Tissue-type Plasminogen Activator Research Articles

Synergism of red blood cells and tranexamic acid in the inhibition of fibrinolysis

BackgroundPostpartum hemorrhage (PPH) is the leading cause of maternal death worldwide. The World Maternal Antifibrinolytic trial showed that antifibrinolytic tranexamic acid (TXA) reduces PPH deaths. Maternal anemia increases the risk of PPH. The World Maternal Antifibrinolytic-2 trial is now assessing whether TXA can prevent PPH in women with anemia. Low red blood cell (RBC) counts promote fibrinolysis by altering fibrin structure and plasminogen activation. ObjectivesWe explored interactions between RBCs and TXA in inhibiting fibrinolysis. MethodsWe used global fibrinolytic assays (ball sedimentation and viscoelasticity) to monitor the lysis of fibrin containing plasminogen and tissue-type plasminogen activator. We applied a fluorogenic kinetic assay to measure plasmin generation in fibrin clots and scanning electron microscopy to study fibrin structure. ResultsAccording to parallel-line bioassay analysis of the fibrin lysis-time data, the antifibrinolytic potency of 4-128 μM TXA was increased in the presence of 10% to 40% (v/v) RBCs. Global fibrinolysis assays showed that the joint effect of RBCs and TXA was about 15% larger than the sum of their individual effects in the inhibition of fibrinolysis. In plasminogen activation, TXA added the same increment of inhibition to the effect of RBCs at any cell count in the fibrin clot. Regarding fibrin structure, TXA thickened fibrin fibers, which impaired plasminogen activation, whereas RBCs promoted fine fibers that were more resistant to plasmin. ConclusionsThe antifibrinolytic potency of TXA is enhanced in fibrin formed in the presence of RBCs through inhibition of plasminogen activation and fibrin lysis, which correlates with modifications of fibrin structures.

Development of a Hypercoagulable-Hypofibrinolytic State Early After Spinal Cord Injury

ObjectivesTo determine whether spinal cord injury (SCI) is associated with adverse changes in coagulation and fibrinolytic factors that underlie thrombogenesis and contribute to atherothrombotic events such as myocardial infarctions (MIs) and strokes. DesignCross-sectional study. SettingNeurorehabilitation hospital and general community. ParticipantsThirty young and middle-aged (20-58 years) adults (N=30) were studied: 14 non-injured community dwelling adults. (11M/4F) and 16 with subacute tetraplegic motor complete SCI during initial inpatient rehabilitation (13M/3F; time since injury: 11.8±5.3 wk). InterventionsNot applicable. Main Outcome MeasuresCirculating markers of coagulation [von Willebrand factor (vWf) and factors VII, VIII, and X], the fibrinolytic system [tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1) antigen and activity], and fibrin formation (D-dimer) were determined by enzyme immunoassay. ResultsThirty young and middle-aged (20-58 years) adults were studied: 14 non-injured (11M/4F) and 16 with subacute tetraplegic motor complete SCI (13M/3F; time since injury: range 4-25 wk). Circulating levels of coagulation factors VII, VIII, and X were significantly higher (∼20%-45%; P<.05) in the adults with SCI than non-injured adults, whereas vWf was similar between groups. Fibrinolytic markers were adversely disrupted with SCI with t-PA antigen, PAI-1 antigen and PAI-1 activity were markedly higher (∼50%-800%; P<.05) in adults with SCI compared with non-injured adults. The molar concentration ratio of active t-PA to PAI-1 was significantly higher (∼350%) in adults with SCI. Concordant with coagulation cascade activation and fibrinolytic system inhibition, D-dimer concentrations were markedly ∼70% higher (P<.05) in adults with SCI compared with non-injured adults. ConclusionsSubacute tetraplegic motor complete SCI is associated with a prothrombotic hemostatic profile. Adverse changes in the coagulation cascade and fibrinolytic system appear to occur early after injury and may contribute to the increased atherothrombotic risk in adults living with SCI.

