Release Of Tissue-type Plasminogen Activator Research Articles

Abstract P523: Circulating Endothelial-Cell Derived Microvesicles Associated With Insufficient Sleep Impairs Cerebral Microvascular Fibrinolytic Capacity

Chronic insufficient sleep (< 7 hour/night) is associated with increased risk of ischemic stroke. Endothelial cell dysfunction, specifically impairment endothelial release of tissue-type plasminogen activator (t-PA), represents an important mechanism underlying cerebrovascular events such as ischemic stroke. Endothelial cells are the primary site of synthesis and release of t-PA, the plasminogen activator in fibrinolysis and, thus, a key regulator of endogenous thrombolysis. We have previously demonstrated that insufficient sleep is associated with impaired endothelial capacity to release t-PA, however the mechanisms for the sleep-related fibrinolytic impairment are not well understood. Clinical interest in endothelial cell-derived microvesicles (EMVs) has intensified due to their involvement in the development of endothelial dysfunction and, in turn, cardiovascular and cerebrovascular events. The aim of this study was to determine, in vitro, the effects of circulating EMVs isolated from adults with chronic insufficient sleep on brain endothelial cell t-PA release. Considering EMVs are known to cross the blood brain barrier and are associated with ischemic stroke incidence, severity and outcome; circulating EMVs associated with insufficient sleep may be a mediator of stroke risk. Twenty-four healthy, non-obese, normotensive, sedentary adults (age: 23-69 years) were studied: 12 normal nightly sleep duration (7M/5F; age: 47±5 yr; nightly sleep: 7.4±0.1 hour/night) and 12 with short nightly sleep duration (9M/3F; 53±4 yr; 5.9±0.2 hour/night). EMV identification (CD144+) and isolation from peripheral blood was performed by flow cytometry. Human cerebral microvascular endothelial cells (hCMECs) were cultured and separately treated with EMVs from each subject. Cultured hCMECs were treated with EMVs in the absence and presence of thrombin (1 unit/mL) for 24 hours. Intracellular expression of t-PA was significantly lower (~30%) in hCMECs treated with EMVs from adults with insufficient sleep (31±3 AU) compared with cells treated with EMVs from normal sleep adults (40±3 AU). Although the effect of EMVs on basal t-PA release was not different between the groups; thrombin-stimulated t-PA release was significantly lower in cells treated with EMVs from adults with insufficient sleep (from 57±3 to 59±4 ng/mL) compared with EMVs from adults with normal sleep (from 53±3 to 66±4 ng/mL). The capacity of hCMECs to release t-PA in response to thrombin was ~75% lower when treated with EMVs from adults with insufficient sleep. These results indicate that circulating EMVs associated with chronic insufficient sleep diminishes brain endothelial cell t-PA production and impairs t-PA release. Circulating EMVs represent a mechanistic factor in insufficient sleep-related fibrinolytic dysfunction and ischemic stroke risk.

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.

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.

Aging Is Associated with a Circulating Endothelial Cell-Derived Microvesicle Phenotype that Impairs Cerebral Fibrinolytic Capacity

