Effect of Nitric Oxide on the Cryopreservation of Platelets

Jae Hyeon Lee; Jeong Tae Kim; Yong Gon Cho

doi:10.3343/kjlm.2008.28.2.136

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Lee, Kim, and Cho: Effect of Nitric Oxide on the Cryopreservation of Platelets

Original Article

Korean J Leg Med 2008;28(2):136-143.

Published online: 14 February 2008

DOI: https://doi.org/10.3343/kjlm.2008.28.2.136

Effect of Nitric Oxide on the Cryopreservation of Platelets

Jae Hyeon Lee, M.D.¹, Jeong Tae Kim, M.D.¹, Yong Gon Cho, M.D.^1,²

¹Department of Laboratory Medicine, Chonbuk National University Medical School, Jeonju, Korea

²Research Institute of Clinical Medicine, Chonbuk National University, Jeonju, Korea

Received 16 January 20085 February 2008 Accepted 12 February 2008

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

To determine whether nitric oxide (NO) could inhibit activation of platelets stored in a cold or frozen state, we measured platelet P-selectin expression and platelet-bound fibrinogen in platelet-rich plasma (PRP) with S-nitrosoglutathione (GSNO) (Sigma, USA) by flow cytometry.

Methods

PRP was prepared by centrifuging venous blood collected in a 3.2% sodium citrate tube from 10 healthy donors. It was aliquotted into 4 groups (no cryoprotectant, GSNO, GSNO/dimethyl sulfoxide [DMSO] [Sigma], and DMSO), and stored at room, cold and freezing temperatures for 24 hrs. We performed a flow cytometric analysis of all specimens stained with FITC-fibrinogen and PE-CD62P monoclonal antibodies (Becton Dickinson, USA). The results were compared according to the storage temperature and agonist among 4 groups.

Results

GSNO inhibited significantly the activation of frozen platelets, but not in the presence of DMSO. GSNO was also shown to preserve the aggregability of frozen platelets because in the presence of GSNO the delta percent change of P-selectin expression and fibrinogen binding of frozen platelets increased significantly irrelevant to DMSO.

Conclusions

GSNO inhibited the activation of frozen platelets and preserved the platelet aggregability; therefore, it may be used as a protectant for platelet cryopreservation.

Keywords: Platelets, Cryopreservation, Nitric oxide

REFERENCES

1.Liu JH., Ouyang XL., Shi Q., Luo Q., Li XJ., Wang HB, et al. CD62p expression in platelet during the preparation course of Cryopreservated platelet-rich plasma. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2002. 10:253–6.

2.Armitage WJ. Osmotic stress as a factor in the detrimental effect of glycerol on human platelets. Cryobiology. 1986. 23:116–25.

3.Arnaud FG., Hunt CJ., Pegg DE. Some effects of propane-1,2-diol on human platelets. Cryobiology. 1990. 27:119–29.

4.Dayian G., Pert JH. A simplified method for freezing human blood platelets in glycerol-glucose using a statically controlled cooling rate device. Transfusion. 1979. 19:255–60.

5.Smillie JA., Munro AC., Wood GC., Mitchell R. Cryopreservation of human platelets with polyvinylpyrrolidone. Transfusion. 1981. 21:552–6.

6.Angelini A., Dragani A., Berardi A., Iacone A., Fioritoni G., Torlontano G. Evaluation of four different methods for platelet freezing. In vitro and in vivo studies. Vox Sang. 1992. 62:146–51.

7.Gao DY., Neff K., Xiao HY., Matsubayashi H., Cui XD., Bonderman P, et al. Development of optimal techniques for cryopreservation of human platelets. I. Platelet activation during cold storage (at 22 and 8 degrees C) and cryopreservation. Cryobiology. 1999. 38:225–35.

8.Lozano ML., Rivera J., Corral J., Gonzalez-Conejero R., Vicente V. Platelet cryopreservation using a reduced dimethyl sulfoxide concentration and second-messenger effectors as cryopreserving solution. Cryobiology. 1999. 39:1–12.

9.Borzini P., Lazzaro A., Mazzucco L., Schiavo R., Connor J., Siena S. Platelet cryopreservation using second-messenger effector and low-dose (2%) dimethyl sulfoxide. In vitro evaluation of post-thawing platelet activity with the platelet function analyzer. Haematologica. 2000. 85:885–7.