Plasma from patients with vaccine-induced immune thrombotic thrombocytopenia displays increased fibrinolytic potential and enhances tissue-type plasminogen activator but not urokinase-mediated plasminogen activation

BackgroundVaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication of adenovirus vector-based COVID-19 vaccines. VITT is associated with markedly raised levels of D-dimer; yet, how VITT modulates the fibrinolytic system is unknown. ObjectivesWe aimed to compare changes in fibrinolytic activity in plasma from patients with VITT, patients diagnosed with venous thromboembolism (VTE) after vaccination but without VITT (VTE–no VITT), and healthy vaccinated controls. MethodsPlasma levels of plasmin-antiplasmin (PAP) complexes, plasminogen, and alpha-2-antiplasmin (α2AP) from 10 patients with VITT, 10 patients with VTE–no VITT, and 14 healthy vaccinated controls were evaluated by enzyme-linked immunosorbent assay and/or Western blotting. Fibrinolytic capacity was evaluated by quantitating PAP levels at baseline and after ex vivo plasma stimulation with 50-nM tissue-type plasminogen activator (tPA) or urokinase for 5 minutes. ResultsBaseline PAP complex levels in control and VTE–no VITT individuals were similar but were ∼7-fold higher in plasma from patients with VITT (P < .0001). VITT samples also revealed consumption of α2AP and fibrinogenolysis consistent with a hyperfibrinolytic state. Of interest, VITT plasma produced significantly higher PAP levels after ex vivo treatment with tPA, but not urokinase, compared to the other groups, indicative of increased fibrinolytic potential. This was not due to D-dimer as addition of D-dimer to VTE–no VITT plasma failed to potentiate tPA-induced PAP levels. ConclusionA marked hyperfibrinolytic state occurs in patients with VITT, evidenced by marked elevations in PAP, α2AP consumption, and fibrinogenolysis. An unidentified plasma cofactor that selectively potentiates tPA-mediated plasminogen activation also appears to exist in the plasma of patients with VITT.

Seizures at stroke onset: A case-control study

PurposeSeizures occurring at the immediate onset of a stroke, abbreviated "seizures at onset" (SaO), pose a diagnostic and therapeutic challenge for physicians. In this study, we report on the current clinical practice in managing stroke patients with SaO from a large tertiary stroke center in Germany. MethodsWe selected all patients with SaO and acute ischemic or hemorrhagic stroke admitted to the Department of Neurology at the University Hospital of Cologne between 2019 and 01–01 and 2020–12–31. SaO patients were then compared to patients with acute ischemic or hemorrhagic stroke without SaO from the local stroke registry. Further, we compared SaO patients who received intravenous recombinant tissue-type plasminogen activator (rt-PA) and/or mechanical thrombectomy with matched controls. ResultsOverall, 54 out of 2312 stroke patients (2.3 %) in the examined period presented with SaO. The most prevalent SaO semiology was focal to bilateral tonic-clonic (42.6 %). SaO was associated with hemorrhagic strokes and higher in-hospital mortality in all stroke patients. The rate of acute stroke therapy was not influenced by the occurrence of SaO. In patients that received acute stroke therapy, patients with SaO had higher scores on the National Institutes of Health Stroke Scale (NIHSS) and the modified Rankin Scale (mRS) at admission, and longer door-to-needle times for the administration of rt-PA, while none of the examined outcome parameters revealed a difference between patients with and without SaO after adjusting for potential confounders. ConclusionData show that SaO is rare in stroke patients but associated with more extensive strokes.

Perfusion deficits in thrombolysis-treated acute ischemic stroke patients with negative or positive diffusion-weighted imaging

