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

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Mengyun Xiao ◽  
Stefanie Hammer ◽  
Wissam A Khalel ◽  
Lisann Pelzl ◽  
Bernhard N Bohnert ◽  
...  

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

2017 ◽  
Vol 45 (5) ◽  
pp. 562-568 ◽  
Author(s):  
P. C. A. Kam ◽  
J. P. C. Liou ◽  
K. X. F. Yang

We evaluated the effects of haemodilution with either dextran 40 or 0.9% normal saline on coagulation in vitro using rotational thromboelastometry (ROTEM®, Pentapharm Co., Munich, Germany) and multiple electrode aggregometry (Multiplate® Platelet Function Analyser, Dynabyte, Munich, Germany). Venous blood samples obtained from 20 healthy volunteers were diluted in vitro with dextran 40 or normal saline by 5%, 10% and 15%. Fibrinogen concentration, ROTEM-EXTEM® (screening test for the extrinsic coagulation pathway), FIBTEM® (an EXTEM-based assay of the fibrin component of clot) parameters including coagulation time, clot formation time, alpha angle, maximum clot firmness and lysis index were measured in the undiluted sample and at each level of haemodilution. Dextran 40 at 15% haemodilution significantly prolonged coagulation time, clot formation time and significantly decreased the alpha angle and maximal clot firmness (EXTEM amplitude at five minutes [A5] and ten minutes [A10]) compared with normal saline. The FIBTEM assay (maximal clot firmness and FIBTEM A5 and A10) showed a marked decrease in maximal clot firmness at all dilutions suggesting impaired fibrinogen activity and a risk of bleeding. Multiple electrode aggregometry did not demonstrate any platelet dysfunction. Haemodilution with dextran 40 causes significant impairment in clot formation and strength compared to saline haemodilution and undiluted blood. At the levels of in vitro haemodilution designed to reflect the clinical use of dextran infusions, no significant fibrinolysis or platelet inhibition was observed.


2014 ◽  
Vol 307 (5) ◽  
pp. L347-L354 ◽  
Author(s):  
Sotirios G. Zarogiannis ◽  
Brant M. Wagener ◽  
Susanna Basappa ◽  
Stephen Doran ◽  
Cilina A. Rodriguez ◽  
...  

Chlorine (Cl2) is a highly reactive oxidant gas that, when inhaled, may cause acute lung injury culminating in death from respiratory failure. In this study, we tested the hypothesis that exposure of mice to Cl2causes intra-alveolar and systemic activation of the coagulation cascade that plays an important role in development of lung injury. C57Bl/6 mice were exposed to Cl2(400 for 30 min or 600 ppm for 45 min) in environmental chambers and then returned to room air for 1 or 6 h. Native coagulation (NATEM) parameters such as blood clotting time and clot formation time were measured in whole blood by the viscoelastic technique. D-dimers and thrombin-anti-thrombin complexes were measured in both plasma and bronchoalveolar lavage fluid (BALF) by ELISA. Our results indicate that mice exposed to Cl2gas had significantly increased clotting time, clot formation time, and D-dimers compared with controls. The thrombin-anti-thrombin complexes were also increased in the BALF of Cl2exposed animals. To test whether increased coagulation contributed to the development of acute lung injury, mice exposed to Cl2and returned to room air were treated with aerosolized heparin or vehicle for 20 min. Aerosolized heparin significantly reduced protein levels and the number of inflammatory cells in the BALF at 6 h postexposure. These findings highlight the importance of coagulation abnormities in the development of Cl2-induced lung injury.


