Concentrations Of TPA Research Articles

Comparison of results obtained using clot-fibrinolysis waveform analysis and global fibrinolysis capacity assay with rotational thromboelastography

Global fibrinolysis assays detect the fibrinolysis time of clot dissolution using tissue-type plasminogen activator (tPA). Two such assays, clot-fibrinolysis waveform analysis (CFWA) and global fibrinolysis capacity (GFC) assay, were recently developed. These were compared with rotational thromboelastography (ROTEM). Healthy donor blood samples were divided into four groups based on tPA-spiked concentrations: 0, 100, 500, and 1000 ng/mL. CFWA and GFC fibrinolysis times, including 4.1 µg/mL and 100 ng/mL tPA in the assays, were determined, denoted as CFWA-Lys and GFC-Lys, respectively. Statistical differences were recognized between tPA concentrations of 0 and 500/1000 ng/mL for CFWA-Lys, and 0 and 100/500/1000 ng/mL for GFC-Lys. The correlation coefficients with lysis onset time (LOT) of extrinsic pathway evaluation and intrinsic pathway evaluation in ROTEM were statistically significant at 0.610 and 0.590 for CFWA-Lys, and 0.939 and 0.928 for GFC-Lys, respectively (p-values < 0.0001 for all correlations). Both assays showed significant correlations with ROTEM; however, the GFC assay proved to have better agreement with ROTEM compared with the CFWA assay. These assays have the potential to reflect a hyperfibrinolysis status with high tPA concentrations.

Fibrinolysis as a Causative Mechanism for Bleeding Complications on Extracorporeal Membrane Oxygenation: A Pilot Observational Prospective Study.

Extracorporeal membrane oxygenation (ECMO) is associated with a high risk of bleeding complications. The specific impact of ECMO on fibrinolysis remains unexplored. The objective of the current pilot observational prospective study was to investigate the longitudinal dynamics of fibrinolytic markers-i.e., changes over time-in the context of bleeding events in patients on ECMO. Longitudinal dynamics of contact phase components (kininogen and bradykinin) and fibrinolysis markers (tissue plasminogen activator [tPA], plasminogen activator inhibitor-1 [PAI-1], their complexes [tPA•PAI-1], plasmin-antiplasmin complexes, plasminogen, and D-dimer) were measured in patients undergoing venovenous and venoarterial ECMO, before implantation, at 0, 6, and 12 h after implantation, and daily thereafter. The cohort consisted of 30 patients (214 ECMO days). The concentrations of tPA, D-dimer, plasmin-antiplasmin complexes, PAI-1, and tPA•PAI-1 complexes were increased, whereas plasminogen decreased compared to normal values. A noteworthy divergence was observed between hemorrhagic and nonhemorrhagic patients: in bleeding patients, D-dimer, plasmin-antiplasmin, tPA, PAI-1, and tPA•PAI-1 followed an increasing kinetics before hemorrhage and then decreased to their baseline level; conversely, nonbleeding patients showed a decreasing kinetics in these markers. Also, D-dimer and tPA followed an increasing kinetics in bleeding patients compared to nonbleeding patients (median values for D-dimer dynamics: 1,080 vs. -440 ng/ml, P = 0.05; tPA dynamics: 0.130 vs. 0.100 nM, P = 0.038), and both markers significantly increased the day before hemorrhage. A tPA concentration above 0.304 nM was associated with bleeding events (odds ratio, 4.92; 95% CI, 1.01 to 24.08; P = 0.049). Contact activation induces fibrinolysis in ECMO patients, especially in patients experiencing bleeding. This finding supports the role of this mechanism as a possible causal factor for hemorrhages during ECMO and open new avenues for novel therapeutic perspectives.

Internal fibrinolysis of fibrin clots is driven by pore expansion

Blood clots, which are composed of blood cells and a stabilizing mesh of fibrin fibers, are critical in cessation of bleeding following injury. However, their action is transient and after performing their physiological function they must be resolved through a process known as fibrinolysis. Internal fibrinolysis is the degradation of fibrin by the endogenous or innate presence of lytic enzymes in the bloodstream; under healthy conditions, this process regulates hemostasis and prevents bleeding or clotting. Fibrin-bound tissue plasminogen activator (tPA) converts nearby plasminogen into active plasmin, which is bound to the fibrin network, breaking it down into fibrin degradation products and releasing the entrapped blood cells. It is poorly understood how changes in the fibrin structure and lytic protein ratios influence the biochemical regulation and behavior of internal fibrinolysis. We used turbidity kinetic tracking and microscopy paired with mathematical modeling to study fibrin structure and lytic protein ratios that restrict internal fibrinolysis. Analysis of simulations and experiments indicate that fibrinolysis is driven by pore expansion of the fibrin network. We show that this effect is strongly influenced by the ratio of fibrin:tPAwhen compared to absolute tPA concentration. Thus, it is essential to consider relative protein concentrations when studying internal fibrinolysis both experimentally and in the clinic. An improved understanding of effective internal lysis can aid in development of better therapeutics for the treatment of bleeding and thrombosis.

