Correlations of inhaled NO with the cTnI levels and the plasma clotting factor in rabbits with acute massive pulmonary embolism

Purpose: To investigate the correlation of inhaled nitric oxide (NO) on plasma levels of cardiac troponin I (cTnI) and von Willebrand factor (vWF), glycoprotein (GP) IIb/IIIa, granule membrane protein 140 (GMP-140) in rabbits with acute massive pulmonary embolism (PE). Methods: Thirty apanese white rabbits were divided into 3 groups, thrombus were injected in model group (n = 10), NO were inhalated for 24 h after massive PE in NO group (n = 10), saline were injected in control group (n = 10). The concentrations of vWF, GP IIb/IIIa, GMP140 and cTnI were tested at 4, 8, 12, 16, 20, and 24 h, Correlation analyses were conducted between cTnI and vWF, GP IIb/IIIa, and GMP-140 by Pearson’s correlation. Results: The concentration of cTnI and vWF, GP IIb/IIIa, and GMP-140 was increased in the model group, compared to control group. In the inhaled group, the concentrations of cTnI, vWF, GP IIb/IIIa, and GMP-140 were reduced compared to model group. There was a positive correlation between cTnI and vWF, GP IIb/IIIa, and GMP-140. Conclusion: Inhaled nitric oxide can lead to a decrease in levels of cardiac troponin I, von Willebrand factor, glycoprotein, and granule membrane protein 140, after an established myocardial damage, provoked by acute massive pulmonary embolism.


Introduction
The mortality rate of acute massive pulmonary thromboembolism was about 30% [3][4] , which was very high 1,2 , particularly in patients with shock or heart failure,, so the risk of mortality should be assessed as early as possible to maintain more effective treatments [5][6][7] .The European Society of Cardiology has divided the prognostic risk into 3 levels.Right ventricle dysfunction in patients with hemodynamic instability are included in high-risk group 5 , in which cardiac troponin is increased in the pulmonary thromboembolism with heart failure.These factors indicate myocardial damage and poor prognosis but are not well-understood 8,9 .
Massive pulmonary embolism (MPE) causes damage to vascular endothelial, induced platelet, and coagulation system activation 10 , as well as some coagulation factors 11,12 , which would lead to further deterioration of pulmonary embolism and resulting in severe hypoxia, as well as myocardial damage.
There were some studies have found that inhaled nitric oxide (NO) was a very important treatment in the MPE 13,14 , which can reduce pulmonary arterial pressure as well as reduce neutrophil chelation, promote lung endothelial integrity, improve lung ventilation/ perfusion, increase blood vessels density, and repair vascular endothelial cells to alleviate lung injury 15,16 .In the past 20 years, NO has become one of the most important signaling molecules in the cardiovascular system and is considered as a heart protection mediator 17 .
Above all, we supposed that, since inhaled NO was used to treat MPE through von Willebrand factor (vWF), glycoprotein (GP) IIb/ IIIa and granule membrane protein 140 (GMP-140), it may alleviate the myocardial damage.
In the study, a rabbit model of massive pulmonary thromboembolism was established by emboli injection to detect changes in cardiac troponin I (cTnI), vWF, GP IIb/IIIa, and removed) in syringe, and was form into striplike clot into normal saline inaseptic disk by hard and slow press through the needle, the clot was φ 1mm and was cut into 3-4 mm length, which was mixed with NS to make suspensions when used.Homemade pulmonary artery catheters and microvascular catheters (5Fr TI; Tyler & Company, Atlanta, GA, USA) were inserted into the right jugular vein and left carotid artery via the anterior chest approach through a transverse incision of 2 cm.Pulmonary artery catheter (homemade) insertion was monitored by an oscilloscope to control the insertion site and saline infusion was maintained at 0.3 mL/ min with a peristaltic pump.The arterial mean pressure and pulmonary artery pressure were measured synchronously with a multi-channel physiological parameter analysis recorder (MP150, BIOPA Systems, Inc., Goleta, CA, USA) through a pressure sensor.The left femoral vein was separated and a microcatheter was inserted for rehydration and blood collection.0.5 ml thrombus suspension (3-4 clots) was injected through the right jugular vein every 3 min and washed by 2-3 ml normal saline, until the average arterial blood pressure decreased to 40% of the base.Diastolic pressure was maintained at 55-60 mmHg for 40 min, and the massive pulmonary thrombosis with myocardial damage model was successfully established.Mechanical ventilation was not essential during modeling, only when the rabbit was in danger, such as the respiratory rates >40 times/min or the SO 2% <80% to control the respiratory rate of 28 times/min and 20 cm H 2 O of 50% O 2 .The control group was injected with saline.The inhaled group inhaled NO (20ppm) after 2 h of modeling for 24 h.During the experiment, all the rabbits in 3 group took mechanical ventilation, which was performed with a SERVO-ibaby ventilator (Rontgenvagen 2, SE-17154 Solna, Maquet Critical Care AB Company, Solna, Sweden). 2 rabbits were dead during the modeling.

