133110 . OpenUrl CrossRef PubMed ↵ Sharma R , Waller AP , Agrawal S , Wolfgang KJ , Luu H , Shahzad K , Isermann B , Smoyer WE , Nieman MT , Kerlin BA . Thrombin-Induced Podocyte Injury Is Protease-Activated Receptor Dependent . J Am Soc Nephrol . 2017 ; 28 : 2618 – 30 . doi: 10.1681/ASN.2016070789 . OpenUrl Abstract / FREE Full Text ↵ Waller AP , Agrawal S , Wolfgang KJ , Kino J , Chanley MA , Smoyer WE , Kerlin BA , Pediatric Nephrology Research C. Nephrotic syndrome-associated hypercoagulopathy is alleviated by both pioglitazone and glucocorticoid which target two different nuclear receptors . Physiol Rep . 2020 ; 8 : e14515 . doi: 10.14814/phy2.14515 . OpenUrl CrossRef PubMed ↵ Fukuda A , Wickman LT , Venkatareddy MP , Sato Y , Chowdhury MA , Wang SQ , Shedden KA , Dysko RC , Wiggins JE , Wiggins RC . Angiotensin II-dependent persistent podocyte loss from destabilized glomeruli causes progression of end stage kidney disease . Kidney Int . 2012 ; 81 : 40 – 55 . doi: 10.1038/ki.2011.306 . OpenUrl CrossRef PubMed Web of Science ↵ Waller AP , Troost JP , Parikh SV , Wolfgang KJ , Rovin BH , Nieman MT , Smoyer WE , Kretzler M , Kerlin BA , Investigators N . Nephrotic syndrome disease activity is proportional to its associated hypercoagulopathy . Thromb Res . 2021 ; 201 : 50 – 9 . doi: 10.1016/j.thromres.2021.02.007 . OpenUrl CrossRef PubMed ↵ Abdelghani E , Waller AP , Wolfgang KJ , Stanek JR , Parikh SV , Rovin BH , Smoyer WE , Kerlin BA, the PI, the NI. Exploring the Role of Antithrombin in Nephrotic Syndrome-Associated Hypercoagulopathy: A Multi-Cohort Study and Meta-Analysis . Clin J Am Soc Nephrol . 2023 ; 18 : 234 – 44 . doi: 10.2215/CJN.0000000000000047 . OpenUrl CrossRef PubMed ↵ Ueno T , Hirayama S , Ito M , Nishioka E , Fukushima Y , Satoh T , Idei M , Horiuchi Y , Shoji H , Ohmura H , Shimizu T , Miida T . Albumin concentration determined by the modified bromocresol purple method is superior to that by the bromocresol green method for assessing nutritional status in malnourished patients with inflammation . Ann Clin Biochem . 2013 ; 50 : 576 – 84 . doi: 10.1177/0004563213480137 . OpenUrl CrossRef PubMed ↵ Dirkmann D , Gorlinger K , Gisbertz C , Dusse F , Peters J . Factor XIII and tranexamic acid but not recombinant factor VIIa attenuate tissue plasminogen activator-induced hyperfibrinolysis in human whole blood . Anesth Analg . 2012 ; 114 : 1182 – 8 . doi: 10.1213/ANE.0b013e31823b6683 . OpenUrl CrossRef PubMed ↵ A Practical Guide to Haemostasis: Thromboelastography and Rotational Thromboelastometry . Practical-Haemostasiscom . https://practical-haemostasis.com/Miscellaneous/Global%20Assays/teg_rotem.html : Sang Medicine , 2022 . ↵ Dietrich W , Nicklisch S , Koster A , Spannagl M , Giersiefen H , van de Locht A. CU-2010--a novel small molecule protease inhibitor with antifibrinolytic and anticoagulant properties . Anesthesiology . 2009 ; 110 : 123 – 30 . doi: 10.1097/ALN.0b013e318191408c . OpenUrl CrossRef PubMed Web of Science ↵ Mao SJ , Tucci MA . A colorimetric assay for measuring the lysis of a plasma clot . Anal Biochem . 1991 ; 192 : 6 – 10 . doi: 10.1016/0003-2697(91)90174-r . OpenUrl CrossRef PubMed ↵ Zucker M , Seligsohn U , Salomon O , Wolberg AS . Abnormal plasma clot structure and stability distinguish bleeding risk in patients with severe factor XI deficiency . J Thromb Haemost . 2014 ; 12 : 1121 – 30 . doi: 10.1111/jth.12600 . OpenUrl CrossRef PubMed ↵ Machlus KR , Cardenas JC , Church FC , Wolberg AS . Causal relationship between hyperfibrinogenemia, thrombosis, and resistance to thrombolysis in mice . Blood . 