ObjectiveMagnetic resonance imaging (MRI) and CT perfusion may provide diagnostic information for intravenous tissue-type plasminogen activator (IV t-PA) administration in acute ischemic stroke (AIS) patients. We aimed to compare the clinical features and perfusion deficits of diffusion weighted imaging (DWI)-negative and DWI-positive AIS patients.MethodsThis retrospective and observational study included thrombolysis-treated AIS patients undergoing multimodel CT imaging before treatment and DWI after treatment between 2021 and 2022. Two experienced neuroradiologists blindly and independently examined the images to identify perfusion deficits in AIS patients. The patients were divided into DWI-positive and DWI-negative groups based on visible hyperintense lesions on DWI. A modified Rankin scale (mRS) score of ≤ 2 indicated good functional outcomes at discharge. Sensitivity analysis was conducted to determine whether CT perfusion was an independent predictor of positive DWI imaging on follow-up.ResultsThis study included 151 patients, of whom 35 (23.2%) patients were DWI-negative on follow-up. These DWI-negative patients were less likely to have a medical history of atrial fibrillation; they had lower triglyceride levels, a shorter admission time, lower National Institutes of Health Stroke Scale (NIHSS) scores after IV t-PA and lower mRS scores at discharge, and had better functional outcomes. A total of 37.1% of DWI-positive and 25.7% of DWI-negative patients had vascular stenosis (P = 0.215). A total of 47.4% of DWI-positive and 37.1% of DWI-negative patients had CT perfusion deficits (P = 0.284). A total of 73.5% of patients with normal CT perfusion had positive DWI, while 19.1% of patients with perfusion deficits had negative DWI. The sensitivity and specificity of NCCT were 14.8% and 97.1% (Kappa = 0.061, P = 0.074), CTP was 47.4% and 62.9% for predicting DWI lesion (Kappa = 0.069, P = 0.284).ConclusionsAbout 23.2% of AIS patients who received intravenous thrombolysis treatment did not have a relevant DWI-MRI lesion on follow-up. Over one-third of patients in the DWI-MRI negative group showed CT perfusion deficits, with a sensitivity of 47.4% for predicting DWI lesions in non-mechanical thrombectomy patients.

Phenotypes of Disseminated Intravascular Coagulation.

Two phenotypes of disseminated intravascular coagulation (DIC) are systematically reviewed. DIC is classified into thrombotic and fibrinolytic phenotypes characterized by thrombosis and hemorrhage, respectively. Major pathology of DIC with thrombotic phenotype is the activation of coagulation, insufficient anticoagulation with endothelial injury, and plasminogen activator inhibitor-1-mediated inhibition of fibrinolysis, leading to microvascular fibrin thrombosis and organ dysfunction. DIC with fibrinolytic phenotype is defined as massive thrombin generation commonly observed in any type of DIC, combined with systemic pathologic hyperfibrinogenolysis caused by underlying disorder that results in severe bleeding due to excessive plasmin formation. Three major pathomechanisms of systemic hyperfibrinogenolysis have been considered: (1) acceleration of tissue-type plasminogen activator (t-PA) release from hypoxic endothelial cells and t-PA-rich storage pools, (2) enhancement of the conversion of plasminogen to plasmin due to specific proteins and receptors that are expressed on cancer cells and endothelial cells, and (3) alternative pathways of fibrinolysis. DIC with fibrinolytic phenotype can be diagnosed by DIC diagnosis followed by the recognition of systemic pathologic hyperfibrin(ogen)olysis. Low fibrinogen levels, high fibrinogen and fibrin degradation products (FDPs), and the FDP/D-dimer ratio are important for the diagnosis of systemic pathologic hyperfibrin(ogen)olysis. Currently, evidence-based treatment strategies for DIC with fibrinolytic phenotypes are lacking. Tranexamic acid appears to be one of the few methods to be effective in the treatment of systemic pathologic hyperfibrin(ogen)olysis. International cooperation for the elucidation of pathomechanisms, establishment of diagnostic criteria, and treatment strategies for DIC with fibrinolytic phenotype are urgent issues in the field of thrombosis and hemostasis.

Tissue-type plasminogen activator (tPA) homozygous Tyr471His mutation associates with thromboembolic disease.

Tissue-type plasminogen activator (tPA) encoded by PLAT is a major mediator that promotes fibrinolysis and prevents thrombosis. Pathogenetic mutations in PLAT associated with venous thromboembolism have rarely been reported. Here, we report the first case of a homozygous point mutation c.1411T>C (p.Y471H) in PLAT leading to thromboembolic events and conduct related functional studies. The corresponding tPA mutant protein (tPA-Y471H) and wild-type tPA (tPA-WT) were synthesized in vitro, and mutant mice (PLATH/H mice) were constructed. The molecular docking and surface plasmon resonance results indicated that the mutation impeded the hydrogen-bonding interactions between the protease domain of tPA and the kringle 4 domain of plasminogen, and the binding affinity of tPA and plasminogen was significantly reduced with a difference of one order of magnitude. mRNA half-life assay showed that the half-life of tPA-Y471H was shortened. The inferior vena cava thrombosis model showed that the rate of venous thrombosis in PLATH/H mice was 80% compared with 53% in wild-type mice. Our data suggested a novel role for the protease domain of tPA in efficient plasminogen activation, and demonstrated that this tPA mutation could reduce the fibrinolysis function of the body and lead to an increased propensity for thrombosis.

