Pulmonary Thromboembolism Model Research Articles

Pulmonary Microcirculation in Experimental Model of Pulmonary Thromboembolism under Conditions of α-Adrenoceptor Blockade

Changes in the pulmonary microcirculation in isolated perfused rabbit lungs during modeling of pulmonary thromboembolism were studied in control animals and against the background of α-adrenoceptors blockade with phentolamine. Intravenous injection of emboli to control animals was followed by an increase in pressure in the pulmonary artery, mean capillary hydrostatic pressure, capillary filtration coefficient, pulmonary vascular resistance, as well as precapillary and postcapillary resistances. Against the background of α-adrenoceptor blockade, the increase in most parameters was less pronounced than in control animals, while capillary filtration coefficient increased more drastically. Thus, adrenergic mechanisms are involved in the constrictor reactions of both arterial and venous pulmonary vessels under conditions of pulmonary thromboembolism.

Макро- и микрогемодинамика легких при экспериментальной тромбоэмболии легочной артерии в условиях блокады альфа- и бета-адренорецепторов

The pulmonary microhemodynamics changes in the experimental pulmonary thromboembolism model were studied in acute experiments on anesthetized rabbits with isolated dual channels lungs perfusion in control and after the blockade of α-adrenoceptors with phentolamine and β-adrenoceptors with propranolol. In control animals, pulmonary embolism caused the increasing of the pulmonary artery pressure, pulmonary vascular resistance, and precapillary resistance as well as elevating of the capillary filtration coefficient and postcapillary resistance. Phentolamine caused the decrease of the pulmonary artery pressure, pulmonary vascular resistance, and precapillary resistance. After the propranolol injection, the changes in most of the investigated parameters were opposite comparing with their shifts after the blockade of α-adrenoceptors. In the last case, pulmonary thromboembolism caused less increasing of the pulmonary microcirculatory parameters. However, the capillary filtration coefficient was elevated more, then in the control animals. In case of pulmonary thromboembolism after the blockade of the β-adrenoceptors, the most of the hemodynamic parameters increased more, than after the blockade of α-adrenoceptors; in both cases the capillary filtration coefficient increased to the same level. The value of its changes is determined of the reciprocal interaction of the α- and β-adrenergic mechanisms of the regulation of the permeability of the endothelium of the pulmonary vessels.

N-Acetylcysteine potentiates the haemodynamic-improving effect of sildenafil in a rabbit model of acute pulmonary thromboembolism via the p38 MAPK pathway.

The current study aimed to investigate the effects of sildenafil and N-acetylcysteine (NAC) on the haemodynamics in a rabbit model of acute pulmonary thromboembolism (APT). We developed an APT model using healthy male China big-ear rabbits (2.7±0.4kg). The rabbits were divided into five groups subjected to various interventions. We recorded the haemodynamic parameters and assessed the oxidative stress and lipid peroxidation response in the groups. Additionally, we detected apoptosis-associated molecules, FoxO1, Bad and Bcl-2, in the lung tissue. Gelatine zymography was used to detect matrix metalloproteinase (MMP) activity in bronchoalveolar lavage (BLA). Pulmonary artery endothelial cells were isolated, and their apoptosis rates and MMP activity were assayed. N-acetylcysteine potentiated the haemodynamic-improving effect of sildenafil and significantly inhibited the oxidative stress response. N-acetylcysteine combined with sildenafil decreased MMP-2 and MMP-9 activity and NO consumption and inhibited apoptosis of pulmonary arterial endothelial cells. Moreover, NAC combined with sildenafil inhibited the expression of MCP-1 and p-p38 MAPK. Thus, NAC potentiates the haemodynamic-improving effect of sildenafil in a rabbit model of acute pulmonary thromboembolism via the MCP-1 and p38 MAPK signalling pathway. This study may provide a promising treatment method for APT.

Efficacy evaluation of reteplase in a novel canine acute pulmonary thromboembolism model developed by minimally invasive surgery and digital subtraction angiography.

