Journal of Clinical Neuroscience 22 (2015) 1123–1127
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Clinical Study
Dynamic changes in plasma tissue plasminogen activator, plasminogen activator inhibitor-1 and beta-thromboglobulin content in ischemic stroke Ping Zhuang b, Da Wo a, Zeng-guang Xu a, Wei Wei a, Hui-ming Mao a,⇑ a b
Department of Central Laboratory, Shanghai East Hospital, Tongji University, 150 Jimo Road, Pudong New District, Shanghai 200120, People’s Republic of China Shanghai International Medical Center, Shanghai, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 31 March 2014 Accepted 20 December 2014
Keywords: Beta–thromboglobulin Ischemic stroke Plasma Plasminogen activator inhibitor-1 Tissue plasminogen activator
a b s t r a c t The aim of this paper is to investigate the corresponding variations of plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) activities, and beta–thromboglobulin (b-TG) content in patients during different stages of ischemic stroke. Ischemic stroke is a common disease among aging people and its occurrence is associated with abnormalities in the fibrinolytic system and platelet function. However, few reports focus on the dynamic changes in the plasma fibrinolytic system and b-TG content in patients with ischemic stroke. Patients were divided into three groups: acute, convalescent and chronic. Plasma t-PA and PAI-1 activities were determined by chromogenic substrate analysis and plasma b-TG content was detected by radioimmunoassay. Patients in the acute stage of ischemic stroke had significantly increased levels of t-PA activity and b-TG content, but PAI-1 activity was significantly decreased. Negative correlations were found between plasma t-PA and PAI-1 activities and between plasma t-PA activity and b-TG content in patients with acute ischemic stroke. There were significant differences in plasma t-PA and PAI-1 activities in the aged control group, as well as in the acute, convalescent and chronic groups. It can be speculated that the increased activity of t-PA in patients during the acute stage was the result of compensatory function, and that the increase in plasma b-TG level not only implies the presence of ischemic stroke but is likely a cause of ischemic stroke. During the later stages of ischemic stroke, greater attention is required in monitoring levels of PAI-1. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Stroke is one of the leading causes of death around the world as well as being the primary cause of adult disability [1]. China has a high incidence of stroke, greater than 336 per 100,000 population in 2010 [2]. The occurrence of ischemic stroke is associated with abnormalities in fibrinolysis and platelet function [1,2]. Coagulation and fibrinolysis are precisely regulated by a combination of substrates, activators, inhibitors, cofactors and receptors under physiological conditions. Activation of coagulation ultimately produces thrombin resulting in the formation of thrombus by the conversion of fibrinogen to fibrin and by platelet activation [3]. Although plasminogen can also be converted to plasmin by urokinase in plasma [3], tissue plasminogen activator (t-PA), a serine protease, is the most important activator of fibrinolysis in vivo and converts plasminogen into fibrinolytically active plasmin. It is ⇑ Corresponding author. Tel.: +86 21 61569681; fax: +86 21 58763830. E-mail address: [email protected] (H.-m. Mao). http://dx.doi.org/10.1016/j.jocn.2014.12.027 0967-5868/Ó 2015 Elsevier Ltd. All rights reserved.
produced mainly in vascular endothelial cells but also in smooth muscle cells, monocytes and megakaryocytes. Plasminogen activator inhibitor-1 (PAI-1) is a single chain glycoprotein which is synthesized mainly in endothelial and hepatic cells but also in smooth muscle cells and adipose tissue. It has an inhibitive effect on serine proteases and is considered to be the major inhibitor of fibrinolysis and a marker of vascular endothelial dysfunction. Both t-PA and PAI-1 are regulative substances of the fibrinolytic system in vivo [4,5]. Both clinical and animal studies have demonstrated that during cerebral ischemia, plasma t-PA and/or PAI-1 levels change significantly [6–8]. Beta–thromboglobulin (b-TG) is the most abundant specific protein within the platelet a-granule and its presence in serum reflects platelet activation [9]. An earlier study found that in ischemic cerebrovascular diseases, plasma b-TG levels are significantly elevated [10]. These blood biomarkers play an important role in thrombogenesis and their measurement has been widely adopted in the field of cardiovascular and cerebrovascular diseases [11–14]. However, there are very few studies evaluating the simultaneous determination and correlation of these
1124
P. Zhuang et al. / Journal of Clinical Neuroscience 22 (2015) 1123–1127
three blood biomarkers in patients with ischemic stroke. In addition, few studies have examined the relationship during different stages of ischemic stroke and the fibrinolytic system. In order to investigate dynamic changes in the fibrinolytic system and in platelet function in patients with ischemic stroke, we measured the plasma activities of t-PA and PAI-1, and b-TG content in patients with acute ischemic stroke and analyzed the correlation between the three biomarkers. We then evaluated the plasma activities of t-PA and PAI-1 in patients during the convalescent and chronic stages of ischemic stroke.