Postoperative Coagulation Changes in Patients after Epicardial Left Atrial Appendage Occlusion Varies Based on the Left Atrial Appendage Size.

Left atrial appendage occlusion affects systemic coagulation parameters, leading to additional patient-related benefits. The aim of this study was to investigate the differences in coagulation factor changes 6 months after epicardial left atrial appendage occlusion in patients with different LAA morphometries. This is the first study to analyze these relationships in detail. A prospective study of 22 consecutive patients was performed. Plasminogen, fibrinogen, tPA concentration, PAI-1, TAFI and computed tomography angiograms were performed. Patients were divided into subgroups based on left atrial appendage body and orifice diameter enlargement. The results of blood tests at baseline and six-month follow-up were compared. In a population with normal LAA body size and normal orifice diameter size, a significant decrease in analyzed clotting factors was observed between baseline and follow-up for all parameters except plasminogen. A significant decrease between baseline and follow-up was observed with enlarged LAA body size in all parameters except TAFI, in which it was insignificant and plasminogen, in which a significant increase was observed. Occlusion of the left atrial appendage is beneficial for systemic coagulation. Patients with a small LAA may benefit more from LAA closure in terms of stabilizing their coagulation factors associated with potential thromboembolic events in the future.

Ziqi Dihuang decoction ameliorates thrombosis in septic rats by inhitbiting plasminogen activator inhibitor-1.

Sepsis is now a global medical burden with high morbility and mortality. The focus of this study was to evaluate the effects of Ziqi Dihuang (ZQDH) decoction on inflammatory and thrombosis-related parameters in septic rats. A rat model of sepsis was established by cecal ligation and puncture (CLP). Male Sprague-Dawley rats were randomly divided into Sham group, CLP group, ZQDH-1ow group (0.735g/kg) and ZQDH-high group (1.47g/kg). Rats in ZQDH groups were given ZQDH decoction by gavage for 7 days before CLP. White blood cells (WBC), inflammatory cell infiltration of liver, kidney and lung, as well as serum levels of tumor necrosis factor (TNF-α), interleukin-6 (IL-6) and reactive oxygen species (ROS) were used to assess systemic inflammatory response. Coagulation and fibrinolytic indexes included platelet count, coagulation function, fibrin deposition, and levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) in serum, liver, kidney and lung. LPS rats showed significant changes in inflammatory and thrombosis-related parameters such as increased WBC and inflammatory factors, decreased platelet counts, and increased tPA and PAI-1 concentrations in serum and organs. ZQDH decoction pretreatment can significantly inhibit the infiltration of inflammatory cells in the lung, and inhibit the production of TNF-α, IL-6 and ROS in a dose-dependent manner. ZQDH decoction also ameliorated thrombocytopenia, renal fibrin deposition, and tPA and PAI-1 levels in serum and organs. These results suggest that ZQDH decoction can dose-dependently relieve systemic inflammatory injury and regulate fibrinolysis system in septic rats, which may be mediated by PAI-1.

Long-term assessment of cutaneous inflammation and treatment using optical resolution photoacoustic microscopy.

Cutaneous inflammation is an acute skin disease characterized by edema and vascular hyperplasia. Longitudinal monitoring of vasculature is crucial for studying the development of inflammation and evaluating the therapeutic efficacy of drugs. Optical-resolution photoacoustic microscopy (OR-PAM) is a hybrid imaging tool for non-invasive and label-free visualization of microcirculations with a capillary-scale spatial resolution. In this study, we assess the feasibility of OR-PAM for long-term monitoring of vascular changes in 12-O-Tetradecanoylphorbol-13-Acetate (TPA)-induced mouse models, as well as the corresponding treatment process. Quantitative vascular evaluation is conducted based on derived key parameters, including vessel length, branchpoint number, vessel area fraction, vessel diameter, fractal dimension, vessel tortuosity and ear thickness, which reveal that vascular morphological changes are highly dependent on the concentration of TPA and existence of therapeutic drugs. Furthermore, the results show the potential of OR-PAM in the clinical management of inflammation and as an effective tool to evaluate vascular responses to pharmacological interventions in vivo.