PurposeIn order to evaluate the thrombolytic effects of reteplase in pulmonary thromboembolism (PTE), we developed a novel canine PTE model. The efficacy of reteplase against PTE in comparison to alteplase was clarified for the first time, and this PTE model could be further applied to studies of novel thrombolytic therapies.Patients and methodsTwenty-four dogs were divided into four groups: sham operation, vehicle, alteplase, and reteplase. Autologous thrombi/saline were injected into the pulmonary artery, and thrombolytic agents were administrated. Thrombus formation and dissolution were monitored by real-time digital subtraction angiography (DSA), and pulmonary pressures were measured simultaneously. Blood coagulation, blood gas, hematology, and histopathologic examinations were used as subsidiary methods.ResultsThe canine PTE model was established with a significant decrease of blood flow and ~75% blocking area. Administration of reteplase (0.6 mg/kg) resulted in effective thrombus dissolution with a recovery of over 80% blood flow, as effective as alteplase (1.6 mg/kg). Correspondingly, the elevated pulmonary systolic, diastolic, and mean arterial pressures declined to the normal level. Blood coagulation was changed by reteplase, with a dramatic elongation of prothrombin time, activated partial thromboplastin time, and thrombin time, even longer than alteplase. In contrast to the vehicle group, no obvious pathological changes were found in the two thrombolytic groups. Hematological, blood biochemical, and blood gas results also indicated that reteplase had no adverse reactions in this PTE model.ConclusionReteplase proved to be an effective and safe therapy for PTE for the first time, and a small dosage of reteplase exerted an efficacy comparable to the routine dosage of alteplase. Our findings indicated the potential of reteplase as clinical treatment against PTE. This technically innovative, stability- and validity-proved canine PTE model developed by minimally invasive surgery and DSA resembled major clinical features. This may further facilitate our understanding of thrombotic disorders and development of prophylactic and therapeutic approaches.

Therapeutic effects of JLX001 on cerebral ischemia through inhibiting platelet activation and thrombus formation in rats

(3β,5α,16α,20S)-4,4,14-trimethyl-3,20-bis(methylamino)-9,19-cyclopregnan-16-ol-dihydrochloride (JLX001), a derivative of cyclovirobuxine D (CVB-D), is a novel compound from synthesis. This study aims to confirm the therapeutic effect of JLX001 on cerebral ischemia and researchits antiplatelet and antithrombosis activities via thromboxane (TXA2)/phospholipase C-β-3(PLCβ3)/protein kinase C (PKC) pathway suppression. The therapeutic effects of JLX001 was evaluated by infarct sizes, brain edema and neurological scores in Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO). Brain TXA2 and prostacyclin (PGI2) were measured by enzyme-linked immunosorbentassay (ELISA). P-PLCβ3and activated PKC were detected by immunohistochemical method. Adenosine diphosphate (ADP) or 9, 11-dieoxy-11α, 9α-epoxymethanoeprostaglandin F2α (U46619) was used as platelet agonist in the in vivo and in vitro platelet aggregation experiments. Clotting time and bleeding time were determined. Besides, two whole-animal experiments including arteriovenous shunt thrombosis and pulmonary thromboembolism model were conducted. Results showed that JLX001 treatment markedly alleviated cerebral infarcts, edema, and neurological scores in permanent middle cerebral artery occlusion (pMCAO) rats. Brain TXA2 level, p-PLCβ3and activated PKC were decreased, while PGI2level had no significant change. Besides, JLX001 inhibited platelet aggregation induced by ADP or U46619 and exhibited anti-coagulation effects with a minor bleeding risk. In the two whole-animal experiments, JLX001 inhibited thrombus formation. In summary, JLX001 attenuates cerebral ischemia injury and the underlying mechanisms relate to inhibiting platelet activation and thrombus formation via TXA2/PLCβ3/PKC pathway suppression.

Ginsenoside-Rp3 inhibits platelet activation and thrombus formation by regulating MAPK and cyclic nucleotide signaling