2020 ◽  
pp. 1098612X2095961
Author(s):  
James Mack Fudge ◽  
Bernie Page ◽  
Amy Mackrell ◽  
Inhyung Lee ◽  
Unity Jeffery

Objectives The aims of this study were to determine if there is increased risk of intraoperative bleeding in pregnant cats undergoing elective ovariohysterectomy (OHE), and to compare coagulation in queens in various stages of estrus and pregnancy subjected to elective OHE using a whole-blood viscoelastic assay. Methods Intraoperative blood loss was compared between non-pregnant and pregnant cats undergoing elective OHE. Viscoelastic evaluations of whole blood drawn pre- and postoperatively were performed using a point-of-care device measuring clot time (CT), clot formation time (CFT), alpha angle, maximum clot formation (MCF), amplitude at 10 and 20 mins (A10 and A20, respectively), and lysis index at 30 and 45 mins after MCF (LI30 and LI45, respectively). Results One hundred and ninety-three cats underwent OHE by a ventral midline approach. Median blood loss was greater for pregnant cats (2.0 ml, range <0.5–13 ml) than non-pregnant cats (<0.5 ml, range <0.5–15 ml; P <0.0001). Preoperatively, pregnant cats had a shorter median CFT (165 s vs 190.5 s), increased median A10 (31 from 25.5 VCM units) and A20 (38 from 35 VCM units), and a lower median LI45 (99% from 100%) than non-pregnant cats. Postoperatively, A10 and A20 increased, and LI30 and LI45 decreased in both non-pregnant and pregnant queens. In pregnant queens, mean CT also increased postoperatively. Conclusions and relevance Pregnant cats were relatively hypercoagulable and had an increased rate of clot lysis than non-pregnant cats. Intraoperative blood loss was increased in pregnant vs non-pregnant cats, but no clinically relevant bleeding conditions occurred.


2021 ◽  
Vol 10 (15) ◽  
pp. 3397
Author(s):  
Andreas G. Tsantes ◽  
Dimitrios V. Papadopoulos ◽  
Ioannis G. Trikoupis ◽  
Stavros Goumenos ◽  
Daniele Piovani ◽  
...  

Introduction: Coronavirus disease 2019 (COVID-19) in patients with hip fractures is associated with increased incidence of venous thromboembolism (VTE). The purpose of this study was to evaluate the hemostatic alterations of COVID-19 that are associated with a higher thrombotic risk using rotational thromboelastometry (ROTEM). Methods: A retrospective observational study was performed including 20 COVID-19 patients with hip fractures. To compare the coagulopathy of patients with mild COVID-19 and hip fractures with the coagulopathy associated with each of these two conditions separately, we used two previously recruited groups of patients; 198 hip fracture patients without COVID-19 and 21 COVID-19 patients without hip fractures. The demographics, clinical parameters, conventional coagulation parameters and ROTEM findings of the three groups were analyzed and compared. Results: COVID-19 hip fracture patients had higher amplitude of clot firmness at 10 min (p < 0.001), higher alpha angle (p < 0.001), higher lysis index at 60 min (p < 0.001), and shorter clot formation time (p < 0.001) than non-COVID-19 hip fracture patients, indicating increased clot strength and impaired fibrinolysis due to COVID-19. The value of lysis index at 60 min (99%) in COVID-19 patients with hip fractures was consistent with fibrinolysis shut down. Multivariable linear regression analysis further confirmed that COVID-19 resulted in increased amplitude of clot firmness at 10 min (p < 0.001), increased maximum clot firmness (p < 0.001), increased lysis index at 60 min (p < 0.001) and increased alpha angle (p < 0.001), but significantly shortened clot formation time (p < 0.001). Discussion: The higher thrombotic risk in COVID-19 patients with hip fractures is characterized by increased clot strength and fibrinolysis shutdown, as shown by ROTEM findings. Further prospective studies are warranted to evaluate the need for modification of thromboprophylaxis to balance the hemostatic derangements of COVID-19 patients with hip fractures.


2010 ◽  
Vol 103 (02) ◽  
pp. 344-350 ◽  
Author(s):  
Tzipi Strauss ◽  
Yael Levy-Shraga ◽  
Bruria Ravid ◽  
Irit Schushan-Eisen ◽  
Ayala Maayan-Metzger ◽  
...  

