Study Blue From Where Are Additional Platelets First Derived When the Circulating Count Decreases

July 05, 2022 Post a Comment

Endothelium-derived NO inhibits platelet adhesion and aggregation via a cGMP-dependent mechanism.¹ ² NO activates soluble guanylate cyclase, which catalyzes the formation of cGMP from GTP.² The amount of NO available for the regulation of platelet function primarily depends on the synthesis and release of this free radical from vascular endothelium. Decreased formation of NO by dysfunctional endothelium can lead directly to platelet-rich thrombosis, which has been implicated in the pathogenesis of atherosclerosis, primary pulmonary hypertension, and acute respiratory distress syndrome.³ ⁴ ⁵ ⁶

Thrombosis is the most commonly encountered obstructive process in pulmonary arteries, leading to pulmonary hypertension with significant morbidity and mortality.⁵ ⁷ ⁸ ⁹ Pulmonary thrombi, whether embolic in origin or developed in situ, may occlude a large portion of the pulmonary arterial bed and increase pulmonary artery pressure and pulmonary vascular resistance.⁸ ⁹ Platelet activation is an important factor leading to thrombosis, and pharmacological interventions aimed at reducing platelet activation may reduce pulmonary thrombosis and subsequent pulmonary hypertension.¹⁰ ¹¹ Anticoagulant therapy indeed significantly improves survival in primary pulmonary hypertension patients, who frequently present with in situ thrombosis.¹²

In experimental animal models, increased concentrations of NO reduce platelet aggregation, adhesion, and platelet-rich thrombus formation following endothelial injury.¹³ ¹⁴ In these models, increased levels of NO were obtained by intravenous administration of an NO donor or by stimulation of endogenous NO synthesis or release with l-arginine or endotoxin,¹⁴ ¹⁵ which may, however, be associated with systemic side effects.¹³ ¹⁴ ¹⁶

Inhaled NO gas is a safe and selective pulmonary vasodilator in patients with pulmonary hypertension.¹⁷ ¹⁸ NO readily diffuses across the alveolar wall to neighboring precapillary pulmonary vascular smooth muscle cells but is rapidly inactivated in the capillary bed by hemoglobin, thereby reducing potential systemic hypotensive effects.¹⁹ NO inhalation also significantly decreased platelet-mediated coronary artery occlusion in dogs²⁰ and reduced neointimal formation in response to peripheral vascular injury in rats,²¹ suggesting an effect of inhaled NO on circulating platelets and/or leukocytes.

In the present study, we evaluated the effect of inhaled NO on ex vivo collagen-induced platelet aggregation and intraplatelet cGMP levels and on in vivo antithrombotic activity in a rat model of platelet-mediated pulmonary thrombosis. To compare the antithrombotic effects of inhaled NO with conventional pharmacological antiplatelet therapy, a separate group of rats was treated with G4120, a cyclic RGD–containing synthetic pentapeptide that binds to the platelet GPIIb/IIIa receptor.²² The same variables were measured in the ex vivo and in vivo study protocols.

Materials and Methods

Experimental Animals

Male Wistar rats (330 to 360 g) were anesthetized with pentobarbital 50 mg/kg IP, orally intubated, and mechanically ventilated with room air or a mixture of air with different concentrations of NO (tidal volume of 7 mL/kg and respiratory rate of 60/min) following institutional guidelines for animal experimentation. During mechanical ventilation, arterial blood gases were analyzed on a blood gas system 288 (CIBA Corning). NO was released from a NO tank containing 800 ppm, mixed with room air (3 L/min), and delivered to the animals via a rodent ventilator (model 683, Harvard Apparatus). NO and NO₂ concentrations were monitored continuously (Inhaled NO Therapy Monitor, Bedfont Scientific Ltd). NO flow was adjusted to obtain NO concentrations of 20, 40, and 80 ppm. Generation of NO₂ from NO and O₂ was <2 ppm at the highest NO concentrations administered.