Background & purposeGinseng (Panax ginseng C.A. Mayer) contains saponin fractions called ginsenosides, which are thought to be the main components responsible for its various pharmacological activities. Ginsenosides have cardioprotective and antiplatelet effects. In the present study, we evaluated the effects of ginsenoside Rp3 (G-Rp3) on platelet function. MethodsThe in vitro effects of G-Rp3 were evaluated on agonist-induced human and rat platelet aggregation, while [Ca2+]i mobilization, granule secretion, integrin αIIbβ3 activation, and clot retraction were assessed in rat platelets. Its effects on vasodilator-stimulated phosphoprotein (VASP) expression, phosphorylation of MAPK signaling molecules, and PI3K/Akt activation were also studied. Moreover, the tyrosine phosphorylation of components of the P2Y12 receptor downstream signaling pathway was also examined. The in vivo effects of G-Rp3 were studied using an acute pulmonary thromboembolism model and lung histopathology. Key resultsG-Rp3 significantly inhibited collagen, ADP, and thrombin-induced platelet aggregation. G-Rp3 elevated cAMP levels and VASP phosphorylation and suppressed agonist-induced [Ca2+]i mobilization, ATP release, and P-selectin expression along with fibrinogen binding to integrin αIIbβ3, fibronectin adhesion, and clot retraction. G-Rp3 also attenuated the phosphorylation of MAPK, Src, and PLCγ2 as well as PI3K/Akt activation. Furthermore, it inhibited tyrosine phosphorylation of the Src family kinases (Src, Fyn, and Lyn) and PLCγ2 and protected mice from thrombosis. Conclusion and implicationG-Rp3 modulates agonist-induced platelet activation and thrombus formation by inhibiting granule secretion, integrin αIIbβ3 activation, MAPK signaling, and Src, PLCγ2, and PI3K/Akt activation, and VASP stimulation. Our data suggest that G-Rp3 has therapeutic potential as a treatment for platelet-related cardiovascular disorders.

Gintonin modulates platelet function and inhibits thrombus formation via impaired glycoprotein VI signaling.

Panax ginseng (P. ginseng), one of the most valuable medicinal plants, is known for its healing and immunobooster properties and has been widely used in folk medicine against cardiovascular diseases, including stroke and heart attack. In this study, we explored the anti-platelet activity of gintonin (a recently discovered non-saponin fraction of ginseng) against agonist-induced platelet activation. In vitro effects of gintonin on agonist-induced human and rat platelet aggregation, granule secretion, integrin αIIbβ3 activation, and intracellular calcium ion ([Ca2+]i) mobilization were examined. Western blot analysis and immunoprecipitation techniques were used to estimate the expression of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and interaction of glycoprotein VI (GPVI) signaling pathway molecules such as Src family kinases (SFK), tyrosine kinase Syk, and PLCγ2. In vivo effects were studied using acute pulmonary thromboembolism model in mice. Gintonin remarkably inhibited collagen-induced platelet aggregation and suppressed granule secretion, [Ca2+]i mobilization, and fibrinogen binding to integrin αIIbβ3 in a dose-dependent manner and clot retraction. Gintonin attenuated the activation of MAPK molecules and PI3K/Akt pathway. It also inhibited SFK, Syk, and PLCγ2 activation and protected mice from thrombosis. Gintonin inhibited agonist-induced platelet activation and thrombus formation through impairment in GPVI signaling molecules, including activation of SFK, Syk, PLCγ2, MAPK, and PI3K/Akt; suggesting its therapeutic potential against platelet related CVD.

Pharmacological actions of miltirone in the modulation of platelet function.

Salvia miltiorrhiza Bunge contains various active constituents, some of which have been developed as commercially available medicine. Moreover, some other ingredients in Salvia miltiorrhiza play roles in anti-platelet activity. The aim of the present study was to investigate the effects and the underlying mechanism of miltirone, a lipophilic compound of Salvia miltiorrhiza Bunge. The ability of miltirone to modulate platelet function was investigated by a variety of in vitro and in vivo experiments. Platelet aggregation and dense granule secretion induced by various agonists were measured with platelet aggregometer. Clot retraction and spreading were imaged by digital camera and fluorescence microscope. Ferric chloride-induced carotid injury model and pulmonary thromboembolism model were used to check miltirone antithrombotic effect in vivo. To elucidate the mechanisms of anti-platelet activity of miltirone, flow cytometry and western blotting were performed. Miltirone (2, 4, 8 µM) was shown to suppress platelet aggregation, dense granule, and α granule secretion in a dose-dependent manner. Meanwhile, miltirone inhibited the clot retraction and spreading of washed platelets. It reduced the phosphorylation of PLCγ2, PKC, Akt, GSK3βand ERK1/2 in the downstream signal pathway of collagen receptor. It also reduced the phosphorylation of Src and FAK in the integrin αIIbβ3-mediated "outside-in" signaling, while it did not suppress the phosphorylation of β3. In addition, miltirone prolonged the occlusion time and reduced collagen/epinephrine-induced pulmonary thrombi. Miltirone suppresses platelet "inside-out" and "outside-in" signaling by affecting PLCγ2/PKC/ERK1/2, PI3K/Akt, and Src/FAK signaling. Therefore, miltirone might represent a potential anti-platelet candidate for the prevention of thrombotic disorders.