SummaryEvaluation of clot formation in neonates is troublesome. Our aim was to investigate cord blood clot formation of pre-term versus full-term infants and adults, using rotating thromboelastogram (ROTEM®, Pentafarm, Munich, Germany). ROTEM was investigated in cord blood of 184 full-term and 47 pre-term infants. Measurements of the clotting time (CT), clot formation time (CFT) and maximal clot firmness (MCF) were obtained in order to asses reference values for this age group, and compare between full-term and pre-term neonates and compared to adult controls. For each infant demographic information and data regarding pregnancy and delivery were gathered. Infants were prospectively followed until discharge. CT and CFT were significantly shorter among pre-term and term infants as compared to adults [median CT: 185, 194, 293 seconds respectively, p≤0.001, CFT: 80, 76, 103 seconds respectively, p≤0.001). MCF was lower in pre-term and term as compared to adults (p≤0.001) with significantly lower values in pre-term as compared to full-term neonates (p=0.004). Clotting time and MCF correlated with gestational age (R=0.132, p=0.045, R= 0.259, p<0.001, respectively). No association was found between any ROTEM values and the occurrence of post-natal complications in infants of our study group. This is the first study assessing clot formation by ROTEM in pre-term infants. Clot formation parameters of term and premature infants correlated with gestational age. The predictive value of clot formation tests in neonates deserves further attention.


Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3738-3738
Author(s):  
Raul Justo Sanz ◽  
Elena Monzón Manzano ◽  
Ihosvany Fernandez-Bello ◽  
Teresa Álvarez-Roman ◽  
Mónica Martín ◽  
...  

Abstract Background: The treatment goal for patients with immune thrombocytopenia (ITP) is to raise platelet counts to levels that minimize or stop bleeding. Thrombopoietin receptor agonists (TPO-RAs) have been successfully and extensively employed as second-line therapy for ITP. TPO-RAs, however, have a small but significant increase in the risk of thrombosis. Aim: The aim of this study was to elucidate the mechanisms involved in the procoagulant effect of TPO-RAs. Methods: This is a prospective, observational and transversal study. Eighty-two patients with chronic primary ITP, 40 without treatment for at least six months (UT-ITP) and 42 responders to TPO-RA therapy (64.3% with eltrombopag and 35.7 % with romiplostim) were recruited. One hundred and twelve healthy participants were also included. ROTEM® (naTEM test: only recalcification) was performed on platelet rich plasma adjusted to a platelet count of 25 x 109/L. Clotting time (CT, time from start of measurement until 2 mm of amplitude [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2 mm amplitude [in degrees]), maximum clot firmness, which reflects the maximum tensile strength of the thrombus (MCF, [in mm]) and LI60, which describes the percentage of maximum clot strength present at 60 min (in %), were recorded. Surface exposure of phosphatidylserine (PS), active caspase-3, -8 or -9 and prothrombinase complex binding to platelets were assessed by flow cytometry. Plasma and platelet levels of PAI-1 were determined by ELISA (eBioscience Ltd., Hatfield, United Kingdom). The effect of TPO and romiplostim on PAI-1 content of MEG-01 cells was evaluated by Western blot. Three MEG-01 cell cultures were initiated simultaneously: control without drugs and treated with either TPO (100 ng/mL) or romiplostim (53 μg/mL). Samples were collected at the start and after 24, 48 and 72 hours to determine the PAI-1 content. The statistical analysis was performed using SPSS 9.0 software (SPSS Inc., Chicago, Illinois, USA). Results: The ROTEM® studies showed significant differences in the dynamics of clot formation when comparing the control with ITP samples. There was a delay in clot formation in the UT-ITP group, as observed by a prolonged CT [expressed as median (p25-p75): control: 516 (490- 633) s; UT-ITP: 938 (914-1348) s, p<0.001], and a diminished alpha angle (mean±SD; control: 61.7±5.6 degrees; UT-ITP: 49.2±7.3 degrees, p<0.05). Nevertheless, samples from patients with UT-ITP reached the same MCF as those from healthy controls (control: 45.3±2.4 mm; UT-ITP: 46.9±3.7 mm). On the other hand, patients with ITP undergoing TPO-RA therapy presented an initial clot formation similar to that of the control group [expressed as median (p25-p75): CT, 672 (598-928) s; alpha angle, 55.8±5.8 degrees] but achieved a higher MCF (53.1±4.5 mm, p<0.05) and a reduced clot lysis after 60 min (control: 91.8±4.0%; UT-ITP: 93.7±4.0%, TPO-RA ITP: 97.6±1.7, p<0.05). Higher values of MCF observed with platelets from ITP patients treated with TPO-RAs might be a consequence of their augmented apoptosis signs: platelets from this group exposed more PS than controls and this situation was accompanied by an increased activity of caspases-3,7, -8 and -9 (Figure 1 A and B). Moreover, platelets from ITP patients on treatment with TPO-RAs bound more prothrombinase complex than platelets from UT-ITP patients and healthy controls (Figure 1 C). Reduced clot lysis observed in ITP patients treated with TPO-RA was due, at least in part, to increased plasma and platelet levels of PAI-1 (Table 1). Increase in platelet content of PAI-1 might be the result of the effect of TPO-RAs during megakaryopoiesis since treatments of MEG-01 cells with TPO or romiplostim induced a 3-fold increase in their endogenous PAI-1 content after an incubation period of 48 hs. Conclusion: The patients with ITP undergoing TPO-RAs therapy presented a procoagulant profile due to the formation of a more fibrinolysis-resistant clot because of increased platelet and plasma PAI-1 levels. Moreover, platelets from this group of patients showed more signs of apoptosis that causes a higher exposure of PS and, consequently, a larger surface for the binding of the prothrombinase complex. Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). Disclosures Álvarez-Roman: SOBI: Consultancy; NovoNordisk: Consultancy; Shire: Consultancy. Jimenez-Yuste:Grifols: Consultancy, Research Funding; Octapharma: Consultancy, Research Funding; CSL Behring: Consultancy; Bayer: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Shire: Consultancy, Research Funding; Sobi: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; NovoNordisk: Consultancy, Research Funding. Butta:FIS-Fondos FEDER: Research Funding.