10.Xiao H., Harvey K., Labarrere CA., Kovacs R. Platelet cryopreservation using a combination of epinephrine and dimethyl sulfoxide as cryoprotectants. Cryobiology. 2000. 41:97–105.

11.Ekmekci H., Isler I., Sonmez H., Gurel C., Ciftci O., Ulutin T, et al. Comparison of platelet fibronectin, ADP-induced platelet aggregation and serum total nitric oxide (NOx) levels in angiographically determined coronary artery disease. Thromb Res. 2006. 117:249–54.

12.Marcondes S., Cardoso MH., Morganti RP., Thomazzi SM., Lilla S., Murad F, et al. Cyclic GMP-independent mechanisms contribute to the inhibition of platelet adhesion by nitric oxide donor: a role for alpha-actinin nitration. Proc Natl Acad Sci USA. 2006. 103:3434–9.

13.Stojanovic A., Marjanovic JA., Brovkovych VM., Peng X., Hay N., Skidgel RA, et al. A phosphoinositide 3-kinase-AKT-nitric oxide-cGMP signaling pathway in stimulating platelet secretion and aggregation. J Biol Chem. 2006. 281:16333–9.

14.Hagberg IA., Solvik UO., Opdahl H., Roald HE., Lyberg T. Inhalation of nitric oxide inhibits ADP-induced platelet aggregation and alpha-granule release. Platelets. 1999. 10:382–90.

15.Buyukkagnici DI., Ilhan O., Kavas GO., Arslan O., Arat M., Dalva K, et al. Effects of plasma nitric oxide levels on platelet activation in single donor apheresis and random donor concentrates. Transfus Apher Sci. 2007. 36:73–8.

16.Albert J., Daleskog M., Wallen NH. A comparison of the antiplatelet effect of S-nitrosoglutathione in whole blood and platelet-rich plasma. Thromb Res. 2001. 102:161–5.

17.Xiao HY., Matsubayashi H., Bonderman DP., Bonderman PW., Reid T., Miraglia CC, et al. Generation of annexin V-positive platelets and shedding of microparticles with stimulus-dependent procoagulant activity during storage of platelets at 4 degrees C. Transfusion. 2000. 40:420–7.

18.Kim SW., Lim YA. Establishment of reference values for platelet activation markers by flow cytometry. Korean J Lab Med. 2006. 26:323–8. (김신원 및 임영애. 유세포분석기를 이용한 혈소판 활성화 표지자 참고치 선정. 대한진단검사의학회지 2006;26: 323-8.).

19.Phillips DR., Charo IF., Parise LV., Fitzgerald LA. The platelet membrane glycoprotein IIb-IIIa complex. Blood. 1988. 71:831–43.

20.Brecher ME, editor. Technical manual. 15th ed.Bethesda, MD.: AABB;2005.

21.Owens M., Cimino C., Donnelly J. Cryopreserved platelets have decreased adhesive capacity. Transfusion. 1991. 31:160–3.

22.Fratantoni JC., Poindexter BJ. Dimethyl sulfoxide: effects on function of fresh platelets and on the viability of platelets in storage. Transfusion. 1983. 23:109–13.

23.Cetin M., Eser B., Er O., Unal A., Kilic E., Patiroglu T, et al. Effects of DMSO on platelet functions and P-selectin expression during storage. Transfus Apher Sci. 2001. 24:261–7.

24.Borzini P., Assali G., Riva MR., Bramante M., Sciorelli G. Platelet cry-opreservation using dimethylsulfoxide/polyethylene glycol/sugar mixture as cryopreserving solution. Vox Sang. 1993. 64:248–9.

25.Pedrazzoli P., Noris P., Perotti C., Schiavo R., Ponchio L., Belletti S, et al. Transfusion of platelet concentrates cryopreserved with ThromboSol plus low-dose dimethylsulphoxide in patients with severe thrombocytopenia: a pilot study. Br J Haematol. 2000. 108:653–9.

26.Wong K., Li X. Nitric oxide infusion alleviates cellular activation during preparation, leukofiltration and storage of platelets. Transfus Apher Sci. 2004. 30:29–39.