Aging is associated with increased risk and prevalence of ischemic stroke. Endothelial cell dysfunction is considered to be a central mechanism underlying the age-related increase in cerebrovascular accidents (CVA). However, the direct, and indirect, effects of aging on endothelial cell biology are diverse and not completely understood. Reduced endogenous fibrinolytic activity is a central etiologic factor underlying ischemic stroke. Endothelial cells are the primary site of synthesis and release of tissue-type plasminogen activator (t-PA), the main plasminogen activator in fibrinolysis and central regulator. Clinical interest in circulating extracellular vesicles, particularly endothelial cell-derived microvesicles (EMVs), has intensified due to their involvement in the development and progression of endothelial dysfunction and CVA. The aim of this study was to determine, in vitro, the effect of circulating EMVs isolated from young and older adults on brain endothelial cell fibrinolytic capacity. We hypothesized that EMVs from older adults would reduce brain endothelial cell t-PA production and release and increase PAI-1 production compared with EMVs from young adults. Twenty-two healthy, non-obese, normotensive, sedentary adults were studied: 10 young (6M/4F; age: 25±1 yr) and 12 older (7M/5F; 64±2 yr). EMV identification (CD144 + ) and isolation from peripheral blood were performed by flow cytometry. Human cerebral microvascular endothelial cells (hCMECs) were cultured and separately treated with EMVs from each subject. Cultured hCMECs were treated with EMVs in the absence and presence of thrombin (1 unit/mL) for 24 hours. Basal expression of t-PA (25.2±1.5 vs 39.8±1.8 AU) and the t-PA response to thrombin (32.5±1.5 vs 57.2±2.2 AU) were significantly lower (~35%) in hCMECs treated with EMVs from older compared with young adults. Concordant with changes in cell protein expression, basal t-PA production (55.2±3.5 vs 70.1±3.7 ng/mL) and thrombin-stimulated t-PA production (61.9±3.7 vs 88.4±3.8 ng/mL) was significantly lower in cells treated with EMVs from older adults. In conclusion, circulating EMVs may contribute to the increased risk of ischemic stroke with aging by impairing brain endothelial cell fibrinolytic capacity. Diminished capacity of the endothelium to release t-PA is a key mechanistic factor underlying cerebral thrombosis. Nothing to Disclose This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tissue-type plasminogen activator induces TNF-α-mediated preconditioning of the blood-brain barrier

Ischemic tolerance is a phenomenon whereby transient exposure to a non-injurious preconditioning stimulus triggers resistance to a subsequent lethal ischemic insult. Despite the fact that not only neurons but also astrocytes and endothelial cells have a unique response to preconditioning stimuli, current research has been focused mostly on the effect of preconditioning on neuronal death. Thus, it is unclear if the blood-brain barrier (BBB) can be preconditioned independently of an effect on neuronal survival. The release of tissue-type plasminogen activator (tPA) from perivascular astrocytes in response to an ischemic insult increases the permeability of the BBB. In line with these observations, treatment with recombinant tPA increases the permeability of the BBB and genetic deficiency of tPA attenuates the development of post-ischemic edema. Here we show that tPA induces ischemic tolerance in the BBB independently of an effect on neuronal survival. We found that tPA renders the BBB resistant to an ischemic injury by inducing TNF-α-mediated astrocytic activation and increasing the abundance of aquaporin-4-immunoreactive astrocytic end-feet processes in the neurovascular unit. This is a new role for tPA, that does not require plasmin generation, and with potential therapeutic implications for patients with cerebrovascular disease.

Negative Influence of Insufficient Sleep on Endothelial Vasodilator and Fibrinolytic Function in Hypertensive Adults.

[Figure: see text].

Thromboplasminflammation in COVID-19 Coagulopathy: Three Viewpoints for Diagnostic and Therapeutic Strategies.