2. Patients and methods 2.1. Patients Blood samples were collected from 24 patients with ischemic stroke (middle cerebral artery occlusion) during different stages of the disease. The acute stage is within 2 days of ischemic stroke onset, the convalescent stage two weeks after the onset of ischemic stroke and the chronic stage 6 months following onset. Patients were taken to the Emergency Department at Shanghai East Hospital, Tongji University School of Medicine eleven patients were men and 13 were women. Mean age was 63 years (range: 55–86). The inclusion criterion was patients with ischemic stroke confirmed by head CT scan. Exclusion criteria were cerebral hemorrhage, hemorrhagic transformation, progressive ischemic stroke or repeated stroke and cardio embolism. Among the patients with ischemic stroke, six had hypertension, 11 had hyperlipidemia and five had diabetes (Table 1). All patients were discharged from hospital, had recovery of limb function, limb function and could walk independently. We had 35 healthy volunteers consisting of 21 men and 14 women from Shanghai Geriatric University with a mean age of 62 years (range: 55–80). Subjects from the control group did not suffer from liver, kidney, blood, cardiac, cerebrovascular or thrombotic diseases. The patient control group (non-ischemic stroke) consisted of 20 patients (12 men and eight women) with a mean age of 61 years (range: 54–83). Among the patients, eight had hypertension, six had hyperlipidemia and six had diabetes.
Table 1 Details of patients with acute ischemic stroke Characteristic
Patients
Mean age (years) Sex (%) Weight (kg) NIHSS score (mean)* Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Hyperlipidemia (%) Diabetes (%) Previous use of aspirin or antiplatelet drugs (%) History of stroke (%)
63.4 ± 8.3 45.8 M, 54.2F 64.1 ± 13.2 12.0 ± 6.3 153.6 ± 18.4 84.8 ± 14.5 45.8 20.8 12.2
Smoking status (%) Never smoked Ex-smoker Current smoker Type of stroke (%)
All patients and healthy controls received no statins, calcium channel blockers or other drugs that may influence the level of blood biomarkers for the 2 weeks prior to study entry. The protocol was approved by the Local Ethics Committee of Shanghai East Hospital, Tongji University School of Medicine. 2.2. Methods 2.2.1. Sample processing Plasma was obtained by venous blood collection from all subjects using size 8 needles. The first 2 ml of blood was added to a test tube containing anti-coagulant agent (0.13 M natrium citricum; anti-coagulant agent to blood 1:9 v/v) to be used for the determination of the plasma t-PA and PAI-1 activities. Next, the syringe was detached from the needle and 2 ml venous blood was freely dripped into a separate plastic test tube containing anti-coagulant agent (ethylenediaminetetraacetic acid, theocin and prostaglandin E1; anti-coagulant agent to blood 1:9 v/v) for the determination of plasma b-TG content. Blood and anti-coagulant agent were mixed gently. Plasma was collected by centrifugation (3000 rpm, 20 min) at 4 °C before being subpackaged. Acetic acid buffer (pH 3.9) and a portion of plasma were mixed in equal volumes for the determination of t-PA activity. All samples were stored at 70 °C until analysis. 2.2.2. Determination The plasma activities of t-PA and PAI-1 were determined by chromogenic substrate analytical method. The kits used in this study were obtained from the Laboratory of Molecule Heredity at Fudan University School of Medicine. Under the action of an enzyme such as fibrin, t-PA rapidly activates plasminogen to form plasmin which undergoes hydrolysis as But-CHT-Lys-pNA generating free paranitroaniline (pNA). pNA has a strong absorption peak at 405 nm wavelength. Therefore, plasma activity of t-PA can be measured indirectly. Briefly, the plasma sample was diluted 1:60 with buffer, t-PA standard (100 International units [IU]/ml) was diluted to 1.0 IU/ml with acidified and t-PA-free plasma, a series of t-PA standards were prepared by buffer solution (unit of activity is 0–0.04 IU/ml). The standard and sample wells for the ELISA were set up and 100 ll of t-PA standard and sample were added to the wells, respectively. Next, 100 ll of plasminogen chromogenic substrate mixture was added to each well and the ELISA plates were incubated in a 37 °C water bath for 5 h. The sample absorbance was then measured on a microplate reader at 405 nm. The measurement of plasma PAI-1 activity was identical to that of t-PA according to the instructions [15]. The plasma activities of t-PA and PAI-1 were expressed in IU and arbitrary units (Au), respectively. The 1.0 Au of PAI-1 was defined as the amount required for 1.0 IU activity of t-PA to be inhibited by PAI-1. The plasma content of b-TG was determined by radioimmunoassay according to the instruction manual [16]. The kits used were obtained from the Ruijin Hospital, Shanghai Jiaotong University School of Medicine. The plasma content of b-TG was expressed as ng/ml.