27.Langford EJ., Parfitt A., de Belder AJ., Marrinan MT., Martin JF. A study of platelet activation during human cardiopulmonary bypass and the effect of S-nitrosoglutathione. Thromb Haemost. 1997. 78:1516–9.

28.Wallen NH., Andersson A., Hjemdahl P. Effects of treatment with oral isosorbide dinitrate on platelet function in vivo; a double-blind placebo-controlled study in patients with stable angina pectoris. Br J C Pharmacol. 1994. 38:63–70.

29.Karlberg KE., Torfgard K., Ahlner J., Sylven C. Dose-dependent effect of intravenous nitroglycerin on platelet aggregation, and correlation with plasma glyceryl dinitrate concentration in healthy men. Am J Cardiol. 1992. 69:802–5.

30.Karlberg KE., Ahlner J., Henriksson P., Torfgard K., Sylven C. Effects of nitroglycerin on platelet aggregation beyond the effects of acetyl-salicylic acid in healthy subjects. Am J Cardiol. 1993. 71:361–4.

31.Albert J., Wallen NH., Broijersen A., Frostell C., Hjemdahl P. Effects of inhaled nitric oxide compared with aspirin on platelet function in vivo in healthy subjects. Clin Sci (Lond). 1996. 91:225–31.

32.Albert J., Norman M., Wallen NH., Frostell C., Hjemdahl P. Inhaled nitric oxide does not influence bleeding time or platelet function in healthy volunteers. Eur J Clin Invest. 1999. 29:953–9.

33.Radomski MW., Rees DD., Dutra A., Moncada S. S-nitroso-glutathione inhibits platelet activation in vitro and in vivo. Br J Pharmacol. 1992. 107:745–9.

34.Kelm M., Schrader J. Control of coronary vascular tone by nitric oxide. Circ Res. 1990. 66:1561–75.

Fig. 1.

The histogram of flow cytometric results with fibrinogen (FL1) and CD62P (FL2). A and C show the setting of a threshold discriminating between negative and positive platelets. B and D show the percentage of positive platelets above the threshold.

Table 1.

Comparison of P-selectin expression and platelet-bound fibrinogen measured in platelets without treatment of agonists among groups (N=10)

Marker	Temp	None	GSNO	GSNO/DMSO	DMSO	P value
Marker	Temp	None	GSNO	GSNO/DMSO	DMSO	N&G	D&DG	N&D
P-selectin	R	3.1±1.5	8.6±7.6	5.3±7.3	6.1±3.7	0.075	0.344	0.116
P-selectin	C	21.3±14.3	21.3±11.9	29.5±12.5	29.6±16.0	0.463	0.917	0.075
P-selectin	F	38.5±13.9	23.9±9.9	25.3±12.8	26.7±8.5	0.028	0.345	0.173
Fibrinogen	R	5.5±3.1	13.0±12.5	8.5±13.0	11.7±8.4	0.116	0.463	0.173
Fibrinogen	C	23.7±16.7	28.2±14.2	38.7±16.1	42.5±25.5	0.249	0.600	0.046
Fibrinogen	F	40.9±14.3	20.6±13.2	21.8±18.0	36.6±16.4	0.028	0.173	0.345

Abbreviations: R, room temperature; C, cold temperature; F, freezing temperature; N, none; G, GSNO; D, DMSO; DG, GSNO/DMSO.

Table 2.

Comparison of P-selectin expression and platelet-bound fibrinogen measured in platelets with treatment of ADP among groups (N=10)

Marker	Temp	None	GSNO	GSNO/DMSO	DMSO	P value
Marker	Temp	None	GSNO	GSNO/DMSO	DMSO	N&G	D&DG	N&D
P-selectin	R	72.6±3.5	69.6±6.3	28.3±9.7	9.5±5.9	0.075	0.028	0.028
P-selectin	C	45.1±18.9	45.5±20.5	29.8±19.0	24.2±16.3	0.917	0.600	0.028
P-selectin	F	24.5±10.7	29.0±7.5	32.3±7.5	16.5±3.4	0.345	0.028	0.173
Fibrinogen	R	71.4±5.9	70.9±10.1	22.2±10.8	10.1±10.3	0.917	0.116	0.028
Fibrinogen	C	43.6±17.6	44.8±20.0	30.1±20.8	36.3±19.5	0.917	0.600	0.463
Fibrinogen	F	22.8±14.2	30.0±10.7	29.9±6.5	20.0±9.7	0.249	0.116	0.753

Abbreviations: See Table 1.