Ex Vivo Platelet Aggregation Studies

To examine the effects of inhaled NO on platelet activation, groups of 6 rats were ventilated with 20, 40, or 80 ppm NO. After 2 hours, 4 mL blood was taken from the carotid artery in a heparin-containing syringe (0.4 mL of heparin [1000 IU/mL]). To exclude an effect of mechanical ventilation on platelet aggregation, 6 rats were ventilated with room air for 2 hours, and blood samples were collected for platelet aggregation studies. The antiplatelet activity of inhaled NO was compared with the antithrombotic properties of a platelet GPIIb/IIIa antagonistic peptide G4120. Three rats were ventilated with room air and received a bolus injection of G4120 (3 mg/kg IV). Five minutes after the injection of G4120, blood samples were collected for platelet aggregation studies.

Blood samples were processed immediately after withdrawal by centrifugation at 900g for 10 minutes at room temperature to obtain PRP. The platelet count in PRP, which varied from 600 000/μL to 900 000/μL, was adjusted to 500 000/μL by dilution with PPP, obtained by centrifugation at 3000g for 15 minutes. Platelet aggregation was induced by rotating microtiter plates containing 50 μL PRP for 5 minutes at 37°C with various concentrations of collagen (0, 0.5, 1, and 2 μg/mL). Formaldehyde (200 μL, 1%) was added to arrest aggregation, and 200 μL of the platelet suspension was transferred to a second microtiter plate in which aggregation was determined from light scattering at 620 nm in a microtiter plate reader (EAR 400AT, SLT-Lab Instruments). Aggregation was expressed as percent increase of light transmission in PRP. PPP was used as the standard for 100% light transmission.

Effects of Inhaled NO on Intraplatelet cGMP Level

Groups of 4 rats were intubated and ventilated for 2 hours with room air (control) or 20, 40, or 80 ppm NO, respectively. A 2.5F catheter was introduced in the right carotid artery for blood pressure measurements and withdrawal of 4 mL blood in a syringe containing 0.4 mL of 75 mmol/L EDTA and 40 μL of 5 mmol/L isobutyl methylxanthine. Blood was centrifuged at 900g for 10 minutes at room temperature to obtain PRP. PRP was centrifuged at 1500g for 5 minutes, the plasma was removed, and the platelet pellet was resuspended in 0.2 mL of 4 mmol/L EDTA.

For cGMP measurements, samples were sonicated, and cGMP was extracted in ice-cold 50% trichloroacetic acid (pH 4.0) and quantified by a commercial radioimmunoassay after acetylation, according to the manufacturer's instructions (Amersham Life Science).

In Vivo Thrombosis Model

A previously described murine model of pulmonary platelet thromboembolism²³ ²⁴ was used with minor modifications. After 2 hours of mechanical ventilation with room air (control) or 80 ppm NO, in groups of 6 rats, a silastic catheter (inner diameter, 0.30 mm; outer diameter, 0.64 mm) was introduced, via the right jugular vein, into the pulmonary artery for pulmonary artery pressure measurements. Collagen (2.5 mL/kg of 250 μg/mL) was injected into the jugular vein, and the changes in pulmonary artery pressure were recorded continuously to monitor the hemodynamic consequences of collagen-induced pulmonary thrombosis. Similar measurements were obtained from 6 animals that received a bolus injection of G4120 (3 mg/kg IV) 5 minutes before collagen challenge.

Platelet Count

Platelet counts were carried out before and 3 minutes after injection of collagen. Whole blood (1 mL) was collected from the carotid cannula and anticoagulated with heparin (10% [vol/vol] of 1000 IU/mL). After mixing thoroughly, platelets were counted automatically on a cell-DYN 1300 (Abbott Laboratories).