Plasma tissue-type plasminogen activator is associated with lipoprotein(a) and clinical outcomes in hospitalized patients with COVID-19

BackgroundPatients with COVID-19 have a higher risk of thrombosis and thromboembolism, but the underlying mechanism(s) remain to be fully elucidated. In patients with COVID-19, high lipoprotein(a) (Lp(a)) is positively associated with the risk of ischemic heart disease. Lp(a), composed of an apoB-containing particle and apolipoprotein(a) (apo(a)), inhibits the key fibrinolytic enzyme, tissue-type plasminogen activator (tPA). However, whether the higher Lp(a) associates with lower tPA activity, the longitudinal changes of these parameters in hospitalized patients with COVID-19, and their correlation with clinical outcomes are unknown. ObjectivesTo assess if Lp(a) associates with lower tPA activity in COVID-19 patients, and how in COVID-19 populations Lp(a) and tPA change post infection. MethodsEndogenous tPA enzymatic activity, tPA or Lp(a) concentration were measured in plasma from hospitalized patients with and without COVID-19. The association between plasma tPA and adverse clinical outcomes was assessed. ResultsIn hospitalized patients with COVID-19, we found lower tPA enzymatic activity and higher plasma Lp(a) than that in non–COVID-19 controls. During hospitalization, Lp(a) increased and tPA activity decreased, which associates with mortality. Among those who survived, Lp(a) decreased and tPA enzymatic activity increased during recovery. In patients with COVID-19, tPA activity is inversely correlated with tPA concentrations, thus, in another larger COVID-19 cohort, we utilized plasma tPA concentration as a surrogate to inversely reflect tPA activity. The tPA concentration was positively associated with death, disease severity, plasma inflammatory, and prothrombotic markers, and with length of hospitalization among those who were discharged. ConclusionHigh Lp(a) concentration provides a possible explanation for low endogenous tPA enzymatic activity, and poor clinical outcomes in patients with COVID-19.

Sensitivity and specificity of thromboelastography for hyperfibrinolysis: Comparison of TEG 5000 and TEG 6S CK LY30 systems.

The sensitivity and specificity of clot lysis at 30 minutes after maximum clot strength (LY30), as measured by thromboelastography (TEG), for clinically significant hyperfibrinolysis have not been compared across the 2 US Food and Drug Administration-approved instruments (the TEG 5000 and TEG 6s [Haemonetics]). We performed a retrospective, single-center analysis of these 2 instruments using the kaolin (CK) reagent. Local verification studies showed that the TEG 5000 and TEG 6s CK LY30 upper limits of normal (ULNs) were distinct (5.0% and 3.2%, respectively). Retrospective analysis of patient data showed that abnormal LY30 was 6 times more prevalent with the TEG 6s than with the TEG 5000 instrument. LY30 was a significant predictor of mortality with both instruments (TEG 6s: receiver operating characteristic [ROC] area under the curve [AUC] = 0.836, P ≤ .0001; TEG 5000: ROC AUC = 0.779, P = .028). The optimal LY30 cut point was determined based on these mortality data for each instrument. The TEG 6s showed superior mortality prediction than the TEG 5000 at lower LY30 levels (≥10%), with likelihood ratios of 8.22 and 2.62 for the TEG 6s and TEG 5000, respectively. Patients with a TEG 6s CK LY30 of 10% or higher were significantly more likely to die, receive cryoprecipitate, receive transfusions, or receive massive transfusion than patients with a TEG 6s LY30 of 3.3% to 9.9% (all P < .01). Patients with a TEG 5000 LY30 of 17.1% or higher were significantly more likely to die or use cryoprecipitate (P < .05); transfusion and massive transfusion protocol were not significantly different. Whole blood spiking studies showed that 70 ng/mL tissue plasminogen activator (tPA) achieved an average LY30 of approximately 10% for both instruments. CK LY30 above the ULN is a sensitive but not specific cutoff for hyperfibrinolysis. At least moderately elevated CK LY30 carries more clinical portent on the TEG 6s instrument than on the TEG 5000. These TEG instruments are not sensitive to low concentrations of tPA.