Thromboplasminflammation in coronavirus disease 2019 (COVID-19) coagulopathy consists of angiotensin II (Ang II)-induced coagulopathy, activated factor XII (FXIIa)- and kallikrein, kinin system-enhanced fibrinolysis, and disseminated intravascular coagulation (DIC). All three conditions induce systemic inflammation via each pathomechanism-developed production of inflammatory cytokines. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) downregulates angiotensin-converting enzyme 2, leading to an increase in Ang II levels. Ang II-induced coagulopathy comprising platelet activation, thrombin generation, plasminogen activator inhibitor-1 expression and endothelial injury causes thrombosis via the angiotensin II type 1 receptor. SARS-CoV-2 RNA and neutrophil extracellular trap (NET) DNA activate FXII, resulting in plasmin generation through FXIIa- and kallikrein-mediated plasminogen conversion to plasmin and bradykinin-induced tissue-type plasminogen activator release from the endothelium via the kinin B2 receptor. NETs induce immunothrombosis at the site of infection (lungs), through histone- and DNA-mediated thrombin generation, insufficient anticoagulation control, and inhibition of fibrinolysis. However, if the infection is sufficiently severe, immunothrombosis disseminates into the systemic circulation, and DIC, which is associated with the endothelial injury, occurs. Inflammation, and serine protease networks of coagulation and fibrinolysis, militate each other through complement pathways, which exacerbates three pathologies of COVID-19 coagulopathy. COVID-19 coagulopathy causes microvascular thrombosis and bleeding, resulting in multiple organ dysfunction and death in critically ill patients. Treatment targets for improving the prognosis of COVID-19 coagulopathy include thrombin, plasmin, and inflammation, and SARS-CoV-2 infection. Several drugs are candidates for controlling these conditions; however, further advances are required to establish robust treatments based on a clear understanding of molecular mechanisms of COVID-19 coagulopathy.

Tissue-type plasminogen activator regulates p35-mediated Cdk5 activation in the postsynaptic terminal.

Neuronal depolarization induces the synaptic release of tissue-type plasminogen activator (tPA). Cyclin-dependent kinase-5 (Cdk5) is a member of the family of cyclin-dependent kinases that regulates cell migration and synaptic function in postmitotic neurons. Cdk5 is activated by its binding to p35 (also known as Cdk5r1), a membrane-anchored protein that is rapidly degraded by the proteasome. Here, we show that tPA prevents the degradation of p35 in the synapse by a plasminogen-dependent mechanism that requires open synaptic N-methyl-D-aspartate (NMDA) receptors. We show that tPA treatment increases the abundance of p35 and its binding to Cdk5 in the postsynaptic density (PSD). Furthermore, our data indicate that tPA-induced p35-mediated Cdk5 activation does not induce cell death, but instead prevents NMDA-induced ubiquitylation of postsynaptic density protein-95 (PSD-95; also known as Dlg4) and the removal of GluR1 (also known as Gria1)-containing α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors from the PSD. These results show that the interaction between tPA and synaptic NMDA receptors regulates the expression of AMPA receptor subunits in the PSD via p35-mediated Cdk5 activation. This is a novel role for tPA as a regulator of Cdk5 activation in cerebral cortical neurons.

Tissue-type plasminogen activator protects the postsynaptic density in the ischemic brain

Cerebral ischemia causes the presynaptic release of tissue-type plasminogen activator (tPA). The postsynaptic density (PSD) is a postsynaptic structure that provides a matrix where signaling transduction of excitatory synapses takes place. The postsynaptic density protein-95 (PSD-95) is the most abundant scaffolding protein in the postsynaptic density (PSD), where it modulates the postsynaptic response to the presynaptic release of glutamate by regulating the anchoring of glutamate receptors to the PSD. We found that tPA induces the local translation of PSD-95 mRNA and the subsequent recruitment of PSD-95 protein to the PSD, via plasminogen-independent activation of TrkB receptors. Our data show that PSD-95 is removed from the PSD during the early stages of cerebral ischemia, and that this effect is abrogated by either the release of neuronal tPA, or intravenous administration of recombinant tPA (rtPA). We report that the effect of tPA on PSD-95 is associated with inhibition of the phosphorylation and recruitment of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors to the PSD, known to amplify the effect of the excitotoxic injury, and that this is followed by TrkB-mediated protection of dendritic spines from the harmful effects of the hypoxic insult. These data reveal that tPA is a synaptic protector in the ischemic brain.

Tissue-type plasminogen activator is a homeostatic regulator of synaptic function in the central nervous system.