8.9 54.2 16.4 29.4 37.5 (L-MCAO), 62.5 (R-MCAO)
All values are reported as the mean ± standard deviation, unless otherwise specified. L-MCAO = left-middle cerebral artery occlusion, NIHSS = National Institutes of Health Stroke Scale, R-MCAO = right-middle cerebral artery occlusion. * Scores on the NIHSS range from 0 to 42, with higher values reflecting more severe neurological impairment ( 0.05). The plasma activities of t-PA and PAI-1 in patients during the acute, convalescent and chronic stages are shown in Figure 2 and 3, respectively. The increase in plasma t-PA activity during the acute stage was maintained until the convalescent stage, and subsequently decreased during the chronic stage (Fig. 2). Plasma PAI-1 activity initially decreased during acute stage but subsequently there was a notable increase, especially during the chronic stage (Fig. 3). One-way analysis of variance with post hoc multiple comparisons correction indicated that among the healthy controls and the acute, convalescent and chronic stage groups, there were significant differences in plasma t-PA and PAI-1 activities (p < 0.001; Fig. 2, 3). The changes in plasma t-PA and PAI-1 activities in individual patients over time (during the 6 months post stroke period) are displayed in Supplementary Figure 1. The average fold changes of plasma t-PA and PAI-1 activities in patients with ischemic stroke at each stage are shown in Figure 4. There were significant differences between the acute, convalescent and chronic stage groups in average fold changes (p < 0.001). 4. Discussion It is widely understood that the balance between t-PA and its natural, fast-acting inhibitor PAI-1 plays an important role in regulating blood flow and that if that balance is offset a series of severe pathological changes arises [5,17]. t-PA diffuses from the vascular cells and cleaves plasminogen to plasmin. Subsequently, plasmin degrades the fibrin polymer into smaller fragments that are cleared by the monocyte-macrophage scavenger system. Therefore, the normal synthesis and release of t-PA is important for preventing thrombosis in vivo [5,18–19]. Normally, when there is no fibrin in circulating blood, t-PA is secreted at low levels and
Fig. 1. (A) Negative correlation was found between plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) activities in patients with acute ischemic stroke (r = 0.664; p < 0.01); (B) Negative correlation was found between plasma t-PA activity and beta-thromboglobulin (b-TG) content in patients with acute ischemic stroke (r = 0.677; p < 0.01).
Table 2 Plasma levels of t-PA, PAI-1 and b-TG in patients with acute ischemic stroke compared to control groups Patient groups
n
t-PA (IU/ml)
PAI-1 (Au/ml)
b-TG (ng/ml)
Age-matched control Patient control Acute patient
35
3.309 ± 1.878
11.169 ± 3.939
18.56 ± 14.34
20 24
3.101 ± 1.916 9.000 ± 3.803**
13.518 ± 5.072 7.233 ± 5.014*
20.74 ± 16.11 51.09 ± 22.99**
All values are reported as mean ± standard deviation. b-TG = beta-thromboglobulin, Au = arbitrary units, IU = International units, PAI1 = plasminogen activator inhibitor-1, t-PA = tissue plasminogen activator. * Compared to age-matched control p < 0.01. ** Compared to age-matched control p < 0.001.
Fig. 2. Plasma activity of tissue plasminogen activator (t-PA) in patients during different stages of ischemic stroke. *Compared to aged or patient control, p < 0.001; ** Compared to age-matched or patient control, p < 0.01; ***Compared to aged or patient control, p < 0.05; ###Compared to acute stage, p < 0.05.
1126
P. Zhuang et al. / Journal of Clinical Neuroscience 22 (2015) 1123–1127
Fig. 3. Plasma activity of plasminogen activator inhibitor-1 (PAI-1) in patients during different stages of ischemic stroke. *Compared to age-matched or patient control (p < 0.001); **Compared to age-matched or patient control (p < 0.01); # Compared to acute stage (p < 0.001); ##Compared to acute stage (p < 0.01); D Compared to convalescent stage (p < 0.05).