Author(s):  
Alexander A. Blazhko ◽  
Igor I. Shakhmatov ◽  
Igor V. Kovalev ◽  
Valeriy I. Kiselev ◽  
Yuliya A. Bondarchuk ◽  
...  

Suprathreshold physical activity causing distress in the organism can lead to the damage of various organs and systems including the hemostatic system. A modern integrated method of the hemostatic system assessment is thromboelastography. The purpose of the present study was to evaluate the state of the hemostatic system under one-time suprathreshold physical activity of various durations by means of thromboelastography. Experimental groups of rats were exposed to 4-hour and 8-hour physical activities in the form of forced running on a moving platform with the speed of 6-8 m/min. Immediately after the one-time physical activity, blood samples taken from rats were examined using the thrombelastograph in the Natem mode for 35 minutes. The 4-hour physical activity caused a reduction in coagulation time (CT) and an increase in the alpha angle and the maximum clot firmness (MCF). After the 8-hour activity, the thrombelastograph registered a reduction in coagulation time (CT), an increase in the alpha angle, a decrease in the clot formation time (CFT), a decrease in the maximum clot firmness (MCF), and a reduction in the maximum clot lysis (ML). The 4-hour physical activity resulted in partial activation of the hemostatic system without changing the fibrinolytic activity of blood plasm. The changes revealed in thromboelastography parameters indicate a high risk of the development of thrombotic readiness. The 8-hour physical activity causes a shift of the hemostatic system parameters in rats towards the increased clot formation: hypercoagulation, fibrinogen and platelet consumption, inhibition of fibrinolysis. The combination of changes in thromboelastogram parameters is indicative of the development of thrombotic readiness


2020 ◽  
Author(s):  
Brian O’Rourke ◽  
Sunny Nguyen ◽  
Arno W. Tilles ◽  
James A. Bynum ◽  
Andrew P Cap ◽  
...  