Membrane depolarization induces the release of the serine proteinase tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons. Once in the synaptic cleft this tPA promotes the exocytosis and subsequent endocytic retrieval of glutamate-containing synaptic vesicles, and regulates the postsynaptic response to the presynaptic release of glutamate. Indeed, tPA has a bidirectional effect on the composition of the postsynaptic density (PSD) that does not require plasmin generation or the presynaptic release of glutamate, but varies according to the baseline level of neuronal activity. Hence, in inactive neurons tPA induces phosphorylation and accumulation in the PSD of the Ca2+/calmodulin-dependent protein kinase IIα (pCaMKIIα), followed by pCaMKIIα-induced phosphorylation and synaptic recruitment of GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast, in active neurons with increased levels of pCaMKIIα in the PSD tPA induces pCaMKIIα and pGluR1 dephosphorylation and their subsequent removal from the PSD. These effects require active synaptic N-methyl-D-aspartate (NMDA) receptors and cyclin-dependent kinase 5 (Cdk5)-induced phosphorylation of the protein phosphatase 1 (PP1) at T320. These data indicate that tPA is a homeostatic regulator of the postsynaptic response of cerebral cortical neurons to the presynaptic release of glutamate via bidirectional regulation of the pCaMKIIα /PP1 switch in the PSD.

Improving Antiadhesion Effect of Thermosensitive Hydrogel with Sustained Release of Tissue-type Plasminogen Activator in a Rat Repeated-Injury Model.

Intraperitoneal adhesion occurs frequently after pelvic and abdominal surgery, which plays an enormous burden on patients. Various drugs and barrier agents were studied and used to prevent adhesions, but few of them were satisfactory. A thermosensitive hydrogel was developed in our previous work and was effective in preventing adhesions. But in our preliminary experiment, it showed limited activity in a more rigorous rat repeated-injury adhesion model, which was much closer to clinical. In this study, tissue-type plasminogen activator (tPA) loaded thermosensitive hydrogel (tPA-hydrogel) was prepared, which combined barrier functions with sustained release of antiadhesion drug. The obtained tPA-hydrogel was injectable and degraded in vivo gradually in four weeks. Both hematoxylin and eosin and Masson trichrome staining confirmed that the tPA-hydrogel exhibited excellent antiadhesion effects on repeated-injury adhesion. Scanning electron microscopy was used to observe the injured abdominal wall, and cecum remesothelialized after treated with tPA-hydrogel for 14 d. In addition, the PAI-1 and tPA levels were measured by enzyme-linked immunosorbent assay. Results showed the PAI-1 concentrations in peritoneal lavage fluids of tPA-hydrogel treated rats were lower than that of other groups, leading to decreased fibrin formation, while there were no significant differences observed in tPA blood levels at any point in time (P > 0.05). This study demonstrated that the effectiveness of thermosensitive hydrogel in preventing adhesions could be enhanced by delivering antiadhesion drugs, and the tPA-hydrogel might be a promising system for clinical application.

Stimulatory effect of an algal fucoidan on the release of vascular endothelial tissue-type plasminogen activator as a mechanism of fucoidan-mediated thrombolysis.

Identifying a pharmacological means for increasing the production of tissue-type plasminogen activator (t-PA) is always desirable to cure impaired production of this enzyme. An algal fucoidan has been shown to exhibit both novel thrombolytic and synergistic stimulatory effects in a mouse thrombosis model. The plasma levels of active t-PA were measured in mouse arterial thrombus models that were treated with various fucoidans to investigate the mechanism of thrombolysis. The mean plasma level of active t-PA after the infusion of fucoidan was 2.136 ± 0.231 ng/ml for nonthrombolytic Fucus fucoidan and 3.917 ± 0.0.529 ng/ml for thrombolytic Undaria fucoidan, which resulted in a 1.56-2.29-fold increase compared with the healthy control group (1.706 ± 0.194 ng/ml) and the untreated thrombus group (2.506 ± 0.301 ng/ml) (P < 0.01). An algal fucoidan has demonstrated to exert a thrombolytic and stimulatory effect via the induction of t-PA release in a dose-dependent manner in an arterial thrombosis model.