Abstract 051: A New Model of Murine Stasis Pulmonary Thromboembolism in vivo With Assessment by Noninvasive Multimodal Molecular-Structural Imaging

Objective: Pulmonary embolism (PE) is a life-threatening cardiovascular disease that urgently requires improved diagnostic, prognostic and therapeutic options. Here we established and investigated a novel murine PE model based on embolization of a stasis-induced venous thrombus (VT). We further utilized in vivo molecular-structural imaging of fibrin accessibility to assess the healing status of the PE. Methods: Stasis VT was induced in the femoral veins of donor C57BL6 mice. VT was resected 24 hours (h) after induction and then intravenously injected into recipient mice to create PE. Pulse-wave Doppler of pulmonary artery flow was utilized to evaluate the pulmonary arterial pressure (PAP) before and 24h after embolization. One day after embolization, the fibrin-specific probe FTP11-CyAm7 (750/767nm ex/em, 150 nmoles/kg i.v.) assessed the FTP binding and spatial localization of PE via integrated fluorescence-mediated tomography and computed tomography (FMT-CT) pulmonary angiography. Mice were sacrificed 1, 4, 8, or 21 days following stasis-VT embolization, and underwent histological analysis of the PE. Results: Femoral vein stasis-VT exhibited similar temporal inflammatory neutrophil and monocyte/macrophage infiltration and FTP11 binding (fibrin accessibility) kinetics compared to inferior vena cava stasis-VT models. The mean VT width and length were 2.4±0.2mm and 0.65±0.02mm, respectively (n=29 thrombi). Noninvasive ultrasound assessment of the mouse PAP, assessed via the pulmonary acceleration time (PAT), showed a 22% decrease (p=0.03) in pre-to-post-PE PAT times, consistent with increased PAP due to PE. FMT-CT imaging using FTP11 demonstrated accessible fibrin in day 1 PE and visualized PE confined to the lungs, primarily (91%) in the right lung. Ex vivo fluorescence reflectance imaging and histological analysis verified the location PE reported by FMT-CT. Conclusions: These data validate the first in vivo model of PE based on stasis VT formed in vivo. The model reproducibly generates a survival PE that harbors accessible fibrin in recent stasis VT lodged in the lung, and acute increases PAP. This new clinically relevant model of PE is anticipated to facilitate research of acute PE and chronic thromboembolic pulmonary hypertension.

Abstract 301: A New Model of Murine Stasis Pulmonary Thromboembolism in vivo With Assessment by Noninvasive Multimodal Molecular-Structural Imaging

Objective: Pulmonary embolism (PE) is a life-threatening cardiovascular disease that urgently requires improved diagnostic, prognostic and therapeutic options. Here we established and investigated a novel murine PE model based on embolization of a stasis-induced venous thrombus (VT). We further utilized in vivo molecular-structural imaging of fibrin accessibility to assess the healing status of the PE. Methods: Stasis VT was induced in the femoral veins of donor C57BL6 mice. VT was resected 24 hours (h) after induction and then intravenously injected into recipient mice to create PE. Pulse-wave Doppler of pulmonary artery flow was utilized to evaluate the pulmonary arterial pressure (PAP) before and 24h after embolization. One day after embolization, the fibrin-specific probe FTP11-CyAm7 (750/767nm ex/em, 150 nmoles/kg i.v.) assessed the FTP binding and spatial localization of PE via integrated fluorescence-mediated tomography and computed tomography (FMT-CT) pulmonary angiography. Mice were sacrificed 1, 4, 8, or 21 days following stasis-VT embolization, and underwent histological analysis of the PE. Results: Femoral vein stasis-VT exhibited similar temporal inflammatory neutrophil and monocyte/macrophage infiltration and FTP11 binding (fibrin accessibility) kinetics compared to inferior vena cava stasis-VT models. The mean VT width and length were 2.4±0.2mm and 0.65±0.02mm, respectively (n=29 thrombi). Noninvasive ultrasound assessment of the mouse PAP, assessed via the pulmonary acceleration time (PAT), showed a 22% decrease (p=0.03) in pre-to-post-PE PAT times, consistent with increased PAP due to PE. FMT-CT imaging using FTP11 demonstrated accessible fibrin in day 1 PE and visualized PE confined to the lungs, primarily (91%) in the right lung. Ex vivo fluorescence reflectance imaging and histological analysis verified the location PE reported by FMT-CT. Conclusions: These data validate the first in vivo model of PE based on stasis VT formed in vivo. The model reproducibly generates a survival PE that harbors accessible fibrin in recent stasis VT lodged in the lung, and acute increases PAP. This new clinically relevant model of PE is anticipated to facilitate research of acute PE and chronic thromboembolic pulmonary hypertension.