Blood tissue Plasminogen Activator (tPA) of liver origin contributes to neurovascular coupling involving brain endothelial N-Methyl-D-Aspartate (NMDA) receptors

BackgroundRegulation of cerebral blood flow (CBF) directly influence brain functions and dysfunctions and involves complex mechanisms, including neurovascular coupling (NVC). It was suggested that the serine protease tissue-type plasminogen activator (tPA) could control CNV induced by whisker stimulation in rodents, through its action on N-methyl-d-Aspartate receptors (NMDARs). However, the origin of tPA and the location and mechanism of its action on NMDARs in relation to CNV remained debated.MethodsHere, we answered these issues using tPANull mice, conditional deletions of either endothelial tPA (VECad-CreΔtPA) or endothelial GluN1 subunit of NMDARs (VECad-CreΔGluN1), parabioses between wild-type and tPANull mice, hydrodynamic transfection-induced deletion of liver tPA, hepatectomy and pharmacological approaches.ResultsWe thus demonstrate that physiological concentrations of vascular tPA, achieved by the bradykinin type 2 receptors-dependent production and release of tPA from liver endothelial cells, promote NVC, through a mechanism dependent on brain endothelial NMDARs.ConclusionsThese data highlight a new mechanism of regulation of NVC involving both endothelial tPA and NMDARs.

Деструкция фталевых кислот штаммом Paenarthrobacter sp. SA101, выделенным из сточных вод химического предприятия

Phthalic and terephthalic acids (PA and TPA) are widely used in the chemical industry as plasticizers, for the production of polymer materials, and therefore are common eco-pollutants. The ability of the strain Paenarthrobacter sp. SA101, isolated from a sample of a chemical plant wastewater (Perm), to growth on high concentrations of PA and TPA was studied. It was shown that strain SA101 is capable of efficient growth on these substrates at concentrations up to 20 g/l, which is shown for the representatives of the genus Paenarthrobacter for the first time. Higher strain growth parameters were recorded in media with PA, than in media with TPA: the maximum value of OD600 (1.64), the highest specific growth rate (0.073±0.003 h-1) and the shortest lag phase were observed in a medium with 10 g/l of PA. The highest specific growth rate (0.04±0.002 h‑1) and the maximum value of OD600 (1.6) were observed with 20 g/l of TPA, however, the lag phase of growth was longer than in the medium with 10 g/l of TPA. The strain SA101 was able to utilize 76% of PA in 39 hours and 74.2% of TPA in 71 hours of cultivation, when using these substrates at a concentration of 10 g/l. The cells of the strain SA101 contain a plasmid with a molecular weight of ~ 100 bp. The strain SA101 is promising for the development of biotechnological methods for cleaning phthalate-contaminated soils and industrial effluents.

Congruent identification of imbalanced fibrinolysis by 2 distinct clot lysis time assays

BackgroundA plasma-based clot lysis time (CLT) assay is an established research test to assess plasma fibrinolytic potential, with application in hyperfibrinolytic or hypofibrinolytic conditions. Interprotocol variations make comparisons between laboratories challenging. The aim of this study was to compare the results of 2 different CLT assays performed by 2 distinct research laboratories by using their own protocol. MethodsWe evaluated fibrinolysis in the plasma of 60 patients undergoing hepatobiliary surgery and in plasma from a healthy donor that was spiked with commonly used anticoagulant drugs (enoxaparin, dabigatran, and rivaroxaban) in 2 distinct laboratories (Aarhus and Groningen) by using 2 different assays that differ, among others, in tissue plasminogen activator (tPA) concentration. ResultsOverall conclusions on fibrinolytic potential in patients undergoing hepatobiliary surgery were similar between the 2 CLT assays, with hyperfibrinolytic and hypofibrinolytic profiles identified at the same time points during and after surgery. Severe hypofibrinolysis was less commonly reported in the Aarhus assay (36/319 samples; 11%) than in the Groningen assay (55/319 samples; 17%). No clot formation was observed in 31 of 319 samples in the Aarhus assay vs 0 of 319 samples in the Groningen assay. Clotting times increased much more profoundly on the addition of all 3 anticoagulants in the Aarhus assay. ConclusionsDespite the differences in laboratory, protocol, reagents, operator, data processing, and analysis, overall conclusions on fibrinolytic capacity are similar between the 2 laboratories. With a higher concentration of tPA in the Aarhus assay, the test becomes less sensitive for the detection of hypofibrinolysis and is more sensitive to the addition of anticoagulants.

Influenza A(H1N1)pdm09 Virus Alters Expression of Endothelial Factors in Pulmonary Vascular Endothelium in Rats.