Tissue-type plasminogen activator induces synaptic vesicle endocytosis in cerebral cortical neurons

The release of the serine proteinase tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons plays a central role in the development of synaptic plasticity, adaptation to metabolic stress and neuronal survival. Our earlier studies indicate that by inducing the recruitment of the cytoskeletal protein βII-spectrin and voltage-gated calcium channels to the active zone, tPA promotes Ca2+-dependent translocation of synaptic vesicles (SVs) to the synaptic release site where they release their load of neurotransmitters into the synaptic cleft. Here we used a combination of in vivo and in vitro experiments to investigate whether this effect leads to depletion of SVs in the presynaptic terminal. Our data indicate that tPA promotes SV endocytosis via a mechanism that does not require the conversion of plasminogen into plasmin. Instead, we show that tPA induces calcineurin-mediated dynamin I dephosphorylation, which is followed by dynamin I-induced recruitment of the actin-binding protein profilin II to the presynaptic membrane, and profilin II-induced F-actin formation. We report that this tPA-induced sequence of events leads to the association of newly formed SVs with F-actin clusters in the endocytic zone. In summary, the data presented here indicate that following the exocytotic release of neurotransmitters tPA activates the mechanism whereby SVs are retrieved from the presynaptic membrane and endocytosed to replenish the pool of vesicles available for a new cycle of exocytosis. Together, these results indicate that in murine cerebral cortical neurons tPA plays a central role coupling SVs exocytosis and endocytosis.

Dependence of Proximal GC Boxes and Binding Transcription Factors in the Regulation of Basal and Valproic Acid-Induced Expression of t-PA.

Objective. Endothelial tissue-type plasminogen activator (t-PA) release is a pivotal response to protect the circulation from occluding thrombosis. We have shown that the t-PA gene is epigenetically regulated and greatly induced by the histone deacetylase (HDAC) inhibitor valproic acid (VPA). We now investigated involvement of known t-PA promoter regulatory elements and evaluated dependence of potential interacting transcription factors/cofactors. Methods. A reporter vector with an insert, separately mutated at either the t-PA promoter CRE or GC box II or GC box III elements, was transfected into HT-1080 and HUVECs and challenged with VPA. HUVECs were targeted with siRNA against histone acetyl transferases (HAT) and selected transcription factors from the Sp/KLF family. Results. An intact VPA-response was observed with CRE mutated constructs, whereas mutation of GC boxes II and III reduced the magnitude of the induction by 54 and 79% in HT-1080 and 49 and 50% in HUVECs, respectively. An attenuated induction of t-PA mRNA was observed after Sp2, Sp4, and KLF5 depletion. KLF2 and p300 (HAT) were identified as positive regulators of basal t-PA expression and Sp4 and KLF9 as repressors. Conclusion. VPA-induced t-PA expression is dependent on the proximal GC boxes in the t-PA promoter and may involve interactions with Sp2, Sp4, and KLF5.

Endogenous plasminogen activators mediate progressive intracerebral hemorrhage after traumatic brain injury in mice

AbstractPersistent intracerebral hemorrhage (ICH) is a major cause of death and disability after traumatic brain injury (TBI) for which no medical treatment is available. Delayed bleeding is often ascribed to consumptive coagulopathy initiated by exposed brain tissue factor. We examined an alternative hypothesis, namely, that marked release of tissue-type plasminogen activator (tPA) followed by delayed synthesis and release of urokinase plasminogen activator (uPA) from injured brain leads to posttraumatic bleeding by causing premature clot lysis. Using a murine model of severe TBI, we found that ICH is reduced in tPA−/− and uPA−/− mice but increased in PAI-1−/− mice compared with wild-type (WT) mice. tPA−/−, but not uPA−/−, mice developed a systemic coagulopathy post-TBI. Tranexamic acid inhibited ICH expansion in uPA−/−mice but not in tPA−/− mice. Catalytically inactive tPA-S481A inhibited plasminogen activation by tPA and uPA, attenuated ICH, lowered plasma d-dimers, lessened thrombocytopenia, and improved neurologic outcome in WT, tPA−/−, and uPA−/− mice. ICH expansion was also inhibited by tPA-S481A in WT mice anticoagulated with warfarin. These data demonstrate that protracted endogenous fibrinolysis induced by TBI is primarily responsible for persistent ICH and post-TBI coagulopathy in this model and offer a novel approach to interrupt bleeding.