Association between ACE2/ACE balance and pneumocyte apoptosis in a porcine model of acute pulmonary thromboembolism with cardiac arrest.

Acute pulmonary embolism (APE) is frequently reported in patients with cardiac arrest (CA) in emergency care. Pneumocyte apoptosis is commonly observed in the lungs following an APE. An important pathological mechanism evoking apoptosis during a lipopolysaccharide-induced acute lung injury is the angiotensin-converting enzyme 2 (ACE2)/ACE imbalance. The present study uses a porcine model to examine the anti-apoptotic effects of captopril on APE-CA and the return of spontaneous circulation (ROSC). Pigs were randomly assigned into four groups: Control, APE-CA, ROSC-saline, and ROSC-captopril. Surviving pigs were euthanized at 6 h and lungs were isolated for analysis using several biochemical assays. Compared with the control group, the ACE2/ACE ratio was lower in the APE-CA and ROSC pigs. In addition, APE-CA pigs had higher Bcl-2-associated X protein (Bax) and cleaved caspase-3 levels, and lower B-cell lymphoma-2 (Bcl-2) level compared to control pigs. Captopril treatment reduced lung apoptosis, as demonstrated by lower TUNEL-positive cells, higher Bcl-2, and lower cleaved caspase-3 protein levels in the lung. Notably, the ACE2/ACE ratio was positively correlated with Bcl-2 protein levels and Bcl-2/Bax ratio. In conclusion, captopril has a protective effect against lung apoptosis following ROSC and that maintaining the balance of the ACE2/ACE axis is important for inhibiting pulmonary apoptosis during APE.

Platelet amyloid precursor protein is a modulator of venous thromboembolism in mice

AbstractThe amyloid precursor protein (APP), primarily known as the precursor of amyloid peptides that accumulate in the brain of patients with Alzheimer disease, is abundant in platelets, but its physiological function remains unknown. In this study, we investigated the role of APP in hemostasis and thrombosis, using APP knockout (KO) mice. Ex vivo aggregation, secretion, and integrin αIIbβ3 inside-out activation induced by several agonists were normal in APP-deficient platelets, but the number of circulating platelets was reduced by about 20%, and their size was slightly increased. Tail bleeding time was normal, and in vivo, the absence of APP did not alter thrombus formation in the femoral artery. In contrast, in a model of vein thrombosis induced by flow restriction in the inferior vena cava, APP-KO mice, as well as chimeric mice with selective deficiency of APP in blood cells, developed much larger thrombi than control animals, and were more sensitive to embolization. Consistent with this, in a pulmonary thromboembolism model, larger vessels were occluded. APP-KO mice displayed a shorter APTT, but not PT, when measured in the presence of platelets. Moreover, the activity of factor XIa (FXIa), but not FXIIa, was higher in APP-KO mice compared with controls. APP-KO mice presented a higher number of circulating platelet–leukocyte aggregates, and neutrophils displayed a greater tendency to protrude extracellular traps, which were more strongly incorporated into venous thrombi. These results indicate that platelet APP limits venous thromboembolism through a negative regulation of both fibrin formation and neutrophil function.