Method of NOI
Following successful modeling, mechanical ventilation was initiated when mPAP reached 40% and the rabbit exhibited breathlessness or breathing difficulties for 2 h.NO was composed of 800 x 10-6 g/l decompressed NO, nitrogen (N2).When the concentration of NOI reached 20ppm, a ventilator pipe was connected to the NOI pipe for invasive mechanical ventilation (pressure, 20 cm water column; respiratory rate, 28 bpm; oxygen concentration with mechanical ventilation, 50%).The concentrations of NO and nitrogen dioxide (NO 2 ) were continuously monitored using a nitrogen oxide analyzer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the level of methemoglobin was also monitored throughout and did not exceed 0.3 g/L.

Pulmonary perfusion and pathology of lung and heart
After anesthesia and fixation on the rabbit platform, the abdominal aorta of the rabbit was flushed with heparin saline until the liver became white and was then injected with acetone until the color of lung changed.Pulmonary perfusion was conducted with an ABS perfusion agent at 18-25 mmHg using a BIOPAC pressure conditioner.The lung artery was detected with a MicroCT X-ray-3D system for laboratory (A00001514J, PerkinElmer, Waltham, MA, USA) (Figure 1A, B) and both the lung and heart were sampled for pathology.

Detection of cTnI, vWF, GP IIb/IIIa, and GMP-140
cTnI was detected by microparticle chemiluminescence method using automatic immune analyzers, vWF and GMP-140 were tested by enzyme-linked immunosorbent assay (Shanghai Sun Biotechnology Company, Shanghai, China), and GP IIb/IIIa was examined by flow cytometry.The monoclonal antibody was supplied by BD Biosciences (San Jose, CA, USA).Blood samples were collected before and at 2, 4, 8, 12, 16, 20, and 24 h while monitoring mean pressure and mean pulmonary artery pressure.

Statistical analysis
All data were processed using the statistics software SPSS20.0(SPSS, Inc., Chicago, IL, USA).The missing data of the study has been deleted in the statistical analysis.Variations in the normal distribution were shown as mean±standard deviation(χ ± s), and one-way analysis of variance and the q test were used for comparison.The distribution was tested by Kolmogorov-Smirnov test (KS), if K-S showed the p>0.05, we say the variables distributed normally.P<0.05 was considered as with statistical differences.

Pathological results
Lung embolization is shown in Figure 1C and the heart is shown in Figure 1D, E. The right ventricular wall of the model group was thinner than that of the control and inhaled groups; myocardial necrosis and embolism in the heart of the model group were higher than those in the heart of inhaled group in cardiomyopathy (Figure 1F, G).

Changes in plasma cardiac troponin concentration
The plasma cardiac troponin concentration of acute massive area pulmonary thromboembolism was increased at 2 h, which was clear at 4 h and reached a peak at 16 h (0.46 ± 0.10 µg/L).These values were significantly higher than those in the inhaled group at each time point (P< 0.05), and extremely higher than those in the control group from 4 to 20 h (P< 0.05) (Table 1).

Change in plasma vWF
Plasma vWF increased from 2 h and reached a peak at 8 h, which was higher than in the control group from 2 to 16 h (P< 0.05); compared with model group, there was a decrease significantly of vWF in the inhaled group at 4 and 8 h (P< 0.05) (Table 2).

Change in plasma GP IIb/IIIa
The plasma GP IIb/IIIa concentration of acute massive area pulmonary thromboembolism increased at 2 h and reached a peak at 16 h, which was significantly higher than in the control group (P< 0.05) at each time point; compared with model group, , there was a decrease significantly in the inhaled group at 16 h (P< 0.05) (Table 2).

Change in plasma GMP-140
The plasma GMP-140 concentration of acute massive area pulmonary thromboembolism increased at 2 h and reached a peak at 16 h, which was significantly higher than that in the control group at each time point (P< 0.05);, this value was extremely higher than that in the inhaled NO group from 4 to 20 h (P< 0.05) (Table 2).