Impaired endogenous fibrinolytic capacity in prehypertensive men.

Prehypertension (BP 120–139/80–89 mmHg) is associated with an increased risk for future atherothrombotic events. Although the mechanisms underlying this elevated risk are not completely understood, one possibility is that prehypertension is associated with impaired endothelial fibrinolytic capacity. We tested the hypothesis that vascular endothelial release of t-PA is impaired in prehypertensive men. Net endothelial release of t-PA was determined, in vivo, in response to intrabrachial infusions of bradykinin (12.5, 25, 50 ng/100 mL tissue/min) and sodium nitroprusside at (1.0, 2.0, 4.0 µg/100 mL tissue/min) in 42 middle-age and older men: 16 normotensive (BP range: 100–119/57–79 mmHg); 16 prehypertensive (BP range: 120–139/76–89 mmHg); and 10 hypertensive (BP range: 140–150/74–100 mmHg). Net release of t-PA antigen was ~25% lower (P < 0.05) in the prehypertensive (−0.9 ± 0.8 to 42.4 ± 5.3 ng/100 mL tissue/min) compared with the normotensive (0.5 ± 1.0 to 53.9 ± 6.5 ng/100 mL tissue/min) men. There was no significant difference in t-PA release between the hypertensive (−1.8±1.6 to 40.8±6.6 ng/100 mL tissue/min) and prehypertensive groups. Sodium nitroprusside did not significantly alter t-PA release in any group. These data indicate that endothelial t-PA release is diminished in prehypertensive men. Further, the level of impairment in t-PA release seen with clinical hypertension is already apparent in the prehypertensive state. Impaired endothelial fibrinolytic function may underlie the increased atherothrombotic risk associated with blood pressure in the prehypertensive range.

Influence of Dietary Saturated Fat Intake on Endothelial Fibrinolytic Capacity in Adults

Approximately 50% of middle-aged and older adults in the United States regularly consume a diet high in saturated fat. High dietary saturated fat intake has been linked to promote atherothrombotic vascular disease. We tested the hypothesis that endothelial fibrinolytic function is diminished in middle-aged and older adults who habitually consume a diet high in saturated fat. Twenty-four healthy, sedentary middle-aged, and older adults (54 to 71years) were studied: 10 (8 men and 2 women) with a dietary saturated fat intake <10% (lower saturated fat) of total calories and 14 (9 men and 5 women) with a dietary saturated fat intake ≥10% of total calories (high saturated fat). Net endothelial release of tissue-type plasminogen activator (t-PA), the primary activator of fibrinolysis, was determined, invivo, in response to intrabrachial infusions of bradykinin (12.5 to 50.0ng/100ml tissue/min) and sodium nitroprusside (1.0 to 4.0μg/100ml tissue/min). Capacity of the endothelium to release t-PA in response to bradykinin was ∼30% less (p <0.05) in the high (from-0.7 ± 0.6 to 36.9 ± 3.3ng/100ml tissue/min) compared with the lower (from-0.3 ± 0.3 to 53.4 ± 7.8ng/100ml tissue/min) dietary saturated fat group. Moreover, total amount of t-PA released was significantly less (∼30%) (201 ± 22 vs 274 ± 29ng/100ml tissue) in the adults who reported consuming a diet high in saturated fat. These results indicate that the capacity of the endothelium to release t-PA is lower in middle-aged and older adults who habitually consume a diet high in saturated fat. In conclusion, endothelial fibrinolytic dysfunction may underlie the increased atherothrombotic disease risk with a diet high in saturated fat.