Influenza virus infection may cause endothelial activation and dysfunction. However, it is still not known to what extent the influenza virus can dysregulate the expression of various endothelial proteins. The aim of the study is to identify the level of expression of endothelial nitric oxide synthase (eNOS), plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator (tPA) in the pulmonary vascular endothelium, as well as the concentration of PAI-1 and tPA in the blood plasma in Wistar rats. Animals were intranasally infected with rat-adapted influenza A(H1N1)pdm09 virus. The expression of eNOS, PAI-1 and tPA in the pulmonary vascular endothelium was determined by immunohistochemistry; the concentration of PAI-1 and tPA was analyzed by ELISA at 24 and 96 h post infection (hpi). Thus, the expression of eNOS in the pulmonary vascular endothelium decreased by 1.9-fold at 24 hpi and increased by 2-fold at 96 hpi. The expression of PAI-1 in the pulmonary vascular endothelium increased by 5.23-fold and 6.54-fold at 24 and 96 hpi, respectively. The concentration of PAI-1 in the blood plasma of the rats decreased by 3.84-fold at 96 hpi, but not at 24 hpi. The expression of tPA in the pulmonary vascular endothelium was increased 2.2-fold at 96 hpi. The obtained data indicate the development of endothelial dysfunction that is characterized by the dysregulation of endothelial protein expression in non-lethal and clinically non-severe experimental influenza virus infection.

Ionic modification on COF with rare earth ions for the selective optical sensing and removal of picronitric acid.

In this work, we reported a modified COF material for trinitrophenol (TPA) ratiometric sensing and removal. Here a cationic covalent organic framework (C-COF) was prepared as host, while two Tb(III)-based ions were doped into C-COF as probe by ionic exchange reaction with probe loading level of ∼15%. In the absence of TPA, weak Tb(III) emission (489nm, 545nm, 585nm) and bright red COF emission were observed (633nm). The addition of TPA increased Tb(III) emission and decreased COF emission, following linear response within TPA concentration region of 0-9μM. Their limit of detection values were determined as 0.9μM and 4.5μM, respectively. Corresponding working equations were fitted as I/I0=1.225+6.914×105M-1[TPA], R2=0.997 for TbCF3-COF and I/I0=1.063+9.222×104M-1 [TPA], R2=0.993 for TbDBM-COF. TbCF3-COF showed better sensing performance than TbDBM-COF, due to its suitable ligand triplet energy level. Their sensing mechanism was revealed as dopant "replacement", where dopant molecules loaded in COF micropore were replaced by TPA molecules, accompanied with energy competing on Tb(III) 5D4 level, showing ratiometric signals. Good selectivity and removal capacity (∼7.4wt%) for TPA were achieved.

Premature delivery impacts the concentration of plasminogen activators and a plasminogen activator inhibitor and the plasmin activity in human milk.

Background and aimsPlasmin in human milk partially hydrolyzes milk proteins within the mammary gland and may enhance the hydrolysis of milk proteins within the infant’s stomach. This study examined the effects of extremely preterm (EP)-, very preterm (VP)-, and term-delivery on plasmin activity and the concentrations of plasminogen activators [urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA)], plasminogen activator inhibitor type 1 (PAI-1) and the complexes of PAI-1/uPA and PAI-1/tPA in human milk.Materials and methodsHuman milk samples were collected from mothers who delivered extremely preterm infants [24–27 weeks gestational age (GA), n = 20], very preterm infants (28–32 weeks GA, n = 12), and term infants (38–39 weeks GA, n = 8) during 2–72 days postnatally. Plasmin activity was determined using fluorometric substrate assay, whereas concentrations of uPA, tPA, PAI-1, the PAI-1/uPA complex and the PAI-1/tPA complex were quantified by ELISA.ResultsPlasmin activity, uPA and tPA were detected in all human milk samples, PAI-1 and the PAI-1/uPA complex were present in 42.5 and 32.5% of milk samples, respectively, and the PAI-1/tPA complex was not detected. Plasmin activity was correlated negatively with postnatal age and postmenstrual age (PMA) in the VP group and positively with postnatal age in the term group. uPA and tPA concentrations decreased with increasing postnatal age in both EP and VP groups but did not correlate in the term group. uPA concentration was correlated positively with GA in the VP group and tended to be elevated with increasing GA in the combined three groups. In contrast, tPA concentrations were correlated negatively with GA and PMA in the combined three groups (P < 0.008) and with PMA in the EP and VP groups. PAI-1 concentration tended to be correlated positively with postnatal age in the combined three groups. No correlation was detected with the PAI-1/uPA complex.ConclusionPremature delivery impacted the plasmin activity and the concentrations of uPA, tPA, and PAI-1 in human milk. Whether these changes in milk plasminogen activators and inhibitors have a role in balancing the proteolytic digestion of premature infants remains to be investigated.