ELMO1 Is a Novel Negative Regulator of Glycoprotein VI Signaling in Platelets

PI3-kinase (phosphoinositide 3-kinase) is an important signaling molecule that is activated downstream of various receptors upon platelet activation. PI3-kinase activation leads to the generation of PIP3 (Phosphatidylinositol (3,4,5)-trisphosphate) subsequently leading to the recruitment of PH (pleckstrin homology) domain containing proteins to the plasma membrane. Our laboratory screened for proteins that interacted with PIP3 (Phosphatidylinositol (3,4,5)-trisphosphate) using PIP3 beads. One of the proteins that interacted with PIP3 was ELMO1 (Engulfment and cell motility-1). ELMO1 is a scaffold protein with no catalytic activity and is well known to regulate actin cytoskeletal rearrangement via Rac1 in other cells. However, it is not known whether ELMO1 is expressed in platelets and if so, does it regulate platelet functional responses. Here, we show that ELMO1 is present in both human and murine platelets. We used ELMO1-deficient (ELMO1-/-) mice to study its role in platelets. ELMO1-/- murine platelets showed enhanced platelet aggregation and dense granule secretion in response to the GPVI agonist, CRP (Figure 1 A & B), compared to the wildtype controls although there was no difference in GPVI expression levels between the two. There was no difference observed in response to AYPGKF- or 2-MeSADP. These data suggest that ELMO1 plays a specific role downstream of GPVI pathway but GPCRs. Moreover, ELMO1-/- platelets exhibited enhanced clot retraction and spreading indicating its role in Glycoprotein IIb/IIa (GPIIb/IIIa) mediated outside-in signaling. Furthermore, whole blood from ELMO1-/- mice perfused over collagen under arterial shear conditions exhibited enhanced thrombus formation. In an in vivo pulmonary thromboembolism model, ELMO1-/- mice showed reduced survival compared to the wildtype control. ELMO1-/- mice also showed shorter time to occlusion and increased thrombus stability using the ferric-chloride injury model indicating the role of ELMO1 in thrombus formation in vivo. At the molecular level, Rac1 activity was enhanced in ELMO1-/- murine platelets compared to the wildtype control in response to CRP (Figure 1C). Together, these data suggest that ELMO1 regulates Rac1 activity upon GPVI-mediated thrombus formation and it may play a negative regulator role in both inside-out and outside-in signaling, which might involve Rac1. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Antiplatelet and Antithrombotic Effects of the Extract of Lindera obtusiloba Leaves.

Lindera obtusiloba has been used in traditional herbal medicine for the treatment of blood stasis and inflammation. The leaves of Lindera obtusiloba have been reported to exhibit various physiological activities. However, there is little information available on their antiplatelet and antithrombotic activities. Thus, the present study aimed to evaluate the effect of Lindera obtusiloba leaf extract (LLE) on platelet activities, coagulation and thromboembolism. In a platelet aggregation study, LLE significantly inhibited various agonist-induced platelet aggregations in vitro and ex vivo. Furthermore, LLE significantly inhibited collagen-induced thromboxane A2 (TXA2) production in rat platelets. In addition, oral administration of LLE was protective in a mouse model of pulmonary thromboembolism induced by intravenous injection of a mixture of collagen and epinephrine. Interestingly, LLE did not significantly alter prothrombin time (PT) and activated partial thromboplastin time (aPTT). This study indicates that the antithrombotic effects of LLE might be due to its antiplatelet activities rather than anticoagulation. Taken together, these results suggest that LLE may be a candidate preventive and therapeutic agent in cardiovascular diseases associated with platelet hyperactivity.

Gene Expression Profiling of Pulmonary Artery in a Rabbit Model of Pulmonary Thromboembolism

Acute pulmonary thromboembolism (PTE) refers to the obstruction of thrombus in pulmonary artery or its branches. Recent studies have suggested that PTE-induced endothelium injury is the major physiological consequence of PTE. And it is reasonal to use PTE-induced endothelium injury to stratify disease severity. According to the massive morphologic and histologic findings, rabbit models could be applied to closely mimic the human PE. Genomewide gene expression profiling has not been attempted in PTE. In this study, we determined the accuracy of rabbit autologous thrombus PTE model for human PTE disease, then we applied gene expression array to identify gene expression changes in pulmonary arteries under PTE to identify potential molecular biomarkers and signaling pathways for PTE. We detected 1343 genes were upregulated and 923 genes were downregulated in PTE rabbits. The expression of several genes (IL-8, TNF-α, and CXCL5) with functional importance were further confirmed in transcript and protein levels. The most significantly differentially regulated genes were related to inflammation, immune disease, pulmonary disease, and cardiovascular diseases. Totally 87 genes were up-regulated in the inflammatory genes. We conclude that gene expression profiling in rabbit PTE model could extend the understanding of PTE pathogenesis at the molecular level. Our study provides the fundamental framework for future clinical research on human PTE, including identification of potential biomarkers for prognosis or therapeutic targets for PTE.