Cortical‐layer‐specific effects of PACAP and tPA on interneuron migration during post‐natal development of the cerebellum

During early post-natal development of the cerebellum, granule neurons (GN) execute a centripetal migration toward the internal granular layer, whereas basket and stellate cells (B/SC) migrate centrifugally to reach their final position in the molecular layer (ML). We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates in vitro the expression and release of the serine protease tissue-type plasminogen activator (tPA) from GN, but the coordinated role of PACAP and tPA during interneuron migration has not yet been investigated. Here, we show that endogenous PACAP is responsible for the transient arrest phase of GN at the level of the Purkinje cell layer (PCL) but has no effect on B/SC. tPA is devoid of direct effect on GN motility in vitro, although it is widely distributed along interneuron migratory routes in the ML, PCL, and internal granular layer. Interestingly, plasminogen activator inhibitor 1 reduces the migration speed of GN in the ML and PCL, and that of B/SC in the ML. Taken together, these results reveal for the first time that tPA facilitates the migration of both GN and fast B/SC at the level of their intersection in the ML through degradation of the extracellular matrix. Crucial role of tissue plasminogen activator (tPA) in interneuron migration. Interneuron migration is a critical step for normal establishment of neuronal network. This study indicates that, in the post-natal cerebellum, tPA facilitates the opposite migration of immature excitatory granule neurons (GN) and immature inhibitory basket/stellate cells (B/SC) along the same migratory route. These data show that tPA exerts a pivotal role in neurodevelopment.

Coagulopathy, catecholamines, and biomarkers of endothelial damage in experimental human endotoxemia and in patients with severe sepsis: A prospective study

PurposeThe aim of this study was to investigate associations between circulating catecholamines, endothelial damage, and coagulopathy in experimental human endotoxemia and septic patients. Materials and MethodsNine healthy male volunteers undergoing endotoxemia (4-hour 0.5 ng/kg/hour infusion of E. coli lipopolysaccharide, blood sampling at 0, 4, and 6 hours) and 20 patients with severe sepsis. Analysis of plasma biomarkers (adrenaline, noradrenaline, thrombomodulin, syndecan-1, soluble vascular endothelial cadherin, histone-complexed DNA fragments, soluble CD40 ligand [sCD40L], protein C, tissue-type plasminogen activator, plasminogen activator inhibitor 1) and routine coagulation tests. ResultsEndotoxemia increased heart rate, temperature, white blood cell count, C-reactive protein and procalcitonin, decreased blood pressure and induced a hemostatic response with platelet consumption, reduced protein C and sCD40L levels and enhanced tissue-type plasminogen activator release (all P < .05). Septic patients had increased levels of noradrenaline, syndecan-1, thrombomodulin, histone-complexed DNA and sCD40L but reduced soluble vascular endothelial cadherin and plasminogen activator inhibitor 1 (all P < .05) and plasma catecholamines correlated positively with syndecan-1 (adrenaline and noradrenaline) and sTM (only noradrenaline) (all P < .05), biomarkers reflecting endothelial damage. Furthermore, noradrenaline, syndecan-1 and thrombomodulin levels correlated with INR and disease severity scores (noradrenaline and thrombomodulin) (all P < .05). ConclusionsExperimental endotoxemia induced a discrete hemostatic response without sympathoadrenal activation or endothelial damage. Septic patients had high levels of catecholamines and endothelial damage biomarkers that correlated with each other and with markers of hypocoagulability and disease severity.