Lung Histology

Animals were killed 10 minutes after the injection of collagen. The chest was opened, the pulmonary vessels were perfused with saline at a pressure of 80 cm H₂O, and the lungs were instilled with 10% formalin via the trachea. The trachea was ligated and removed together with the lungs and immersed in 10% formalin for 24 hours. Two transverse sections through the right lower lobe and the left lower lobe were paraffin-embedded and cut into 7-μm sections. The presence of platelet-rich thrombi in pulmonary vessels was determined via immunohistochemical staining using a murine monoclonal antibody, G28E5, raised in our laboratory with human GPIbα, which cross-reacted with rat GPIbα. After overnight incubation at 4°C with G28E5 (10 μg/mL), sections were exposed to normal goat serum to block nonspecific sites and incubated with a secondary goat anti-mouse antibody conjugated with horseradish peroxidase (1/100, Dako A/S). Bound antibody was visualized with diaminobenzidine and examined by light microscopy. For every experimental condition, two sections from each lobe were examined, and at least 20 microscopic fields were studied at ×400 magnification. The total number of identifiable small lung vessels with a diameter <100 μm was counted, and the percentage of vessels filled with platelet-rich thrombi was determined. All sections were encoded and analyzed by investigators blinded to the experimental treatment.

Statistical Analysis

All values are given as mean±SEM. ANOVA and subsequent multiple comparison using Fisher's test were used to determine differences between groups. Paired Student's t tests were used when appropriate. Significance in all cases was defined as two-sided P<.05.

Results

Arterial blood gases were similar in control rats and in rats ventilated with room air or 80 ppm NO (Pao ₂, 96±3 mm Hg; Paco ₂, 39±2 mm Hg; pH 7.40±0.02). Inhalation of different concentrations of NO for 2 hours did not affect systolic blood pressure (153±3 mm Hg after 80 ppm NO versus 154±4 mm Hg in the control group).

Inhaled NO exerted a dose-dependent effect on intraplatelet cGMP levels. Platelet cGMP levels 2 hours after inhalation of 40 and 80 ppm NO were significantly increased compared with control values (68±13 and 81±13 fmol/10⁸ platelets, respectively, versus 39±6 fmol/10⁸ platelets; P<.05; Fig 1). Ex vivo platelet aggregation with various concentrations of collagen was identical between spontaneously breathing rats and rats ventilated with room air (data not shown). Inhalation with 40 and 80 ppm NO inhibited ex vivo platelet aggregation induced with 0.5, 1, and 2 μg/mL collagen (from 37±6%, 75±4%, and 97±2% in control to 5±2%, 22±10%, and 62±9% after 40 ppm NO and to 6±4%, 20±7%, and 65±11% after 80 ppm NO, respectively; Fig 2). Inhibition was significant at all collagen concentrations studied. At 80 ppm, NO aggregation was not further reduced, suggesting a plateau at 40 ppm NO. Bolus injection of the platelet GPIIb/IIIa antagonistic peptide G4120 reduced ex vivo platelet aggregation to similar degrees as found with 40 and 80 ppm NO (13±4%, 30±9%, and 51±10% with 0.5, 1, and 2 μg/mL collagen, respectively; Fig 2).

During mechanical ventilation, baseline mPAP before the thrombotic challenge was 16±0.2 mm Hg in control animals and 17±0.2 mm Hg in G4120-treated animals. mPAP rapidly increased after intravenous injection of 250 μg/mL collagen, suggesting that collagen-induced platelet aggregation caused pulmonary thrombosis during the passage of platelets through the pulmonary circulation. Because of fluctuations in mPAP during the first 2 minutes, mPAP was measured after 3 minutes, when the pressure had stabilized. mPAP rose to 32±1 mm Hg in control rats but was significantly lower in rats treated with 80 ppm NO or G4120 (26±1 and 27±1 mm Hg, respectively; P<0.05; Fig 3).

Baseline circulating platelet count was 590 000±13 000/μL in control rats (n=6), 620 000±19 000/μL in rats inhaling 80 ppm NO (n=6, P=NS), and 624 000±2/μL in rats treated with G4120 (n=5, P=NS). Three minutes after the intravenous collagen challenge, the platelet count dropped in control animals by 74±3% to 160 000±18 000/μL. The decrease in circulating platelets in animals ventilated with 80 ppm NO and animals treated with G4120 was significantly smaller than that in control animals (250 000±18 000/μL, a 57±2% reduction, and 223 000±10 000/μL, a 64±1% reduction, respectively; P<.05 versus control).