Fig. 4. Bar graph showing the average fold changes of plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) activities in patients with ischemic stroke at each stage (acute, convalescent, chronic). (A) Average fold change of plasma t-PA; (B) Average fold change of plasma PAI-1. *Kruskal–Wallis H test (p < 0.001).
has very weak activity. However, once thrombosis occurs along with vascular endothelial cell activation, t-PA is abundantly secreted and has higher activity to dissolve the thrombus. As we observed, the plasma t-PA activity in patients with acute ischemic stroke was significantly higher than in the age-matched healthy controls. Essentially, it can be speculated that this was a result of a compensatory function and indicated a decrease in fibrinolytic activity.
PAI-1, a fast-acting inhibitor of t-PA, can rapidly combine with t-PA forming a 1:1 ratio compound which inactivates t-PA. When t-PA combines with fibrin it can activate plasminogen. PAI-1 has a strong inhibitive effect on free t-PA. However, it has a very weak effect on t-PA bound to fibrin [19–21]. We found that plasma PAI-1 activity in patients with acute ischemic stroke was significantly lower than that of age-matched healthy controls or non-ischemic stroke patient controls. Furthermore, there was a significant negative correlation between plasma t-PA and PAI-1 activities in patients with acute ischemic stroke suggesting that plasminogen activation is enhanced in patients with acute ischemic stroke. The abnormalities in plasma t-PA and PAI-1 activities in acute ischemic stroke patients may simply be the result of cerebral infarction and hence may not reflect the diseased state during the period of onset. For this reason, we studied the plasma t-PA and PAI-1 activities in patients during convalescent and chronic stages. The results showed that plasma t-PA activity decreased in the later stages together with the functional recovery of limbs whereas the plasma PAI-1 activity increased considerably suggesting plasminogen activation is weakened. Along with the reduction of t-PA activity, plasma PAI-1 activity increased considerably during convalescent and chronic stages. Hence, in the later stages following cerebral thrombosis the activity of plasma t-PA and PAI-1 changed inversely, especially plasma PAI-1 activity which increased significantly during the chronic stage and is therefore a potential cause of recurrent ischemic stroke. Previous research [18] confirmed that the development of thrombotic disease was not only correlated with decreased t-PA activity but also the secretory increase of PAI-1. This means that an increased circulatory activity of PAI-1 is associated with an increased risk of ischemic stroke. Therefore, in order to reduce the incidence of ischemic stroke greater attention should be paid to the circulating activity of PAI-1. t-PA is mainly found in the blood and acts as a central nervous system (CNS) vascular permeability regulator. It is also expressed in the CNS [22,23]. Although t-PA has proven benefits related to vessel recanalization and can be used for the treatment of thromboembolic stroke, in some patients there are reports of adverse reactions, especially intracerebral hemorrhage [24]. Additionally, up to 13% of ischemic stroke patients suffer re-occlusions following treatment with t-PA [25]. Animal studies also suggest that t-PA deficiency increases the transient middle cerebral artery occlusion after vascular fibrous deposition with an increase in brain injury. However, most studies show that excessive t-PA in the CNS leads to neuronal death. The endogenous t-PA in the CNS directly increases vascular permeability causing loss of blood-brain barrier integrity. Thrombolysis with t-PA is associated with increased vascular permeability [23,24] and delayed reperfusion with recombinant t-PA can cause hemorrhagic transformation. In view of the above reasons, the use of t-PA is strictly regulated in a clinical setting [19,26]. Previous research has shown that t-PA plays an inhibitory role in platelet aggregation in vitro. It has been shown that platelets can release b-TG and other substances after aggregation [27]. Therefore, the increase in plasma t-PA activity could lead to the decrease of b-TG level. Similarly, we observed a negative correlation between plasma t-PA activity and b-TG content within the same patient. Regarding the relationship between platelet function and thrombotic disease, tests which merely assess platelet aggregation and adhesion cannot provide direct evidence of platelet activation. Therefore, determining plasma b-TG content as a specific marker is crucial in reflecting platelet activation and release in thrombotic disease [9,28]. In accordance with an earlier study [11], our results showed that plasma b-TG level in patients with ischemic stroke was significantly higher than healthy controls suggesting that
P. Zhuang et al. / Journal of Clinical Neuroscience 22 (2015) 1123–1127