Site-specific PEGylation of recombinant tissue-type plasminogen activator

Tissue-type plasminogen activator (tPA) is the gold standard for emergency treatment of ischemic stroke, which is the third leading cause of death worldwide. Major challenges of tPA therapy are its rapid elimination by plasminogen activator inhibitor-1 (PAI-1) and hepatic clearance, leading to the use of high doses and consequent serious side effects, including internal bleeding, swelling and low blood pressure. In this regard, we developed three polyethylene glycol (PEG)ylated tPA bioconjugates based on the recombinant human tPA drug Alteplase using site-specific conjugation strategies. The first bioconjugate with PEGylation at the N-terminus of tPA performed by reductive alkylation showed a reduced proteolytic activity of 68 % compared to wild type tPA. PEGylation at the single-free cysteine of tPA with linear and branched PEG revealed similar proteolytic activities as the wild-type protein. Moreover, both bioconjugates with PEG-cysteine-modification showed 2-fold slower inhibition kinetics by PAI-1. All bioconjugates increased in hydrodynamic size as a critical requirement for half-life extension.

CD44s-activated tPA/LRP1-NFκB pathway drives lamellipodia outgrowth in luminal-type breast cancer cells.

Some cancer cells migration and metastasis are characterized by the outgrowth of lamellipodia protrusions in which the underlying mechanism remains unclear. Evidence has confirmed that lamellipodia formation could be regulated by various adhesion molecules, such as CD44, and we previously reported that lamellipodia at the leading edge of luminal type breast cancer (BrCa) were enriched with high expression of CD44. In this study, we found that the overexpression of CD44s could promote lamellipodia formation in BrCa cells through inducing tissue type plasminogen activator (tPA) upregulation, which was achieved by PI3K/Akt signaling pathway activation. Moreover, we revealed that tPA could interact with LDL receptor related protein 1 (LRP1) to activate the downstream NFκB signaling pathway, which in turn facilitate lamellipodia formation. Notably, inhibition of the tPA/LRP1-NFkB signaling cascade could attenuate the CD44s-induced lamellipodia formation. Thus, our findings uncover a novel role of CD44s in driving lamellipodia outgrowth through tPA/LRP1-NFkB axis in luminal BrCa cells that may be helpful for seeking potential therapeutic targets.

Augmented Glycemic Gap is a Marker for Predicting the Early Neurological Outcomes in Acute Ischemic Stroke Patients Treated with Intravenous Thrombolysis

Background and Purpose: Glycemic Gap (GG) as an index reflecting the acute fluctuation of glycemia, has been proved to be associated with poor functional outcomes in Acute Ischemic Stroke (AIS) patients. However, it predictive value on post-thrombolysis Early Neurological Outcomes (ENOs) are still controversial. This study aimed to use GG to evaluate the influence of pretreatment relative glucose changes on post-thrombolysis ENOs, and we further explored the predictive value of GG in different glycemic control status. Methods: Early Neurological Deterioration (END) was defined as a National Institutes of Health Stroke Scale Score (NIHSS) =4, Early Neurological Improvement (ENI) was defined as a =4-point decrease in NIHSS score or a complete resolution of neurological deficits, between the time of admission and 24 hours after intravenous recombinant tissue-type plasminogen activator (IV-rtPA). GG was calculated as Admission Blood Glucose level (ABG)- estimated average blood glucose level (eAG), eAG could be derived from HbAlc according to the equation eAG=28.7*HbAlc-46.7 Results: Increased GG was significantly associated with post-thrombolysis END and poor functional outcome at discharge (OR, 1.982; 95% CI, 1.213-3.238; P=0.006) (OR, 2.079; 95% CI, 1.305-3.312; P=0.002). Its predictive value on END was more pronounce in diabetic patients (OR, 2.434; 95% CI, 1.079-5.491; P=0.032), after dichotomizing glycemic control status, its significance was only maintained in diabetic patients with good previous glucose control (OR, 6.946; 95% CI, 1.217-39.636; P=0.029). Conclusion: An evaluated GG was associated with high risk of post-thrombolysis END and poor functional outcome at discharge in AIS patients, and the previous glucose control should be considered when predicting ENOs.

Neurovascular Inflammation and Complications of Thrombolysis Therapy in Stroke.