Expression and function of IL-1R8 (TIR8/SIGIRR): a regulatory member of the IL-1 receptor family in platelets.

Platelets express functional interleukin-1 receptor-1 (IL-1R1) as well as a repertoire of toll-like receptors (TLRs) involved in platelet activation, platelet-leucocyte reciprocal activation, and immunopathology. IL-1R8, also known as single Ig IL-1-related receptor (SIGIRR) or TIR8, is a member of the IL-1R family that negatively regulates responses to IL-1R family members and TLRs. In the present study, we addressed the expression of IL-1R8 in platelets and megakaryocytes and its role in the control of platelet activation during inflammatory conditions and thromboembolism. Here, we show by flow cytometry analysis, western blot, confocal microscopy, and quantitative real-time polymerase chain reaction that IL-1R8 is expressed on human and mouse platelets at high levels and on megakaryocytes. IL-1R8-deficient mice show normal levels of circulating platelets. Homotypic and heterotypic (platelet-neutrophil) aggregation triggered by Adenosine DiPhosphate (ADP) and IL-1 or lipopolysaccharide (LPS) was increased in IL-1R8-deficient platelets. IL-1R8-deficient mice showed increased soluble P-selectin levels and increased platelet-neutrophil aggregates after systemic LPS administration. Commensal flora depletion and IL-1R1 deficiency abated platelet hyperactivity and the increased platelet/neutrophil aggregation observed in Il1r8(-/-) mice in vitro and in vivo, suggesting a key role of IL-1R8 in regulating platelet TLR and IL-1R1 function. In a mouse model of platelet-dependent pulmonary thromboembolism induced by ADP administration, IL-1R8-deficient mice showed an increased frequency of blood vessel complete obstruction. These results show that platelets, which have a large repertoire of TLRs and IL-1 receptors, express high levels of IL-1R8, which plays a non-redundant function as a regulator of thrombocyte activity in vitro and in vivo.

Antiplatelet pyrazolopyridines derivatives: pharmacological, biochemical and toxicological characterization

Platelet aggregation is one of the main events involved in vascular thrombus formation. Recently, N′-substituted-phenylmethylene-3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazides were described as antiplatelet derivatives. In this work, we explore the properties of these antiplatelet agents through a series of pharmacological, biochemical and toxicological studies. The antiplatelet activity of each derivative was confirmed as 3a, 3b and 3 h significantly inhibited human platelet aggregation induced by arachidonic acid, with no detectable effect on clotting factors or healthy erythrocytes. Importantly, mice treated with derivative 3a showed a higher survival rate at an in vivo model of pulmonary thromboembolism with a lower bleeding risk in comparison to aspirin. The in silico studies pointed a series of structural parameters related to thromboxane synthase (TXS) inhibition by 3a, which was confirmed by tracking plasma levels of PGE2 and TXB2 through an in vitro enzyme immunoassay. Derivative 3a showed selective TXS inhibition allied with low bleeding risk and increased animal survival, revealing the derivative as a promising candidate for treatment of cardiovascular diseases.

Quantification of the Blood Platelet Reactivity in the ADP-Induced Model of Non-Lethal Pulmonary Thromboembolism in Mice with the Use of Laser Doppler Flowmetry.

IntroductionThe paper describes an alternative method for quantification of in vivo ADP-induced thromboembolism. The aim of the studies was to develop a method of quantification which would not require either extravasation or labelling of platelets. Our proposed approach is based on the monitoring of changes of blood flow with the use of laser Doppler flowmetry.Materials and MethodsMice of C57Bl strain were used in the study. ADP was injected to the vena cava and blood flow was monitored with the use of a laser Doppler flowmeter in the mesentery. Measurements in platelet-depleted mice, mice pretreated with cangrelor, an ADP receptor antagonist, and eptifibatide, a blocker of fibrinogen binding to GPIIbIIIa, were conducted as the proof-of-concept in the performed experiments. Intravital microscopy and ex vivo imaging of organs was performed to identify the sites of aggregate formation resulting from ADP injection.ResultsThe injection of ADP resulted in a dose-dependent reduction of the blood flow in the mesentery. These responses were fully attributable to blood platelet aggregation, as shown by the lack of the effect in platelet-depleted mice, and significantly reduced responses in mice pretreated with cangrelor and eptifibatide. No platelet aggregate formation in mesenteric vessels was revealed by intravital microscopy, while ex vivo imaging showed accumulation of fluorescent labelled platelets in the lung.ConclusionsInjection of ADP to the venous system results in the formation of platelet aggregates predominantly in the lung. This results in reversible blood flow cessation in peripheral blood vessels. The measurement of this blood flow cessation in the mesentery allows indirect measurement of ADP-induced pulmonary thromboembolism. We suggest that this approach can be useful for in vivo screening for antiplatelet drug candidates.