Double-Gene Copromoting Expression Analysis in tPA/GH Transgenic Goat Mammary Epithelial Cells and Thrombolytic Activity of tPA In Vitro.

Human tissue-plasminogen activator (tPA) is a thrombolytic drug widely used in the treatment of stroke, pulmonary thrombosis, acute myocardial infarction, and other thrombotic diseases. The double genes cointegrated into the organisms and cells can produce a synergistic effect, which will improve the expression level of the target gene. However, the study of the integration of the GH and tPA genes to improve the expression level of tPA has not yet been reported. In order to elucidate this, we generated monoclonal goat mammary epithelial cell lines with tPA/GH double-gene integration and analyzed the tPA expression level in single- and double-gene integrated cells. We selected the mammary gland-specific expressing vectors BLC14/tPA and BLC14/GH with the β-lactoglobulin gene as a regulatory sequence in our previous research. The tPA and GH genes were electronically cotransfected into goat mammary epithelial cells. Resistant cell lines were screened by G418, and transgenic monoclonal cell lines were confirmed by PCR. The tPA expression was induced by prolactin and detected in the cell induction solution after 48 h by ELISA and Western blotting. We detected the tPA biological activity in vitro by fibrin agarose plate assay (FAPA). The results showed that a total of 207 resistant monoclonal cells were obtained, including 126 cell lines with tPA monogenic integration and 51 cell lines with tPA/GH double-gene integration. The rate of double-gene integration was 24.6% (51/207). A total of 48 cells expressed tPA, of which 25.3% (19/75) cells expressed single gene, and 56.9% (29/51) cells expressed double genes. The concentration of tPA in single-gene-expressing cells was 8.0-64.0 μg/mL, and the tPA level in double-gene-expressing cells was significantly higher (200-7200 μg/mL). In addition, the tPA had a relatively strong in vitro thrombolytic activity determined by FAPA. The results showed that goat mammary epithelial cell lines with tPA/GH gene integration were successfully established by electrotransfection, and the expression level of tPA in double-gene integrated cell lines was significantly increased. This study provided a new way for the preparation of a transgenic goat and other animal with high tPA expression by somatic cell nuclear transfer. The findings also laid a foundation for efficient production of pharmaceutical proteins in transgenic animal mammary gland bioreactors in the future.

Breaking the fibrinolytic speed limit with microwheel co‐delivery of tissue plasminogen activator and plasminogen

BackgroundTo reestablish blood flow in vessels occluded by clots, tissue plasminogen activator (tPA) can be used; however, its efficacy is limited by transport to and into a clot and by the depletion of its substrate, plasminogen. ObjectivesTo overcome these rate limitations, a platform was designed to co‐deliver tPA and plasminogen based on microwheels (µwheels), wheel‐like assemblies of superparamagnetic colloidal beads that roll along surfaces at high speeds. MethodsThe biochemical speed limit was determined by measuring fibrinolysis of plasma clots at varying concentrations of tPA (10–800 nM) and plasminogen (1–6 µM). Biotinylated magnetic mesoporous silica nanoparticles were synthesized and bound to streptavidin‐coated superparamagnetic beads to make studded beads. Studded beads were loaded with plasminogen and tPA was immobilized on their surface. Plasminogen release and tPA activity were measured on the studded beads. Studded beads were assembled into µwheels with rotating magnetic fields and fibrinolysis of plasma clots was measured in a microfluidic device. ResultsThe biochemical speed limit for plasma clots was ~15 µm/min. Plasminogen‐loaded, tPA‐immobilized µwheels lyse plasma clots at rates comparableto the biochemical speed limit. With the addition of a corkscrew motion, µwheels penetrate clots, thereby exceeding the biochemical speed limit (~20 µm/min) and achieving lysis rates 40‐fold higher than 50 nM tPA. ConclusionsCo‐delivery of an immobilized enzyme and its substrate via a microbot capable of mechanical work has the potential to target and rapidly lyse clots that are inaccessible by mechanical thrombectomy devices or recalcitrant to systemic tPA delivery.