Three rats in the control group, but none in the NO group or G4120 group, died within 10 minutes after collagen challenge. Survival after collagen challenge in this model constitutes an important marker for the efficiency of antithrombotic agents,²³ ²⁴ although the small group sizes do not allow statistical analysis of mortality.

To investigate whether observed differences in mPAP and circulating platelet count correlated with a protective effect of NO against intrapulmonary thrombosis, specific immunochemical staining of platelets was performed on lung sections from control, NO-treated, and G4120-treated rats using a monoclonal anti-GPIbα antibody (Fig 4a). Vessels filled with platelet thrombi generally had a diameter <75 μm, whereas larger-sized vessels were often filled with red blood cell aggregates, which did not stain with the anti-GPIbα antibody (Fig 4b). In lung sections from control animals injected with collagen, 68±3% of small pulmonary vessels were totally or partially (>50% of lumen) occluded by platelet-rich thrombi. In NO-treated and G4120-treated animals, the number of occluded pulmonary vessels was significantly reduced (56±3% and 50±3%, respectively; P<.05 versus control).

Discussion

In the present study, inhalation of NO gas was found to significantly reduce ex vivo collagen-induced platelet aggregation and to attenuate the rise in the pulmonary artery pressure caused by collagen-induced platelet-mediated pulmonary thrombosis. NO-treated rats had less platelet thrombi in small pulmonary vessels and higher residual circulating platelet counts than did the control animals. The antithrombotic activity of inhaled NO was similar to effects obtained with the platelet GPIIb/IIIa antagonistic peptide G4120, suggesting a direct action of inhaled NO on circulating platelet activation. Intraplatelet cGMP levels increased dose-dependently with inhaled NO concentrations, suggesting that the effects on platelet function were most likely mediated by a cGMP-dependent mechanism.

Endothelial cells constitutively release NO, an important regulator of vessel tone and vascular homeostasis through its effect on platelet and smooth muscle cell function. Incubation of human PRP with NO donors in vitro inhibited platelet aggregation induced by ADP, collagen, and thrombin,²⁵ ²⁶ ²⁷ and administration of NO donor compounds or nitrovasodilators in vivo resulted in transient increases in NO concentration and inhibition of platelet function.¹³ ¹⁴ ¹⁶ In a rabbit thrombosis model consisting of an external constrictor around endothelium-denuded carotid arteries, local infusion of a solution of NO abolished cyclic flow reductions due to recurrent platelet aggregation.¹³ However, cyclic flow reductions were restored spontaneously within 10 minutes after cessation of NO infusion.

Inhalation of NO gas, which allows continuous administration of NO without systemic hypotensive side effects, can also affect platelet function.²⁸ In the present study, 40 or 80 ppm but not 20 ppm of inhaled NO inhibited platelet aggregation and did not affect systolic blood pressure. The effect of inhaled NO on platelet aggregation was found to be associated with concentration-dependent changes in intraplatelet cGMP levels. The platelet cGMP signal transduction system indeed functions as a negative-feedback mechanism regulating the physiological activation of platelets. Sodium nitroprusside, a direct NO donor compound, has a concentration-dependent effect on platelet guanylate cyclase activity in vitro, and the resulting rise in cGMP causes a time-dependent disaggregation.¹⁶

Inhaled NO is also a selective pulmonary vasodilator and effectively reduces pulmonary hypertension.¹⁷ ¹⁸ The vasodilatory action of NO most likely does not contribute to the reduction of pulmonary artery pressure in the present study, because animals ventilated with 80 ppm NO or room air had normal baseline pulmonary pressures before the thrombotic challenge. Moreover, collagen, which is often used to test the antiaggregatory efficacy,²³ ²⁴ has no vasoconstrictor properties. However, we cannot exclude that after collagen-induced platelet activation, platelet-release products, including thromboxane A₂ and serotonin, secondarily affect pulmonary vascular tone.²⁹ Therefore, we tested in this model the efficacy of a platelet GPIIb/IIIa antagonistic peptide, which has no direct effects on pulmonary vascular tone. The synthetic peptide inhibits the binding of fibrinogen via the RGD recognition sequence to the platelet GPIIb/IIIa receptor, an essential step of collagen-induced platelet aggregation,²² but does not block the interaction of vasoactive substances with the vessel wall. High concentrations of G4120, a cyclic RGD–containing pentapeptide (3 mg/kg IV bolus), inhibit ex vivo platelet aggregation in rat PRP to the same extent as inhalation of 80 ppm NO. The effects on pulmonary artery pressure rise and on pulmonary platelet-rich thrombosis were also similar, suggesting that in this experimental model, inhaled NO predominantly acts by modulating platelet function.