Intravenous thrombolysis via tPA (tissue-type plasminogen activator) is the only approved pharmacological treatment for acute ischemic stroke, but its benefits are limited by hemorrhagic transformation. Emerging evidence reveals that tPA swiftly mobilizes immune cells which extravasate into the brain parenchyma via the cerebral vasculature, augmenting neurovascular inflammation, and tissue injury. In this review, we summarize the pronounced alterations of immune cells induced by tPA in patients with stroke and experimental stroke models. We argue that neuroinflammation, triggered by ischemia-induced cell death and exacerbated by tPA, compromises neurovascular integrity and the microcirculation, leading to hemorrhagic transformation. Finally, we discuss current and future approaches to attenuate thrombolysis-associated hemorrhagic transformation via uncoupling immune cells from the neurovascular unit.

Characterization and Usefulness of Clot-Fibrinolysis Waveform Analysis in Critical Care Patients with Enhanced or Suppressed Fibrinolysis.

Recently, clot-fibrinolysis waveform analysis (CFWA), which is a coagulation and fibrinolysis global assay based on assessing the activated partial thromboplastin time with tissue-type plasminogen activator, was developed. This study aimed to investigate the characteristics of CFWA using plasma samples from patients in the critical care unit. The fibrinolysis times using CFWA were measured in 298 plasma samples. These samples were divided into three groups based on the reference interval (RI) of fibrinolysis time using CFWA: shortened group, less than RI; within group, within RI; prolonged group, more than RI. The coagulation and fibrinolysis markers, including D-dimer, plasmin-α2 plasmin inhibitor complex (PIC), fibrin monomer complex (FMC), plasmin-α2 plasmin inhibitor (α2-PI), plasminogen (Plg), and fibrinogen (Fbg) were analyzed and compared among the three groups. The FMC level decreased in the order of shortened, within, and prolonged groups, and the decrease was statistically significant among all three group pairs. The opposite tendency was observed for Fbg and fibrinolysis-related markers of α2-PI and Plg, and significant differences were recognized in all pair comparisons except for between within and prolonged groups in Plg. The mean values of the fibrinolysis markers D-dimer and PIC in all three groups were higher than the cut-off values, and the PIC value differed significantly between the within and prolonged groups. The fibrinolysis reaction was detected in all three groups, but the status differed. CFWA has the potential to reflect the fibrinolysis status in one global assay.

DNA and histones impair the mechanical stability and lytic susceptibility of fibrin formed by staphylocoagulase.

Staphylocoagulase (SCG) is a virulence factor of Staphylococcus aureus, one of the most lethal pathogens of our times. The complex of SCG with prothrombin (SCG/ProT) can clot fibrinogen, and SCG/ProT-induced fibrin and plasma clots have been described to show decreased mechanical and lytic resistance, which may contribute to septic emboli from infected cardiac vegetations. At infection sites, neutrophils can release DNA and histones, as parts of neutrophil extracellular traps (NETs), which in turn favor thrombosis, inhibit fibrinolysis and strengthen clot structure. To characterize the combined effects of major NET-components (DNA, histone H1 and H3) on SCG/ProT-induced clot structure, mechanical and lytic stability. Recombinant SCG was used to clot purified fibrinogen and plasma. The kinetics of formation and lysis of fibrin and plasma clots containing H1 or core histones+/-DNA were followed by turbidimetry. Fibrin structure and mechanical stability were characterized with scanning electron microscopy, pressure-driven permeation, and oscillation rheometry. Histones and DNA favored the formation of thicker fibrin fibers and a more heterogeneous clot structure including high porosity with H1 histone, whereas low porosity with core histones and DNA. As opposed to previous observations with thrombin-induced clots, SCG/ProT-induced fibrin was not mechanically stabilized by histones. Similarly to thrombin-induced clots, the DNA-histone complexes prolonged fibrinolysis with tissue-type plasminogen activator (up to 2-fold). The anti-fibrinolytic effect of the DNA and DNA-H3 complex was observed in plasma clots too. Heparin (low molecular weight) accelerated the lysis of SCG/ProT-clots from plasma, even if DNA and histones were also present. In the interplay of NETs and fibrin formed by SCG, DNA and histones promote structural heterogeneity in the clots, and fail to stabilize them against mechanical stress. The DNA-histone complexes render the SCG-fibrin more resistant to lysis and thereby less prone to embolization.