AbstractWhile mesenchymal stromal cells (MSCs) are an appealing therapeutic option for a range of clinical applications, their potential to induce clotting when used systemically remains a safety concern, particularly in hypercoagulable conditions, such as in patients with severe COVID-19, trauma, or cancers. Here, we tested a novel ex vivo approach aimed at improving the safety of MSC systemic administration by use of a bioreactor. In this device, MSCs are seeded on the outside of a hollow-fiber filter, sequestering them behind a hemocompatible membrane, while still maintaining cross talk with blood cells and circulating signaling molecules. The potential for these bioreactor MSCs to induce clots in coagulable plasma was compared against “free” MSCs, as a model of systemic administration, which were directly injected into the circuit. Our results showed that physical isolation of the MSCs via a bioreactor extends the time necessary for clot formation to occur when compared to “free” MSCs. Measurement of cell surface data indicates the presence of known clot inducing factors, namely tissue factor and phosphatidylserine. Results also showed that recovering cells and flushing the bioreactor prior to use further prolonged clot formation time. Further, application of this technology in two in vivo models did not require additional heparin to maintain target ACT levels relative to the acellular device. Taken together, the use of hollow fiber filters to house MSCs, if adopted clinically, could offer a novel method to control systemic MSC exposure and prolong clot formation time.


Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3153-3153 ◽  
Author(s):  
Seiji Kaku ◽  
Ken-ichi Suzuki ◽  
Toshiyuki Funatsu ◽  
Minori Saitoh ◽  
Hiroyuki Koshio ◽  
...  

Abstract The objective of this study was to evaluate the effects of direct factor Xa inhibitor, YM150 and its major in vivo metabolite, YM-222714, on clot formation and clot lysis compared with other anticoagulants, such as a direct thrombin inhibitor (melagatran), a pentasaccharide (fondaparinux), low molecular weight heparin (enoxaparin) and unfractionated heparin. To assess clot lysis, the tissue plasminogen activator (tPA)-induced clot lysis assay was used with human plasma triggered by low and high levels of tissue factor (TF). Under low TF conditions, clot formation was completely prevented by melagatran at 1 μmol/L, by fondaparinux at all concentrations examined (0.1 to 1 μg/mL), by enoxaparin at 0.3 and 1 IU/mL and by heparin at 0.1 and 0.3 U/mL. Even under high TF conditions, 0.3 U/mL heparin prevented any clot formation. Although melagatran, fondaparinux, enoxaparin, and heparin potently prevented plasma clot formation under low TF conditions, under high TF conditions they were less effective at prolonging the clotting time. Under both low and high TF conditions, YM150 and YM-222714 prolonged the clotting time in a concentration dependent manner at concentrations between 0.3 and 3 μmol/L. YM150 and YM-222714 significantly accelerated clot lysis under both low and high TF conditions, but their effects were most evident under high TF conditions. Lower concentrations of melagatran (0.1 and 0.3 μmol/L) enhanced clot lysis under low TF conditions, but under high TF conditions, enhancement of clot lysis required higher melagatran concentrations (0.3 μmol/L or more). Under high TF conditions, fondaparinux enhanced clot lysis only at the highest concentration tested (1 μg/mL). Enoxaparin and heparin enhanced clot lysis under low TF conditions at the lowest test concentrations (0.1 IU/mL and 0.03 U/mL, respectively). Both also enhanced clot lysis under high TF conditions, but their effect reached statistical significance only at higher concentrations (1 IU/mL and 0.1 U/mL, respectively). These results suggested that direct factor Xa inhibitors, YM150 and YM-222714, exert stable anticoagulant effects independently of TF concentration. Both inhibitors enhanced tPA-induced fibrinolysis in human plasma clotted via the extrinsic coagulation pathway. Useful characteristics of YM150 and YM-222714, such as a linear dose response and reliable anticoagulation independent of TF concentration, may lead to the creation of an anticoagulant that is easier to use in the clinical setting than existing products. Potentially beneficial antithrombotic effects, which can be promoted by accelerating endogenous fibrinolytic pathways, may further aid in the prevention or treatment of thrombosis.


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