It has previously been demonstrated that neointimal lesion formation in balloon-injured rat carotid arteries is significantly inhibited by sustained NO inhalation.²¹ Mitogens released from activated platelets are important in initiating migration of cells into the intima. The present study shows that inhaled NO significantly inhibits platelet activation and adhesion and suggests that modulation of circulating platelet function with NO gas may account for previously reported effects on neointimal formation. Moreover, it has been shown that NO is produced in human platelets and that changes in intraplatelet NO production have important physiological and pathophysiological implications.³⁰ Impaired intraplatelet NO production has been observed in patients with coronary atherosclerosis, and this impairment is associated with increased platelet aggregation,³¹ especially during acute coronary syndromes. Our findings suggest that inhaled NO might compensate for reduced intraplatelet NO production.

In conclusion, NO inhalation significantly reduces circulating platelet activation without systemic side effects. Inhalation of NO may therefore be useful in cardiovascular diseases characterized by platelet activation, such as primary pulmonary hypertension, acute respiratory distress syndrome, unstable angina, or recurrent angina after myocardial infarction. Whether administration and dosing of inhaled NO can be achieved in a fashion that is safe and beneficial to these patients remains to be determined.

Selected Abbreviations and Acronyms

G4120	=	l-cysteine, N-(mercaptoacetyl)-d-tyrosyl-l-arginylglycyl-l-α-aspartyl-cyclic (1-5) sulfide, 5-oxide
GPIbα, GPIIb/IIIa	=	glycoprotein Ibα and IIb/IIIa
mPAP	=	mean pulmonary artery pressure
PPP	=	platelet-poor plasma
PRP	=	platelet-rich plasma
RGD	=	Arg-Gly-Asp

**Figure 1.** Effect of inhaled NO on intraplatelet cGMP levels. Platelets were obtained from arterial blood after inhalation of room air (control) or the indicated concentrations NO for 2 hours. *P<.05 vs control.

**Figure 2.** Ex vivo collagen-induced platelet aggregation in PRP obtained from control rats and from rats treated with G4120 and with different concentrations of inhaled NO for 2 hours. ○ indicates control; •, 20 ppm NO; □, 40 ppm NO; ▪, 80 ppm NO; and ▵, G4120. *P<.05 vs control and 20 ppm NO.

**Figure 3.** mPAP measured before (baseline) and after intravenous collagen challenge. The collagen-induced rise in mPAP was significantly smaller after NO inhalation and G4120 treatment. Open bars indicate room air; hatched bars, G4120 treatment; and solid bars, inhaled NO. *P<.05 vs baseline; †P<.05 vs room air control after collagen injection.

**Figure 4.** a, Platelet-rich thrombus (arrows) in a small pulmonary vessel (<75 μm) after intravenous collagen injection. Platelets were stained with a specific anti-GPIbα antibody. Magnification ×400. b, Red blood cell aggregate (arrow) in a medium-sized pulmonary vessel showing no reactivity with the anti-GPIbα antibody.

This study was supported by the National Fund for Scientific Research (NFWO to Dr Janssens) and by an interuniversity grant from the Belgian government (IUAP No. P4/34 to Dr Hoylaerts). Dr Janssens is the recipient of a chair financed by Zeneca Pharmaceuticals Inc.