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.

71-year Old Male Patient with 20 Hour Onset of Infarct Stroke that was Performed with Intra-Arterial Thrombolysis, Mechanical Thrombectomy, Balloon Angioplasty, and Carotid Stenting: A Case Report

Introduction: For above 2 decades, the definitive management for acute ischemic stroke is intravenous or intra-arterial thrombolysis (IAT), using recombinant tissue-type plasminogen activator. Recently mechanical thrombectomy (MT) was developed to overcome the problem that intravenous thrombolysis is only effective in removing large artery occlusions in the range of 10-30%. Early treatment with intra-arterial thrombolysis, permanent stent insertion and clot extraction devices evolved into the stent-retriever device used in most of the important trials and, recently, emerged aspiration tool. This case report presented 71-year-old male patient with infarct stroke who performed with MT. Case presentation: This case report presented 71-year-old male patient with the main complaint of right limbs weakness. A non-contrast head CT scan found infarction in the cortical-subcortical left parietal lobe, posterior pericornu of the right lateral ventricle and right temporal cornu periventricular; lacunar infarction in the right and left paramedian pons; old lacunar infarction in the left and right centrum semiovale, left corona radiata, right internal capsule, right parietal lobe white matter, left lentiform nucleus, left posterior crus of the internal capsule-thalamus, right thalamus, right lateral ventricular pericornu and left paramedian pons. The patient underwent cerebral digital substraction angiography (DSA), as well as IAT, MT, balloon angioplasty, and carotid stenting with good clinical outcome. Conclusion: With the overwhelming positive results of studies evaluating the safety, efficiency, and efficacy of mechanical thrombectomy; the standard of care for the treatment of patients with anterior circulation vessel occlusion is becoming clear.

Blood Clotting Dissolution in the Presence of a Magnetic Field and Preliminary Study with MG63 Osteoblast-like Cells-Further Developments for Guided Bone Regeneration?

The influence of a magnetic field on the activation of bone cells and remodelling of alveolar bone is known to incite bone regeneration. Guided Bone Regeneration (GBR) aims to develop biomimetic scaffolds to allow for the functioning of the barrier and the precise succession of wound healing steps, including haemostasis. The effect of a magnetic field on blood clot dissolution has not been studied yet. We conducted a methodological study on the clot stability in the presence of a static magnetic field (SMF). Preformed whole blood (WB) clots were treated with either a broad proteolytic enzyme (trypsin) or a specific fibrinolytic agent, i.e., tissue-type plasminogen activator (t-PA). MG63 osteoblast-like cells were added to preformed WB clots to assess cell proliferation. After having experienced a number of clotting and dissolution protocols, we obtained clot stability exerted by SMF when tissue factor (for clotting) and t-PA + plasminogen (for fibrinolysis) were used. WB clots allowed osteoblast-like cells to survive and proliferate, however no obvious effects of the magnetic field were noted. Paramagnetic properties of erythrocytes may have influenced the reduction in clot dissolution. Future studies are warranted to fully exploit the combination of magnetic forces, WB clot and cells in GBR applied to orthodontics and prosthodontics.

Screening of placenta accreta spectrum disorder using maternal serum biomarkers and clinical indicators: a case–control study

BackgroundPlacenta accreta spectrum (PAS) disorder is a major cause of postpartum hemorrhage-associated maternal and fetal death, and novel methods for PAS screening are urgently needed for clinical application.MethodsThe purpose of this study was to develop new methods for PAS screening using serum biomarkers and clinical indicators. A total of 95 PAS cases and 137 controls were enrolled in a case–control study as cohort one, and 44 PAS cases and 35 controls in a prospective nested case–control study were enrolled as cohort two. All subjects were pregnant women of Chinese Han population. Biomarkers for PAS from maternal blood samples were screened based on high-throughput immunoassay and were further validated in three phases of cohort one. Screening models for PAS were generated using maternal serum biomarkers and clinical indicators, and were validated in two cohorts. The expression levels of biomarkers were analyzed using histopathological and immunohistochemical (IHC) techniques, and gene expression was examined by QPCR in the human placenta. Binary logistic regression models were built, and the area under the curve (AUC), sensitivity, specificity, and Youden index were calculated. Statistical analyses and model building were performed in SPSS and graphs were generated in GraphPad Prism. The independent-sample t test was used to compare numerical data between two groups. For nonparametric variables, a Mann–Whitney U test or a X2 test was used.ResultsThe results demonstrated that the serum levels of matrix metalloproteinase-1 (MMP-1), epidermal growth factor (EGF), and vascular endothelial growth factor-A (VEGF-A) were consistently higher, while the level of tissue-type plasminogen activator (tPA) was significantly lower in PAS patients compared with normal term controls and patients with pre-eclampsia (PE) and placenta previa (PP). IHC and QPCR analysis confirmed that the expression of the identified biomarkers significantly changed during the third trimester in human placenta. The generated screening model combining serum biomarkers and clinical indicators detected 87% of PAS cases with AUC of 0.94.ConclusionsSerum biomarkers can be used for PAS screening with low expense and high clinical performance; therefore, it may help to develop a practicable method for clinical prenatal PAS screening.