2011 ; 117 : 4953 – 63 . doi: 10.1182/blood-2010-11-316885 . OpenUrl Abstract / FREE Full Text ↵ Wolberg AS , Monroe DM , Roberts HR , Hoffman M . Elevated prothrombin results in clots with an altered fiber structure: a possible mechanism of the increased thrombotic risk . Blood . 2003 ; 101 : 3008 – 13 . doi: 10.1182/blood-2002-08-2527 . OpenUrl Abstract / FREE Full Text ↵ Byrnes JR , Duval C , Wang Y , Hansen CE , Ahn B , Mooberry MJ , Clark MA , Johnsen JM , Lord ST , Lam WA , Meijers JC , Ni H , Ariens RA , Wolberg AS . Factor XIIIa-dependent retention of red blood cells in clots is mediated by fibrin alpha-chain crosslinking . Blood . 2015 ; 126 : 1940 – 8 . doi: 10.1182/blood-2015-06-652263 . OpenUrl Abstract / FREE Full Text ↵ Aleman MM , Walton BL , Byrnes JR , Wang JG , Heisler MJ , Machlus KR , Cooley BC , Wolberg AS . Elevated prothrombin promotes venous, but not arterial, thrombosis in mice . Arterioscler Thromb Vasc Biol . 2013 ; 33 : 1829 – 36 . doi: 10.1161/ATVBAHA.113.301607 . OpenUrl Abstract / FREE Full Text ↵ Blomback B , Carlsson K , Fatah K , Hessel B , Procyk R . Fibrin in human plasma: gel architectures governed by rate and nature of fibrinogen activation . Thromb Res . 1994 ; 75 : 521 – 38 . doi: 10.1016/0049-3848(94)90227-5 . OpenUrl CrossRef PubMed Web of Science Carr ME , Jr ., Alving BM . Effect of fibrin structure on plasmin-mediated dissolution of plasma clots . Blood Coagul Fibrinolysis . 1995 ; 6 : 567 – 73 . doi: 10.1097/00001721-199509000-00011 . OpenUrl CrossRef PubMed Web of Science Carr ME , Jr ., Dent RM , Carr SL . Abnormal fibrin structure and inhibition of fibrinolysis in patients with multiple myeloma . J Lab Clin Med . 1996 ; 128 : 83 – 8 . doi: 10.1016/s0022-2143(96)90116-x . OpenUrl CrossRef PubMed Web of Science Carrell N , Gabriel DA , Blatt PM , Carr ME , McDonagh J . Hereditary dysfibrinogenemia in a patient with thrombotic disease . Blood . 1983 ; 62 : 439 – 47 . OpenUrl Abstract / FREE Full Text Collet JP , Park D , Lesty C , Soria J , Soria C , Montalescot G , Weisel JW . Influence of fibrin network conformation and fibrin fiber diameter on fibrinolysis speed: dynamic and structural approaches by confocal microscopy . Arterioscler Thromb Vasc Biol . 2000 ; 20 : 1354 – 61 . doi: 10.1161/01.atv.20.5.1354 . OpenUrl Abstract / FREE Full Text ↵ Collet JP , Soria J , Mirshahi M , Hirsch M , Dagonnet FB , Caen J , Soria C . Dusart syndrome: a new concept of the relationship between fibrin clot architecture and fibrin clot degradability: hypofibrinolysis related to an abnormal clot structure . Blood . 1993 ; 82 : 2462 – 9 . OpenUrl Abstract / FREE Full Text Gabriel DA , Muga K , Boothroyd EM . The effect of fibrin structure on fibrinolysis . J Biol Chem . 1992 ; 267 : 24259 – 63 . OpenUrl Abstract / FREE Full Text ↵ Meh DA , Mosesson MW , DiOrio JP , Siebenlist KR , Hernandez I , Amrani DL , Stojanovich L . Disintegration and reorganization of fibrin networks during tissue-type plasminogen activator-induced clot lysis . Blood Coagul Fibrinolysis . 2001 ; 12 : 627 – 37 . doi: 10.1097/00001721-200112000-00003 . OpenUrl CrossRef PubMed ↵ Wolberg AS . Thrombin generation and fibrin clot structure . Blood Rev . 2007 ; 21 : 131 – 42 . doi: 10.1016/j.blre.2006.11.001 . OpenUrl CrossRef PubMed Web of Science ↵ Lau SO , Tkachuck JY , Hasegawa DK , Edson JR . Plasminogen and antithrombin III deficiencies in the childhood nephrotic syndrome associated with plasminogenuria and antithrombinuria . J Pediatr . 