platelet function was in an augmented state and hence its release was enhanced. This is probably the reason why impaired vascular endodermis is more likely to cause platelet adhesion and aggregation and thus induce excessive activation of platelets. Previous research confirmed that b-TG has an inhibitory role on the enzyme prostacyclin (PGI2) synthase, resulting in the decrease of PGI2 synthesis in vascular endothelial cells [28]. On the other hand, because PGI2 has an inhibitive role on platelet aggregation, an increase of plasma b-TG level enhances platelet aggregation and subsequently accelerates thrombosis. Hence, it can be speculated that an increase in plasma b-TG level not only implies the presence of ischemic stroke but is likely to be a cause of ischemic stroke. In summary, because the application of t-PA is strictly limited in a clinical setting, the addition of thrombolytic therapies which could improve platelet function and lower b-TG levels would be invaluable in order to reduce re-occlusions and facilitate the recovery of brain function. Conflicts of Interest/Disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication. Acknowledgments We thank the staff of the Departments of Health Examination and Neurology at Shanghai East Hospital, Tongji University School of Medicine for providing plasma samples. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jocn.2014.12.027. References [1] Lou M. Thrombolysis in acute ischemic stroke: issues and options. Zhejiang Da Xue Xue Bao Yi Xue Ban 2014;43:1–6. [2] Jung S, Stapf C, Arnold M. Stroke unit management and revascularisation in acute ischemic stroke. Eur Neurol 2015;73:98–105. [3] Cesarman-Maus G, Hajjar KA. Molecular mechanisms of fibrinolysis. Brit J Haematol 2005;129:307–21. [4] Chudy´ P, Kotulicová D, Staško J, et al. The relationship among TAFI, t-PA, PAI-1 and F1 + 2 in type 2 diabetic patients with normoalbuminuria and microalbuminuria. Blood Coagul Fibrinol 2011;22:493–8. [5] Yoon BK, Kang YH, Oh WJ, et al. Impact of lysophosphatidylcholine on the plasminogen activator system in cultured vascular smooth muscle cells. J Korean Med Sci 2012;27:803–10. [6] Kario K, Yano Y, Matsuo T, et al. Additional impact of morning haemostatic risk factors and morning blood pressure surge on stroke risk in older Japanese hypertensive patients. Eur Heart J 2011;32:574–80.
1127
[7] Hosomi N, Lucero J, Heo JH, et al. Rapid differential endogenous plasminogen activator expression after acute middle cerebral artery occlusion. Stroke 2001;32:1341–8. [8] Niego B, Medcalf RL. Plasmin-dependent modulation of the blood–brain barrier: a major consideration during tPA-induced thrombolysis? J Cereb Blood Flow Metab 2014;34:1283–96. [9] Ravindran R, Krishnan LK. A biochemical study on the effect of proteolysis of beta-thromboglobulin proteins released from activated platelets on fibroblast proliferation. Pathophysiol Haemost Thromb 2007;36:285–9. [10] Taomoto K, Asada M, Kanazawa Y, et al. Usefulness of the measurement of plasma beta-thromboglobulin (beta-TG) in cerebrovascular disease. Stroke 1983;14:518–24. [11] Iskra T, Turaj W, Stowik A, et al. Hemostatic markers of endothelial injury in ischaemic stroke caused by large or small vessel disease. Pol Merkur Lekarski 2006;21:429–33. [12] Montaner J. Stroke biomarkers: can they help us to guide stroke thrombolysis? Timely Top Med Cardiovasc Dis 2007;11:E11. [13] Soeki T, Tamura Y, Fukuda N, et al. Plasma and platelet plasminogen activator inhibitor-1 in patients with acute myocardial infarction. Jpn Circ J 2000;64:547–53. [14] Vuckovic´ B, Deric´ M, Zarkov M. Influence of fibrinolitic potential reduction in ischemic brain disease development. Med Pregl 2008;61:247–51. [15] Wang JY, Ja HY, Song HY. Determination of plasma tissue type plasminogen activator and its inhibitor activity. Chin J Lab Med 1989;12:163–6. [16] Shi AL. b-TG RIA and clinical application. Chin J Nucl Med 1985;5:98. [17] Abanonu GB, Daskin A, Akdogan MF, et al. Mean platelet volume and bthromboglobulin levels in familial mediterranean fever: effect of colchicine use? Eur J Intern Med 2012;23:661–4. [18] Vaughan DE. PAI-1 antagonists: the promise and the peril. Trans Am Clin Climatol Assoc 2011;122:312–25. [19] Suzuki Y, Nagai N, Umemura K. Novel situations of endothelial injury in stroke – mechanisms of stroke and strategy of drug development: intracranial bleeding associated with the treatment of ischemic stroke: thrombolytic treatment of ischemia-affected endothelial cells with tissue-type plasminogen activator. J Pharmacol Sci 2011;116:25–9. [20] Lou M, Selim M. Does body weight influence the response to intravenous tissue plasminogen activator in stroke patients? Cerebrovasc Dis 2009;27:84–90. [21] Ekmekçi H, Güngör Öztürk Z, Ekmekçi OB, et al. Significance of vitronectin and PAI-1 activity levels in carotid artery disease: comparison of symptomatic and asymptomatic patients. Minerva Med 2013;104:215–23. [22] Samson AL, Medcalf RL. Tissue-type plasminogen activator: a multifaceted modulator of neurotransmission and synaptic plasticity. Neuron 2006;50: 673–8. [23] Yepes M, Sandkvist M, Moore EG, et al. Tissue-type plasminogen activator induces opening of the blood–brain barrier via the LDL receptor-related protein. J Clin Invest 2003;112:1533–40. [24] Nicole O, Docagne F, Ali C, et al. The proteolytic activity of tissue-plasminogen activator enhances NMDA receptor-mediated signaling. Nat Med 2001;7: 59–64. [25] Fernandez-Cadenas I, Del Rio-Espinola A, Rubiera M, et al. PAI-1 4G/5G polymorphism is associated with brain vessel reocclusion after successful fibrinolytic therapy in ischemic stroke patients. Int J Neurosci 2010;120: 245–51. [26] Yamashita T, Abe K. Therapeutic approaches to vascular protectionin ischemic stroke. Acta Med Okayama 2011;65:219–23. [27] Schoorl M, Schoorl M, Nubé MJ, et al. Platelet depletion, platelet activation and coagulation during treatment with hemodialysis. Scand J Clin Lab Invest 2011;71:240–7. [28] Sun LM, Ma ST, Geng M. Study on the relationship between plasmic b-TG, PF4 levels and hemorheology in gerontal patients with the insufficiency of Qi and stagnation of blood. J Pract Tradition Chin Int Med 2007;21:74–5.