Ecotin: Exploring a feasible antithrombotic profile

Ecotin is an Escherichia coli-derived protein that can inhibit serine proteases. It has been suggested that this protein (ecotin-WT) and some of its variants could be used to develop a prototype to treat thrombosis. In this work, the effect of ecotin-WT and a variant of this protein harboring two mutations (Met84Arg and Met85Arg, ecotin-RR) were analyzed to determine their ability to prevent thrombus formation using in vivo models. Ecotins were analyzed in vitro using the coagulation tests. An in vivo venous thrombosis rat model and a pulmonary thromboembolism mouse model were used to investigate the antithrombotic activity. The bleeding time in rats using a tail-transection model was evaluated as a possible side effect caused by the administration of these proteins. Ecotin-RR was more effective in inhibiting thrombin than ecotin-WT. Both ecotins presented similar mechanisms of anticoagulation activity and were able to decrease thrombus formation. In contrast, only ecotin-RR increased survival rates in the in vivo pulmonary thromboembolism model, reinforcing the antithrombotic activity of ecotin-RR. Ecotin-WT and more so ecotin-RR showed potent antithrombotic effects, not associated with bleeding. The presented results indicate that ecotin-WT and ecotin-RR may be new scaffolds that could be used to develop anticoagulation molecules.

Inflammatory response and pneumocyte apoptosis during lung ischemia-reperfusion injury in an experimental pulmonary thromboembolism model.

Lung ischemia–reperfusion injury (LIRI) may occur in the region of the affected lung after reperfusion therapy. The inflammatory response mechanisms related to LIRI in pulmonary thromboembolism (PTE), especially in chronic PTE, need to be studied further. In a PTE model, inflammatory response and apoptosis may occur during LIRI and nitric oxide (NO) inhalation may alleviate the inflammatory response and apoptosis of pneumocytes during LIRI. A PTE canine model was established through blood clot embolism to the right lower lobar pulmonary artery. Two weeks later, we performed embolectomy with reperfusion to examine the LIRI changes among different groups. In particular, the ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO2/FiO2), serum concentrations of tumor necrosis factor-α (TNF-α), myeloperoxidase concentrations in lung homogenates, alveolar polymorphonuclear neutrophils (PMNs), lobar lung wet to dry ratio (W/D ratio), apoptotic pneumocytes, and lung sample ultrastructure were assessed. The PaO2/FiO2 in the NO inhalation group increased significantly when compared with the reperfusion group 4 and 6 h after reperfusion (368.83 ± 55.29 vs. 287.90 ± 54.84 mmHg, P < 0.05 and 380.63 ± 56.83 vs. 292.83 ± 6 0.34 mmHg, P < 0.05, respectively). In the NO inhalation group, TNF-α concentrations and alveolar PMN infiltration were significantly decreased as compared with those of the reperfusion group, 6 h after reperfusion (7.28 ± 1.49 vs. 8.90 ± 1.43 pg/mL, P < 0.05 and [(19 ± 6)/10 high power field (HPF) vs. (31 ± 11)/10 HPF, P < 0.05, respectively]. The amount of apoptotic pneumocytes in the lower lobar lung was negatively correlated with the arterial blood PaO2/FiO2, presented a positive correlation trend with the W/D ratio of the lower lobar lung, and a positive correlation with alveolar PMN in the reperfusion group and NO inhalation group. NO provided at 20 ppm for 6 h significantly alleviated LIRI in the PTE model. Our data indicate that, during LIRI, an obvious inflammatory response and apoptosis occur in our PTE model and NO inhalation may be useful in treating LIRI by alleviating the inflammatory response and pneumocyte apoptosis. This potential application warrants further investigation.