Table 3.

Comparison of P-selectin expression and platelet-bound fibrinogen measured in platelets with treatment of thrombin among groups (N=10)

Marker	Temp	None	GSNO	GSNO/DMSO	DMSO	P value
Marker	Temp	None	GSNO	GSNO/DMSO	DMSO	N&G	D&DG	N&D
P-selectin	R	57.8±34.0	51.8±38.5	46.5±37.9	27.5±22.0	0.075	0.046	0.028
P-selectin	C	53.3±30.3	50.0±33.4	47.6±32.3	37.2±24.8	0.753	0.173	0.046
P-selectin	F	49.4±19.5	50.4±15.1	45.4±29.9	29.3±27.5	0.917	0.028	0.028
Fibrinogen	R	67.3±29.3	64.1±24.1	58.9±33.3	51.7±39.2	0.248	0.028	0.028
Fibrinogen	C	62.3±22.2	61.6±24.1	58.1±26.0	54.8±32.5	0.917	0.753	0.249
Fibrinogen	F	57.6±15.2	58.5±9.8	47.9±32.2	38.7±40.1	0.917	0.116	0.173

Abbreviations: See Table 1.

Table 4.

Comparison of delta percent change of P-selectin expression and platelet-bound fibrinogen measured in platelets with treatment of ADP among groups (N=10)

Marker	Temp	None	GSNO	GSNO/DMSO	DMSO	P value
Marker	Temp	None	GSNO	GSNO/DMSO	DMSO	N&G	D&DG	N&D
P-selectin	R	2,851.7±1,773.4	1,234.1±813.5	1,048.4±772.9	85.1±111.3	0.075	0.046	0.028
P-selectin	C	235.3±296.9	167.6±130.5	26.8±134.5	50.1±163.6	0.463	0.917	0.173
P-selectin	F	-35.2±20.0	43.3±80.6	51.5±75.7	-29.3±34.2	0.028	0.028	0.917
Fibrinogen	R	1,772.6±1,457.2	1,401.1±1,476.6	2,125.2±2,810.5	99.1±264.0	0.753	0.116	0.028
Fibrinogen	C	235.3±371.9	79.4±88.4	27.5±187.0	174.2±447.3	0.173	0.753	0.753
Fibrinogen	F	-45.5±23.7	127.9±176.3	168.6±225.2	-32.5±43.4	0.028	0.028	0.600

Delta percent change is calculated according to next equation. D (%)=([A-B]/B)×100. D, delta percent change; A, results after treatment of agonists; B, results before treatment of agonists.

Abbreviations: See Table 1.

Table 5.

Comparison of delta percent change of P-selectin expression and platelet-bound fibrinogen measured in platelets with treatment of thrombin among groups (N=10)

Marker	Temp	None	GSNO	GSNO/DMSO	DMSO	P value
Marker	Temp	None	GSNO	GSNO/DMSO	DMSO	N&G	D&DG	N&D
P-selectin	R	2,648.7±2,610.8	971.4±1053.0	3,390.0±5,076.4	469.7±497.3	0.046	0.046	0.028
P-selectin	C	316.2±419.5	232.7±265.3	77.5±129.1	249.6±540.3	0.345	0.345	0.345
P-selectin	F	29.9±38.6	135.6±114.4	130.5±230.4	47.6±209.5	0.028	0.028	0.345
Fibrinogen	R	1,885.2±2,182.3	1,256.0±1,476.7	7,238.2±11,580.2	1,295.2±2,177.2	0.753	0.249	0.345
Fibrinogen	C	393.1±543.2	187.8±191.7	91.7±135.6	716.2±1670.8	0.173	0.917	0.345
Fibrinogen	F	47.0±33.2	293.5±227.6	422.8±667.7	108.7±364.7	0.028	0.028	0.463

Delta percent change is calculated according to next equation. D (%)=[(A-B)/B]×100. D, delta percent change; A, results after treatment of agonists; B, results before treatment of agonists.

Abbreviations: See Table 1.

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