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Clinical Study
Dynamic changes in plasma tissue plasminogen activator, plasminogen activator inhibitor-1 and beta-thromboglobulin content in ischemic stroke Ping Zhuang b, Da Wo a, Zeng-guang Xu a, Wei Wei a, Hui-ming Mao a,⇑ a b
Department of Central Laboratory, Shanghai East Hospital, Tongji University, 150 Jimo Road, Pudong New District, Shanghai 200120, People’s Republic of China Shanghai International Medical Center, Shanghai, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 31 March 2014 Accepted 20 December 2014
Keywords: Beta–thromboglobulin Ischemic stroke Plasma Plasminogen activator inhibitor-1 Tissue plasminogen activator
a b s t r a c t The aim of this paper is to investigate the corresponding variations of plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) activities, and beta–thromboglobulin (b-TG) content in patients during different stages of ischemic stroke. Ischemic stroke is a common disease among aging people and its occurrence is associated with abnormalities in the fibrinolytic system and platelet function. However, few reports focus on the dynamic changes in the plasma fibrinolytic system and b-TG content in patients with ischemic stroke. Patients were divided into three groups: acute, convalescent and chronic. Plasma t-PA and PAI-1 activities were determined by chromogenic substrate analysis and plasma b-TG content was detected by radioimmunoassay. Patients in the acute stage of ischemic stroke had significantly increased levels of t-PA activity and b-TG content, but PAI-1 activity was significantly decreased. Negative correlations were found between plasma t-PA and PAI-1 activities and between plasma t-PA activity and b-TG content in patients with acute ischemic stroke. There were significant differences in plasma t-PA and PAI-1 activities in the aged control group, as well as in the acute, convalescent and chronic groups. It can be speculated that the increased activity of t-PA in patients during the acute stage was the result of compensatory function, and that the increase in plasma b-TG level not only implies the presence of ischemic stroke but is likely a cause of ischemic stroke. During the later stages of ischemic stroke, greater attention is required in monitoring levels of PAI-1. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Stroke is one of the leading causes of death around the world as well as being the primary cause of adult disability [1]. China has a high incidence of stroke, greater than 336 per 100,000 population in 2010 [2]. The occurrence of ischemic stroke is associated with abnormalities in fibrinolysis and platelet function [1,2]. Coagulation and fibrinolysis are precisely regulated by a combination of substrates, activators, inhibitors, cofactors and receptors under physiological conditions. Activation of coagulation ultimately produces thrombin resulting in the formation of thrombus by the conversion of fibrinogen to fibrin and by platelet activation [3]. Although plasminogen can also be converted to plasmin by urokinase in plasma [3], tissue plasminogen activator (t-PA), a serine protease, is the most important activator of fibrinolysis in vivo and converts plasminogen into fibrinolytically active plasmin. It is ⇑ Corresponding author. Tel.: +86 21 61569681; fax: +86 21 58763830. E-mail address: [email protected] (H.-m. Mao). http://dx.doi.org/10.1016/j.jocn.2014.12.027 0967-5868/Ó 2015 Elsevier Ltd. All rights reserved.