Footnotes

Correspondence to Stefan Janssens, MD, PhD, Center for Transgene Technology and Gene Therapy and Department of Cardiology, University Hospital Gasthuisberg, 49 Herestraat, B-3000 Leuven, Belgium. E-mail [email protected]

References

1 Radomski MV, Moncada S. The biological and pharmacological role of nitric oxide on platelet function: In: Authi KS, ed. Mechanism of Platelet Activation and Control. New York, NY: Plenum Publishing Corp; 1993:251-264.Google Scholar
2 Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev . 1991; 43:109-143.MedlineGoogle Scholar
3 Chaouat A, Weitzenblum E, Higenbottam T. The role of thrombosis in severe pulmonary hypertension. Eur Respir J . 1996; 9:356-353.CrossrefMedlineGoogle Scholar
4 Chabot F, Dinh-Xuan AT. Pulmonary hypertension: medical treatment and surgical indications. Presse Med . 1995; 24:1574-1576.MedlineGoogle Scholar
5 Malik AB. Pulmonary microembolism and lung vascular injury. Eur Respir J. 1990;3(suppl 11):499s-506s.Google Scholar
6 Hasegawa N, Husari AW, Hart WT, Kandra TG, Raffin TA. Role of the coagulation system in ARDS. Chest . 1994; 105:268-277.CrossrefMedlineGoogle Scholar
7 Wagenvoort CA. Morphological substrate for the reversibility and irreversibility of pulmonary hypertension. Eur Heart J. 1988;9(suppl J):7-12.Google Scholar
8 Edwards WD. Pathology of pulmonary hypertension. Cardiovasc Clin . 1988; 18:321:359.MedlineGoogle Scholar
9 Alert MA, Concannon MD, Mukerji B, Mukerji V. Pharmacotherapy of chronic pulmonary arterial hypertension: value and limitations, I: primary pulmonary hypertension. Angiology . 1994; 45:667-676.CrossrefMedlineGoogle Scholar
10 Heffner JE, Sahn SA, Repine JE. The role of platelets in the adult respiratory distress syndrome. Am Rev Respir Dis . 1987; 135:482-490.MedlineGoogle Scholar
11 Geroge J, Pickett EB, Saucerman S, McEver RP, Kunicki TJ, Kieffer N, Newman PJ. Platelet surface glycoproteins: studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery. J Clin Invest . 1986; 78:340-348.CrossrefMedlineGoogle Scholar
12 Fuster V, Steele PM, Edwads WD, Gersh BJ, McGoon MD, Frye RL. Primary pulmonary hypertension: natural history and the importance of thrombosis. Circulation . 1984; 70:580-587.CrossrefMedlineGoogle Scholar
13 Golino P, Cappelli-Bigazzi M, Ambrosio G, Ragni M, Russolillo E, Condorelli M, Chiariello M. Endothelium-derived relaxing factor modulates platelet aggregation in an in vivo model of recurrent platelet activation. Circ Res. 1992;71;1447-1456.Google Scholar
14 Yao S, Akhtar S, Scott-Burden T, Ober JC, Golino P, Buja M, Casscells W, Willerson JT. Endogenous and exogenous nitric oxide protect against intracoronary thrombosis and reocclusion after thrombolysis. Circulation . 1995; 92:1005-1010.CrossrefMedlineGoogle Scholar
15 Shultz P, Raij L. Endogenously synthesized nitric oxide prevents endotoxin-induced glomerular thrombosis. J Clin Invest . 1992; 90:1718-1725.CrossrefMedlineGoogle Scholar
16 Chirkov YY, Belushkina NN, Tyshchuk IA, Severina IS, Horowitz JD. Increase in reactivity of human platelet guanylate cyclase during aggregation potentiates the disaggregation capacity of sodium nitroprusside. Clin Exp Pharmacol Physiol . 1991; 18:517-524.CrossrefMedlineGoogle Scholar