High Plasma Apolipoprotein(a) Concentration and Low Plasmin Tpa Enzymatic Activity in Hospitalized Patients with COVID-19

Thrombotic and thromboembolic complications in patients diagnosed with coronavirus disease 2019 (COVID-19) are emerging as important sequelae that contribute to mortality, including disseminated intravascular coagulation, pulmonary embolism, deep vein thrombosis, ischemic stroke, and myocardial infraction. Reported incidence of thrombotic and thromboembolic complications in moderate/severe COVID-19 patients is from 21% to 49%, while even higher incidence in non-surviving COVID-19 patients. However, the underlying mechanism between thrombosis and COVID-19 is still unclear.Tissue-type plasminogen activator (tPA) plays an important role on initiating fibrinolysis by converting zymogen plasminogen to plasmin, a serine protease that degrades the fibrin clot, and therefore preventing excessive pathological blood clots. A homologous protein to plasminogen is apolipoprotein(a) [apo(a)], a major component of lipoprotein(a). The apo(a) inhibits fibrinolysis and exacerbates thrombosis through blocking the conversion from Glu-plasminogen to Lys-plasminogen, which has a higher binding affinity to fibrin and is a better substrate to tPA. The population distribution of plasma apo(a) level is positively skewed (most values are clustered around the left tail of the distribution close to zero), and the plasma apo(a) level in most people is less than 300 μg/mL. High plasma concentration of apo(a) (> 300 μg/mL), or genetic variants of LPA, the gene that encodes for apo(a), correlates with thrombotic cardiovascular risk and thromboembolic risk in many population-based clinical or genetic studies. To investigate the potential correlation between infection of SARS-COV-2 and thrombosis, we tested de-identified plasma samples collected from hospitalized patients with or without positivity of SARS-CoV-2 testing results and COVID-19 diagnosis (ICD10CM:U07.1) through the COVD-19 Tissue Bank at the Medical College of Wisconsin. The tPA enzymatic activity was measured by the release of p-nitroaniline chromophore from a plasmin-specific synthetic substrate with exogenous human plasminogen, with the intensity of color proportional to tPA activity. The apo(a) concentration is measured by ELISA capturing total apo(a) antigen.Our results show that the SARS-CoV-2-positive inpatients have higher plasma tPA concentration than the SARS-CoV-2 negative inpatients (6.0 versus 3.0 ng/mL, p<0.05), while plasma tPA enzymatic activity is lower in SARS-CoV-2-positive inpatients than the SARS-CoV-2 negative inpatients (15.2 versus 25.5 ΔA/min/mL/10 4, p<0.0001) (Figure A). The plasma apo(a) concentration is significantly higher in SARS-CoV-2-positive inpatients than in the plasma from SARS-CoV-2-negative inpatients (the median of the two groups are 114.8 versus 34.4 μg/mL, p<0.05) (Figure B). Among the 20 hospitalized patients with COVID-19, 13 survived. The 13 survived patients have one additional plasma sample collected after recovering from COVID-19 (date range between the two blood collections of onset and after recovery is from 19 to 87 days, the mean duration is 42 days). After recovery, 11 out of 13 surviving patients have increased plasma tPA enzymatic activity (the mean value at onset versus recovery is 5.2 versus 7.1 ΔA/min/mL/10 4, p<0.05) (Figure C). Consistently, 11 out of 13 surviving patients have decreased plasma apo(a) concentration compared to the plasma collected during the onset of COVID-19 from the same individuals (the median values of the onset and recovery are 141.1 versus 106.5 μg/mL, p<0.001) (Figure D).In summary, our study shows lower tPA enzymatic activity and higher apo(a) concentration in SARS-CoV-2-positive hospitalized patients compared to SARS-CoV-2-negative hospitalized patients. Among the survived patients, the reduction of apo(a) concentration after recovering from COVID-19 is accordance with the increase of tPA enzymatic activity. Considering the role of apo(a) in inhibiting fibrinolysis through limiting tPA-mediated plasminogen to plasmin conversion, the alteration in apo(a) concentration provide a possible explanation of change of tPA activity in patients with severe COVID-19. [Display omitted] DisclosuresBaumann Kreuziger: CSL Behring: Consultancy; Quercegen Pharmaceuticals: Consultancy; Vaccine Injury Compensation Program: Consultancy.

PGE2 REGULATES THE PLASMINOGEN ACTIVATOR PATHWAY IN HUMAN ENDOMETRIAL ENDOTHELIAL CELLS: DIFFERENCES BETWEEN NORMAL AND HEAVY MENSTRUAL BLEEDERS