produced mainly in vascular endothelial cells but also in smooth muscle cells, monocytes and megakaryocytes. Plasminogen activator inhibitor-1 (PAI-1) is a single chain glycoprotein which is synthesized mainly in endothelial and hepatic cells but also in smooth muscle cells and adipose tissue. It has an inhibitive effect on serine proteases and is considered to be the major inhibitor of fibrinolysis and a marker of vascular endothelial dysfunction. Both t-PA and PAI-1 are regulative substances of the fibrinolytic system in vivo [4,5]. Both clinical and animal studies have demonstrated that during cerebral ischemia, plasma t-PA and/or PAI-1 levels change significantly [6–8]. Beta–thromboglobulin (b-TG) is the most abundant specific protein within the platelet a-granule and its presence in serum reflects platelet activation [9]. An earlier study found that in ischemic cerebrovascular diseases, plasma b-TG levels are significantly elevated [10]. These blood biomarkers play an important role in thrombogenesis and their measurement has been widely adopted in the field of cardiovascular and cerebrovascular diseases [11–14]. However, there are very few studies evaluating the simultaneous determination and correlation of these
1124
P. Zhuang et al. / Journal of Clinical Neuroscience 22 (2015) 1123–1127
three blood biomarkers in patients with ischemic stroke. In addition, few studies have examined the relationship during different stages of ischemic stroke and the fibrinolytic system. In order to investigate dynamic changes in the fibrinolytic system and in platelet function in patients with ischemic stroke, we measured the plasma activities of t-PA and PAI-1, and b-TG content in patients with acute ischemic stroke and analyzed the correlation between the three biomarkers. We then evaluated the plasma activities of t-PA and PAI-1 in patients during the convalescent and chronic stages of ischemic stroke.
2. Patients and methods 2.1. Patients Blood samples were collected from 24 patients with ischemic stroke (middle cerebral artery occlusion) during different stages of the disease. The acute stage is within 2 days of ischemic stroke onset, the convalescent stage two weeks after the onset of ischemic stroke and the chronic stage 6 months following onset. Patients were taken to the Emergency Department at Shanghai East Hospital, Tongji University School of Medicine eleven patients were men and 13 were women. Mean age was 63 years (range: 55–86). The inclusion criterion was patients with ischemic stroke confirmed by head CT scan. Exclusion criteria were cerebral hemorrhage, hemorrhagic transformation, progressive ischemic stroke or repeated stroke and cardio embolism. Among the patients with ischemic stroke, six had hypertension, 11 had hyperlipidemia and five had diabetes (Table 1). All patients were discharged from hospital, had recovery of limb function, limb function and could walk independently. We had 35 healthy volunteers consisting of 21 men and 14 women from Shanghai Geriatric University with a mean age of 62 years (range: 55–80). Subjects from the control group did not suffer from liver, kidney, blood, cardiac, cerebrovascular or thrombotic diseases. The patient control group (non-ischemic stroke) consisted of 20 patients (12 men and eight women) with a mean age of 61 years (range: 54–83). Among the patients, eight had hypertension, six had hyperlipidemia and six had diabetes.
Table 1 Details of patients with acute ischemic stroke Characteristic
Patients
Mean age (years) Sex (%) Weight (kg) NIHSS score (mean)* Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Hyperlipidemia (%) Diabetes (%) Previous use of aspirin or antiplatelet drugs (%) History of stroke (%)
63.4 ± 8.3 45.8 M, 54.2F 64.1 ± 13.2 12.0 ± 6.3 153.6 ± 18.4 84.8 ± 14.5 45.8 20.8 12.2
Smoking status (%) Never smoked Ex-smoker Current smoker Type of stroke (%)
All patients and healthy controls received no statins, calcium channel blockers or other drugs that may influence the level of blood biomarkers for the 2 weeks prior to study entry. The protocol was approved by the Local Ethics Committee of Shanghai East Hospital, Tongji University School of Medicine. 2.2. Methods 2.2.1. Sample processing Plasma was obtained by venous blood collection from all subjects using size 8 needles. The first 2 ml of blood was added to a test tube containing anti-coagulant agent (0.13 M natrium citricum; anti-coagulant agent to blood 1:9 v/v) to be used for the determination of the plasma t-PA and PAI-1 activities. Next, the syringe was detached from the needle and 2 ml venous blood was freely dripped into a separate plastic test tube containing anti-coagulant agent (ethylenediaminetetraacetic acid, theocin and prostaglandin E1; anti-coagulant agent to blood 1:9 v/v) for the determination of plasma b-TG content. Blood and anti-coagulant agent were mixed gently. Plasma was collected by centrifugation (3000 rpm, 20 min) at 4 °C before being subpackaged. Acetic acid buffer (pH 3.9) and a portion of plasma were mixed in equal volumes for the determination of t-PA activity. All samples were stored at 70 °C until analysis. 