1980 ; 96 : 390 – 2 . doi: 10.1016/s0022-3476(80)80678-0 . OpenUrl CrossRef PubMed ↵ Quimby FW . The clinical chemistry of laboratory animals . Philadelphia : Taylor & Francis , 1999 . ↵ Huntington JA , Stein PE . Structure and properties of ovalbumin . J Chromatogr B Biomed Sci Appl . 2001 ; 756 : 189 – 98 . doi: 10.1016/s0378-4347(01)00108-6 . OpenUrl CrossRef PubMed ↵ Hunt LT , Dayhoff MO . A surprising new protein superfamily containing ovalbumin, antithrombin-III, and alpha 1-proteinase inhibitor . Biochem Biophys Res Commun . 1980 ; 95 : 864 – 71 . doi: 10.1016/0006-291x(80)90867-0 . OpenUrl CrossRef PubMed Web of Science ↵ Hugenholtz GC , Macrae F , Adelmeijer J , Dulfer S , Porte RJ , Lisman T , Ariens RA . Procoagulant changes in fibrin clot structure in patients with cirrhosis are associated with oxidative modifications of fibrinogen . J Thromb Haemost . 2016 ; 14 : 1054 – 66 . doi: 10.1111/jth.13278 . OpenUrl CrossRef PubMed Undas A . Prothrombotic Fibrin Clot Phenotype in Patients with Deep Vein Thrombosis and Pulmonary Embolism: A New Risk Factor for Recurrence . Biomed Res Int . 2017 ; 2017 : 8196256 . doi: 10.1155/2017/8196256 . OpenUrl CrossRef PubMed Undas A , Ariens RA . Fibrin clot structure and function: a role in the pathophysiology of arterial and venous thromboembolic diseases . Arterioscler Thromb Vasc Biol . 2011 ; 31 : e88 – 99 . doi: 10.1161/ATVBAHA.111.230631 . OpenUrl Abstract / FREE Full Text Sjoland JA , Sidelmann JJ , Brabrand M , Pedersen RS , Pedersen JH , Esbensen K , Standeven KF , Ariens RA , Gram J . Fibrin clot structure in patients with end-stage renal disease . Thromb Haemost . 2007 ; 98 : 339 – 45 . OpenUrl CrossRef PubMed Web of Science Hudson NE . Biophysical Mechanisms Mediating Fibrin Fiber Lysis . Biomed Res Int . 2017 ; 2017: 2748340 . doi: 10.1155/2017/2748340 . OpenUrl CrossRef Undas A . Fibrin clot properties and their modulation in thrombotic disorders . Thromb Haemost . 2014 ; 112 : 32 – 42 . doi: 10.1160/TH14-01-0032 . OpenUrl CrossRef PubMed Pieters M , Wolberg AS . Fibrinogen and fibrin: An illustrated review . Res Pract Thromb Haemost . 2019 ; 3 : 161 – 72 . doi: 10.1002/rth2.12191 . OpenUrl CrossRef PubMed ↵ Risman RA , Kirby NC , Bannish BE , Hudson NE , Tutwiler V . Fibrinolysis: an illustrated review . Res Pract Thromb Haemost . 2023 ; 7 : 100081 . doi: 10.1016/j.rpth.2023.100081 . OpenUrl CrossRef ↵ Koga S , Okajima K , Inoue M , Okabe H , Araki H , Nagase S , Takatsuki K . Dynamic aspects of thrombus formation in mutant analbuminemic rats . Thromb Res . 1990 ; 58 : 633 – 43 . doi: 10.1016/0049-3848(90)90309-z . OpenUrl CrossRef PubMed ↵ Mutch NJ , Medcalf RL . The fibrinolysis renaissance. J Thromb Haemost . 2023 ; 21 : 3304-16 . doi: 10.1016/j.jtha.2023.09.012 . OpenUrl CrossRef PubMed ↵ Roth S , Jansen C , M’Pembele R , Stroda A , Boeken U , Akhyari P , Lichtenberg A , Hollmann MW , Huhn R , Lurati Buse G , Aubin H . Fibrinogen-Albumin-Ratio is an independent predictor of thromboembolic complications in patients undergoing VA-ECMO . Sci Rep . 2021 ; 11 : 16648 . doi: 10.1038/s41598-021-95689-x . OpenUrl CrossRef ↵ Bi X , Su Z , Yan H , Du J , Wang J , Chen L , Peng M , Chen S , Shen B , Li J . Prediction of severe illness due to COVID-19 based on an analysis of initial Fibrinogen to Albumin Ratio and Platelet count . Platelets . 2020 ; 31 : 674 – 9 . doi: 10.1080/09537104.2020.1760230 . OpenUrl CrossRef PubMed ↵ Zabczyk M , Stachowicz A , Natorska J , Olszanecki R , Wisniewski JR , Undas A . Plasma fibrin clot proteomics in healthy subjects: Relation to clot permeability and lysis time . J Proteomics . 2019 ; 208 : 103487 . doi: 10.1016/j.jprot.2019.103487 . 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