This study seeks to determine if Prostaglandin E2 (PGE2) alters plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) in human endometrial endothelial cells (HEEC) and identify any differences between women with normal and heavy menstrual bleeding. HEEC primary cultures were established from two groups of women: heavy menstrual bleeding (HMB, n=2), and normal menstrual bleeding (NMB, n=3). HEEC were cultured with PGE2 at concentrations of 10-7 10-9, 10-11 M and no PGE2 as a control. We used ELISAs to assess the concentration of PAI-1 and tPA in media; total protein was extracted from the cells and determined using the Bicinchoninic Acid protein assay. There were no differences in patient demographics, the average age in NMB 27.7 and HMB was 31.2 (p=0.44). HMB women had at least one fibroid by ultrasound. There was an increase in PAI-1 concentration in HEEC exposed to both 10-7 and 10-9 M of PGE2 in NMB, 332 ±183 and 248-±70 % of control, respectively. HMB women had a different outcome with PAI-1 where concentrations decreased 68 ±29 and 59-±22 % of control at both 10-7 and 10-9 M of PGE2, respectively. There was no change in tPA concentration 111 ±43 and 103±19% of control in NMB at 10-7 and 10-9 M of PGE2, respectively, but a decrease in tPA concentration when PGE2 was added to the cell cultures of HMB women, 68 ±4, and 64 ±16 % control with 10-9 and 10-11 M of PGE2 respectively (Table 1). PGE2 increased PAI-1 concentration in endothelial cells of NMB, which would lead to more stable clots during menses and an objectively lighter menses. PAI-1 secretion from endothelial cells of HMB women did not respond. This suggests that the endothelium of HMB women may be dysregulated in their PAI-1/tPA pathway in response to PGE2, which may be a central cause of their heavier menses.

Photo-catalytic Ozonation for Degrading Terephthalic Acid in Aqueous Environment

Background &amp; Aims of the Study: Terfetalic Acid (TPA ) is produced in large quantities and used in various industries. Besides, TPA is among the main sources of water pollution in industrialized countries. TPA photo-degradation process was performed in a Circulating Fluidized Bed Reactor (CFBR) by one Ultraviolet type A (UV-A) lamp and ozone generator with MnFe2O4/Willemite photo-catalyst. Materials and Methods: In this research, the nanoparticles of MnFe2O4 and Willemite were synthesized by co-precipitation reactions and wet mixing method, respectively. Then MnFe2O4/ Willemite was synthesized by the immobilization of MnFe2O4 on Willemite by mechanical method. Full factorial experimental design with 4 factors, including the pH, the initial concentration of TPA, the amount of Catalyst (Cat.), and O3 dosage was used for modeling and optimizing the process. Results: In the optimal conditions, the amounts of pH, TPA, Cat., and O3 were obtained equal to 9, 20 ppm, 1.5 g/L, and 2.17 mg/h, respectively. In these conditions, degradation efficiency was obtained to be 98.2695%, and decomposition kinetics was determined as pseudo-first-order with Kapp=0.2707 min-1, kLH=3.729 ppm min-1, and kadd=0.051 ppm-1. Conclusion: Comparing experiments results in different processes, such as UV, UV-Cat, O3, UVO 3, Cat-O3, and UV-Cat-O3 revealed that photo-catalytic ozonation (O3/MnFe2O4/Willemite) in the presence of UV for degradation of TPA in an aqueous environment, present the higher efficiency.

Utility of the Sonoclot analyzer to assess hyperfibrinolysis in dogs.

Coagulation abnormalities, including hyperfibrinolysis, have been documented in sick veterinary patients. Viscoelastic tests, including the Sonoclot Coagulation and Platelet Function Analyzer, are useful in detecting hyperfibrinolysis. Tissue plasminogen activator (tPA) assays have been used to quantify fibrinolysis using thromboelastography. We aimed to document and evaluate changes in the whole blood of healthy dogs exposed to in vitro tPA at varying concentrations using the Sonoclot analyzer. Ten milliliters of blood was collected from healthy adult dogs. Sonoclot tests were run in duplicate and included a control sample and five tPA concentrations: 50, 75, 100, 150, and 200IU/mL of blood. Eleven dogs were enrolled in the study. Based on standard Sonoclot Signature changes, a numeric value fibrinolysis time (FTi) was derived to aid in the quantification of hyperfibrinolysis. Activated clotting time and clot rate Sonoclot values were not significantly affected by any tPA concentration. There was a significant decrease in platelet function (PF) at tPA concentrations equal to and above 75IU/mL on channel 1 and tPA concentrations of 150IU/mL and higher on channel 2. There was a progressive decrease in FTi at increasing tPA concentrations. The Sonoclot analyzer can be used to evaluate hyperfibrinolysis. Predictable changes were seen in the Sonoclot Signature and a decrease in PF and FTi was found with increasing tPA concentrations. The Sonoclot assay with a tPA concentration of 100IU/mL is suggested a baseline measure of hyperfibrinolysis and has a resultant median FTi of 42minutes, which is a practical time for clinical applications.