Response to Letter Regarding Article, “Smoking-Thrombolysis Paradox: Recanalization and Reperfusion Rates After Intravenous Tissue Plasminogen Activator in Smokers With Ischemic Stroke”

HomeStrokeVol. 44, No. 5Response to Letter Regarding Article, “Smoking-Thrombolysis Paradox: Recanalization and Reperfusion Rates After Intravenous Tissue Plasminogen Activator in Smokers With Ischemic Stroke” Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBResponse to Letter Regarding Article, “Smoking-Thrombolysis Paradox: Recanalization and Reperfusion Rates After Intravenous Tissue Plasminogen Activator in Smokers With Ischemic Stroke” Anna Kufner, MSc, Christian H. Nolte, MD and Martin Ebinger, MD Anna KufnerAnna Kufner Klinik und Hochschulambulanz für NeurologieCharité—Universitätsmedizin BerlinBerlin, Germany Search for more papers by this author , Christian H. NolteChristian H. Nolte Klinik und Hochschulambulanz für NeurologieCharité—Universitätsmedizin BerlinBerlin, Germany Search for more papers by this author and Martin EbingerMartin Ebinger Klinik und Hochschulambulanz für NeurologieCharité—Universitätsmedizin BerlinBerlin, Germany Search for more papers by this author Originally published4 Apr 2013https://doi.org/10.1161/STROKEAHA.113.001287Stroke. 2013;44:e59Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 1, 2013: Previous Version 1 Response:We thank Plas et al1 for their interest in our article and for sharing their results about the smoking–thrombolysis paradox in ischemic stroke. A meta-analysis is an important further step to clarify the much debated and often conflicting results on this topic. Plas et al1 performed an analysis of 7 studies including 7494 patients receiving thrombolysis, which revealed an odds ratio of 1.38 (95% confidence interval, 1.24–1.53; P=0.02) for smoking in favor of a good functional outcome (modified Rankin Scale score ≤2) 3 months after stroke. The authors pointed out that the lack of adjustment for age and sex is a limitation of their analysis. This is an essential point, because smokers tend to be significantly younger and more often are men. Age is a strong predictor for outcome and studies have shown gender-specific differences in vascular risk factors and functional recovery.2 Nonetheless, the meta-analysis is reassuring that our observation is not a mere result of chance. An appropriately adjusted meta-analysis is without a doubt still necessary to determine the concrete effects of smoking on ischemic stroke.Two recent studies have shed light on possible pathophysiological mechanisms underlying this peculiar phenomenon. One suggests smoking to have early protective effects and the other suggests an enhanced treatment efficacy in patients with this risk factor. Lisi et al3 provide evidence suggesting that short-term exposure to reactive oxygen species caused by smoking triggers ischemic preconditioning, reducing endothelial susceptibility to ischemia and reperfusion damage. Nielsen et al4 propose that smoking alters the nature of plasmin–antiplasmin–carbon monoxide interactions in blood plasma, consequently modifying fibrin clot kinetics. Earlier studies have demonstrated that smoking decreases endogenous tissue-type plasminogen activator release, causing an increase in fibrinogen levels and more fibrin-rich thrombi, thereby increasing susceptibility to exogenous tissue-type plasminogen activator treatment.5Smoking remains a proven risk factor for stroke and has detrimental effects on the cardiovascular system. Whether smoking leads to increased tissue-type plasminogen activator efficacy deserves further studies. Either way, we continue to believe that no stroke is always better than a recanalized stroke.Anna Kufner, MScChristian H. Nolte, MDMartin Ebinger, MDKlinik und Hochschulambulanz für NeurologieCharité—Universitätsmedizin BerlinBerlin, GermanyDisclosuresNone.FootnotesStroke welcomes Letters to the Editor and will publish them, if suitable, as space permits. Letters must reference a Stroke published-ahead-of-print article or an article printed within the past 3 weeks. The maximum length is 750 words including no more than 5 references and 3 authors. Please submit letters typed double-spaced. Letters may be shortened or edited. Include a completed copyright transfer agreement form (available online at http://stroke.ahajournals.org and http://submit-stroke.ahajournals.org).