■ Discussion
It has been found that increased cardiac troponin during pulmonary thromboembolism leads to higher mortality 9 .In the study, cardiac troponin levels increased and reached a peak concentration of 0.46 ± 0.10 µg/L, meanwhile plasma vWF, GP IIb/IIIa, and GMP-140 increased in rabbits after massive area pulmonary thromboembolism, and the peak concentrations of plasma cardiac troponin with vWF, GP IIb/IIIa, GMP-140, were significantly positively correlated.The increased vWF indicates that platelet adhesion increased and the pulmonary vascular endothelium was impaired 10,11 , which activated platelets when exposed to blood vessels via vWF 12,19 and aggravated thrombosis and increased vWF 20,21 .GP IIb/IIIa is the main glycoprotein in the platelet membrane 22 and mediates platelet and fibrinogen combination, which can be activated by collagen, causing endothelial cell damage by pulmonary thromboembolism, hypoxia, and pulmonary hypertension 22 .
GMP-140 is found in rod cells of the endothelial cells and resting platelets αgranule, which is a component of the receptors of vWF, thrombin, collagen, and fibrinogen (Fg), among others, and is important in platelet activation, adhesion, and aggregation 23,24 .In pulmonary thromboembolism, reactive oxygen species and inflammatory factors stimulate endothelial Correlations of inhaled NO with the cTnI levels and the plasma clotting factor in rabbits with acute massive pulmonary embolism Zhang Z et al.
Acta Cir Bras.2018;33(8):664-672 cells, inducing GMP-140 expression on the cell surface with a combination of the granular membrane of platelet α granule and serous membrane of endothelial cells, activating platelets to form clots [25][26][27] .The activated platelets undergo an irreversible activation reaction with the infarction of pulmonary tissue, and more GMP-140 is released to cause further thrombosis 12,28 .Increased levels of vWF, GP IIb/ IIIa, and GMP-140 lead to local thrombosisinflammation network formation in the lung and cardiac tissue and clot formation, as well as systemic ischemia and hypoxia, which are related to endothelial cell damage.These events cause myocardial damage, as confirmed in this study.
NO inhalation significantly reduced vWF, GP IIb/IIIa, GMP-140 and cardiac troponin levels.The mechanism may involve inhibition of protein kinase C to downregulate the expression of platelet GMP-140 29 and vWF.Additionally, the inhibition of fibrinogen binding to the GP IIb/IIIa receptor by NO, which activated soluble guanylate cyclase in the platelets, may also reduce platelet aggregation and GMP-140 expression.Thus, NO inhalation can inhibit platelet aggregation and reduce myocardial damage in pulmonary thromboembolism.

■ Conclusion
The increased levels of vWF, GP IIb/IIIa and GMP-140 may have played an important role in the myocardial damage, and inhalation of NO would reduce the levels of vWF and GMP-140 and inhibit the expression of GP IIb/ IIIa on the platelet membrane, all these change alleviated myocardial damage in massive area pulmonary thromboembolism.

Figure 1 -
Figure 1 -Pulmonary embolism image and pathology.A,B: Blocked arteries image in MicroCT X-ray-3D system for laboratory; C: Emboli; D: Right ventricle after NO inhalation; E: Right ventricle before NO inhalation; F: Myocardial necrosis pathological changes before NO inhalation; G: Myocardial necrosis pathological changes after NO inhalation.

Figure 2 -
Figure 2 -Correlation between CTnI peak concentration and plasma coagulation system factor.A: Correlation between plasma CTnI peak concentration and GP IIb/IIIa; B: Correlation between plasma CTnI peak concentration and GMP-140; C: Correlation between plasma CTnI peak concentration and vWF.

Table 1 -
Comparison of plasma CTnI ( x ±s, µg/ml).Comparison between the control group and model group.

Table 2 -
Comparison of plasma vWF (ng/ml), GPIIb/IIIa (%), GMP-140l (%) levels ( x ±s).Correlations of inhaled NO with the cTnI levels and the plasma clotting factor in rabbits with acute massive pulmonary embolism Zhang Z et al.
Note: T1M-NO/P1 M-NO: comparison of vWF between Model group and NO group; T2M-NO/P2 M-NO: comparison of GPIIb/IIIa between Model group and NO group;T3M-NO/P3 M-NO: comparison of GMP-140 between Model group and NO group; *p<0.05,***p<0.01