Pretreatment brain CT perfusion thresholds for predicting final infarct volume in distal medium vessel occlusions.

Quantitative CT perfusion (CTP) thresholds for assessing the extent of ischemia in patients with acute ischemic stroke (AIS) have been established; relative cerebral blood flow (rCBF) <30% is typically used for estimating estimated ischemic core volume and Tmax (time to maximum) >6seconds for critical hypoperfused volume in AIS patients with large vessel occlusion (LVO). In this study, we aimed to identify the optimal threshold values for patients presenting with AIS secondary to distal medium vessel occlusions (DMVOs). In this retrospective study, consecutive AIS patients with anterior circulation DMVO who underwent pretreatment CTP and follow-up MRI/CT were included. The CTP data were processed by RAPID (iSchemaView, Menlo Park, CA) to generate estimated ischemic core volumes using rCBF <20%, <30%, <34%, and <38% and critical hypoperfused volumes using Tmax (seconds) >4, >6, >8, and >10. Final infarct volumes (FIVs) were obtained from follow-up MRI/CT within 5days of symptom onset. Diagnostic performance between CTP thresholds and FIV was assessed in the successfully and unsuccessfully recanalized groups. Fifty-five patients met our inclusion criteria (32 female [58.2%], 68.0±12.1years old [mean±SD]). Recanalization was attempted with intravenous tissue-type plasminogen activator and mechanical thrombectomy in 27.7% and 38.1% of patients, respectively. Twenty-five patients (45.4%) were successfully recanalized. In the successfully recanalized patients, no CTP threshold significantly outperformed what is used in LVO setting (rCBF<30%). All rCBF CTP thresholds demonstrated fair diagnostic performances for predicting FIV. In unsuccessfully recanalized patients, all Tmax CTP thresholds strongly predicted FIV with relative superiority of Tmax >10seconds (area under the receiver operating characteristic curve=.875, p=.001). In AIS patients with DMVOs, longer Tmax delays than Tmax >6seconds, most notably, Tmax >10seconds, best predict FIV in unsuccessfully recanalized patients. No CTP threshold reliably predicts FIV in the successfully recanalized group nor significantly outperformed rCBF<30%.

Endothelial-derived extracellular vesicles associated with electronic cigarette use impair cerebral microvascular cell function.