17 Pepke-Zaba J, Higenbottam TW, Dinh-Xuan AT, Stone D, Wallwork J. Inhaled nitric oxide as a cause of selective pulmonary vasodilation in pulmonary hypertension. Lancet . 1991; 338:1173-1174.CrossrefMedlineGoogle Scholar
18 Frostell C, Fratacci MD, Wain JC, Jones R, Zapol WM. Inhaled nitric oxide: a selective pulmonary vasodilator reversing hypoxic pulmonary vasoconstriction. Circulation . 1991; 83:2038-2047.CrossrefMedlineGoogle Scholar
19 Lunn RJ. Inhaled nitric oxide therapy. Mayo Clin Proc . 1995; 70:247-255.CrossrefMedlineGoogle Scholar
20 Adrie C, Bloch KD, Moreno PR, Hurford, WE, Guerrero, Holt R, Zapol WM, Gold HK, Semigran MJ. Inhaled nitric oxide increases coronary artery patency after thrombolysis. Circulation . 1996; 94:1919-1926.CrossrefMedlineGoogle Scholar
21 Lee JS, Adrie C, Jacob HJ, Roberts JD Jr, Zapol WM, Bloch KD. Chronic inhalation of nitric oxide inhibits neointimal formation after balloon-induced arterial injury. Circ Res . 1996; 78:337-342.CrossrefMedlineGoogle Scholar
22 Imura Y, Stassen JM, Bunting S, Stockmans F, Collen D. Antithrombotic properties of L-cysteine, N-(mercaptoacetyl)-D-Tyr-Arg-Gly-Asp-sulfoxide (G4120) in a hamster platelet-rich femoral vein thrombosis model. Blood . 1992; 80:1247-1253.CrossrefMedlineGoogle Scholar
23 Diminno G, Silver MJ. Mouse antithrombotic assay: a simple method for the evaluation of antithrombotic agents in vivo: potentiation of antithrombotic activity by ethyl alcohol. J Pharmacol Exp Ther . 1983; 225:57-60.MedlineGoogle Scholar
24 Gresele P, Corona C. Alberti P, Nenci G. Picotamide protects mice from death in a pulmonary embolism model by a mechanism independent from thromboxane suppression. Thromb Haemost . 1990; 64:80-86.CrossrefMedlineGoogle Scholar
25 Mellion BT, Ignarro LJ, Ohlstein EH, Pontecorvo EG, Hyman AL, Kadowitz PJ. Evidence for the inhibitory role of guanosine 3′,5′-monophosphate in ADP-induced human platelet aggregation in the presence of nitric oxide and related vasodilators. Blood . 1981; 57:946-955.CrossrefMedlineGoogle Scholar
26 Weber AA, Strobach H, Schror K. Direct inhibition of platelet function by organic nitrates via nitric oxide formation. Eur J Pharmacol . 1993; 247:29-37.CrossrefMedlineGoogle Scholar
27 Ivanova K, Schaefe M, Drummer C, Gerzer R. Effects of nitric oxide-containing compounds on increases in cytosolic ionized Ca²⁺ and on aggregation of human platelets. Eur J Pharmacol . 1993; 244:37-47.CrossrefMedlineGoogle Scholar
28 Samama CM, Diaby M, Fellahi J, Mdhafar A, Eyranud D, Arock M, Guillosson J, Coriat P, Rouby J. Inhibition of platelet aggregation by inhaled nitric oxide in patients with acute respiratory distress syndrome. Anesthesiology . 1995; 83:56-65.CrossrefMedlineGoogle Scholar
29 Weyrich AS, Solis GA, Li KS, Tulenko TN, Santamore WP. Platelet amplification of vasospasm. Am J Physiol . 1992; 263:H349-H358.MedlineGoogle Scholar
30 Radomski MW, Palmer RM, Moncada S. An L-arginine/nitric oxide pathway present in human platelets regulates aggregation. Proc Natl Acad Sci U S A . 1990; 87:5193-5197.CrossrefMedlineGoogle Scholar
31 Freedman JE, Hankin B, Alpert C, Loscalzo J, Keaney JF, Vita JA. Impaired platelet production of nitric oxide in patients with coronary artery disease. Circulation . 1996; 94:2340. Abstract.Google Scholar

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