The aim of this study was to determine the effect of circulating endothelial cell-derived microvesicles (EMVs) isolated from e-cigarette users on human cerebral microvascular endothelial cells (hCMECs) nitric oxide (NO) and endothelin (ET)-1 production and tissue-type plasminogen activator (t-PA) release. Circulating EMVs (CD144-PE) were isolated (flow cytometry) from 27 young adults (19-25 yr): 10 nonsmokers (6 M/4 F), 10 e-cigarette users (6 M/4 F), and 7 tobacco cigarette smokers (4 M/3 F). hCMECs were cultured and treated with isolated EMVs for 24 h. EMVs from e-cigarette users and cigarette smokers induced significantly higher expression of p-eNOS (Thr495; 28.4 ± 4.6 vs. 29.1 ± 2.8 vs. 22.9 ± 3.8 AU), Big ET-1 (138.8 ± 19.0 vs. 141.7 ± 19.1 vs. 90.3 ± 18.8 AU) and endothelin converting enzyme (107.6 ± 10.1 and 113.5 ± 11.8 vs. 86.5 ± 13.2 AU), and significantly lower expression of p-eNOS (Ser1177; 7.4 ± 1.7 vs. 6.5 ± 0.5 vs. 9.7 ± 1.6 AU) in hCMECs than EMVs from nonsmokers. NO production was significantly lower and ET-1 production was significantly higher in hCMECs treated with EMVs from e-cigarette (5.7 ± 0.8 µmol/L; 33.1 ± 2.9 pg/mL) and cigarette smokers (6.3 ± 0.7 µmol/L; 32.1 ± 3.9 pg/mL) than EMVs from nonsmokers (7.6 ± 1.2 µmol/L; 27.9 ± 3.1 pg/mL). t-PA release in response to thrombin was significantly lower in hCMECs treated with EMVs from e-cigarette users (from 38.8 ± 6.3 to 37.4 ± 8.3 pg/mL) and cigarette smokers (31.5 ± 5.5 to 34.6 ± 8.4 pg/mL) than EMVs from nonsmokers (38.9 ± 4.3 to 48.4 ± 7.9 pg/mL). There were no significant differences in NO, ET-1, or t-PA protein expression or production in hCMECs treated with EMVs from e-cigarette users and smokers. Circulating EMVs associated with e-cigarette use adversely affects brain microvascular endothelial cells and may contribute to reported cerebrovascular dysfunction with e-cigarette use.NEW & NOTEWORTHY In the present study, we determined the effect of circulating endothelial cell-derived microvesicles (EMVs) isolated from e-cigarette users on human cerebral microvascular endothelial cells (hCMECs) nitric oxide (NO) and endothelin (ET)-1 production and tissue-type plasminogen activator (t-PA) release. EMVs from e-cigarette users reduced brain microvascular endothelial cell NO production, enhanced ET-1 production, and impaired endothelial t-PA release. EMVs are a potential mediating factor in the increased risk of stroke associated with e-cigarette use.

1252-P: Differences in Cardiometabolic and Immune Biomarkers between Novel Subphenotypes of Individuals at Elevated Risk for Type 2 Diabetes

Recent efforts to address heterogeneity among individuals at elevated risk for type 2 diabetes (T2D) have focused on metrics derived from oral glucose tolerance tests, body fat distribution, and hepatic fat content to subphenotype and stratify patients. A novel classification scheme identified six subphenotypes with significantly distinct risks of developing T2D, complications of insulin resistance, and mortality. The aim of our study was to evaluate differences in cardiometabolic and immune biomarkers among these clusters. Fasting serum levels of 5 Olink targeted protein panels (Cardiovascular III, Cardiometabolic, Immune response, Inflammation, Metabolism) comprising 460 oligo-species were measured in 1,597 individuals without diabetes from the Bialystok PLUS study and the Polish Registry of Diabetes. Pairwise differences between subgroups for biomarkers were assessed with generalized linear mixed models, with and without adjustment for the clustering variables. In the unadjusted model, 164 biomarkers showed significantly different levels among clusters (q-value &amp;lt; 0.05), whereas the adjusted model identified 166 significantly different biomarkers. Generally, cluster 2 (lowest T2D and mortality risk) had a more favorable profile of cardiometabolic and immune biomarkers, with clusters 5 and 6 (high-risk) showing more unfavorable profiles. Among significant biomarkers, the greatest differences were observed for tissue-type plasminogen activator and fatty acid binding protein 4, with lowest levels in cluster 2 and highest levels in clusters 5 and 6. Insulin-Like Growth Factor Binding Protein 1 and Paraoxonase 3 showed the greatest differences in the opposite direction. Our study shows that clusters of individuals at elevated risk for T2D stratify across orthogonal axes of cardiometabolic and immune biomarkers, underlining a prominent independent role of these pathways in the pathophysiology of novel subtypes. Disclosure L.Szczerbinski: None. P.Sowa: None. I.Kowalska: None. K.A.Kaminski: Other Relationship; AstraZeneca, Boehringer Ingelheim Inc. J.C.Florez: Consultant; AstraZeneca, Novo Nordisk, Other Relationship; AstraZeneca, Merck &amp; Co., Inc. A.J.Kretowski: None. A.Janucik: None. W.Kwedlo: None. P.Konopka: None. A.Citko: None. A.Paszko: None. M.Taylor: None. M.Czajkowski: None. M.Kondraciuk: None.