2.2.2. Determination The plasma activities of t-PA and PAI-1 were determined by chromogenic substrate analytical method. The kits used in this study were obtained from the Laboratory of Molecule Heredity at Fudan University School of Medicine. Under the action of an enzyme such as fibrin, t-PA rapidly activates plasminogen to form plasmin which undergoes hydrolysis as But-CHT-Lys-pNA generating free paranitroaniline (pNA). pNA has a strong absorption peak at 405 nm wavelength. Therefore, plasma activity of t-PA can be measured indirectly. Briefly, the plasma sample was diluted 1:60 with buffer, t-PA standard (100 International units [IU]/ml) was diluted to 1.0 IU/ml with acidified and t-PA-free plasma, a series of t-PA standards were prepared by buffer solution (unit of activity is 0–0.04 IU/ml). The standard and sample wells for the ELISA were set up and 100 ll of t-PA standard and sample were added to the wells, respectively. Next, 100 ll of plasminogen chromogenic substrate mixture was added to each well and the ELISA plates were incubated in a 37 °C water bath for 5 h. The sample absorbance was then measured on a microplate reader at 405 nm. The measurement of plasma PAI-1 activity was identical to that of t-PA according to the instructions [15]. The plasma activities of t-PA and PAI-1 were expressed in IU and arbitrary units (Au), respectively. The 1.0 Au of PAI-1 was defined as the amount required for 1.0 IU activity of t-PA to be inhibited by PAI-1. The plasma content of b-TG was determined by radioimmunoassay according to the instruction manual [16]. The kits used were obtained from the Ruijin Hospital, Shanghai Jiaotong University School of Medicine. The plasma content of b-TG was expressed as ng/ml.
8.9 54.2 16.4 29.4 37.5 (L-MCAO), 62.5 (R-MCAO)
All values are reported as the mean ± standard deviation, unless otherwise specified. L-MCAO = left-middle cerebral artery occlusion, NIHSS = National Institutes of Health Stroke Scale, R-MCAO = right-middle cerebral artery occlusion. * Scores on the NIHSS range from 0 to 42, with higher values reflecting more severe neurological impairment ( 0.05). The plasma activities of t-PA and PAI-1 in patients during the acute, convalescent and chronic stages are shown in Figure 2 and 3, respectively. The increase in plasma t-PA activity during the acute stage was maintained until the convalescent stage, and subsequently decreased during the chronic stage (Fig. 2). Plasma PAI-1 activity initially decreased during acute stage but subsequently there was a notable increase, especially during the chronic stage (Fig. 3). One-way analysis of variance with post hoc multiple comparisons correction indicated that among the healthy controls and the acute, convalescent and chronic stage groups, there were significant differences in plasma t-PA and PAI-1 activities (p < 0.001; Fig. 2, 3). The changes in plasma t-PA and PAI-1 activities in individual patients over time (during the 6 months post stroke period) are displayed in Supplementary Figure 1. The average fold changes of plasma t-PA and PAI-1 activities in patients with ischemic stroke at each stage are shown in Figure 4. There were significant differences between the acute, convalescent and chronic stage groups in average fold changes (p < 0.001). 4. Discussion It is widely understood that the balance between t-PA and its natural, fast-acting inhibitor PAI-1 plays an important role in regulating blood flow and that if that balance is offset a series of severe pathological changes arises [5,17]. t-PA diffuses from the vascular cells and cleaves plasminogen to plasmin. Subsequently, plasmin degrades the fibrin polymer into smaller fragments that are cleared by the monocyte-macrophage scavenger system. Therefore, the normal synthesis and release of t-PA is important for preventing thrombosis in vivo [5,18–19]. Normally, when there is no fibrin in circulating blood, t-PA is secreted at low levels and
Fig. 1. (A) Negative correlation was found between plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) activities in patients with acute ischemic stroke (r = 0.664; p < 0.01); (B) Negative correlation was found between plasma t-PA activity and beta-thromboglobulin (b-TG) content in patients with acute ischemic stroke (r = 0.677; p < 0.01).
Table 2 Plasma levels of t-PA, PAI-1 and b-TG in patients with acute ischemic stroke compared to control groups Patient groups
n
t-PA (IU/ml)
PAI-1 (Au/ml)
b-TG (ng/ml)
Age-matched control Patient control Acute patient
35
3.309 ± 1.878
11.169 ± 3.939
18.56 ± 14.34
20 24
3.101 ± 1.916 9.000 ± 3.803**
13.518 ± 5.072 7.233 ± 5.014*
20.74 ± 16.11 51.09 ± 22.99**
All values are reported as mean ± standard deviation. b-TG = beta-thromboglobulin, Au = arbitrary units, IU = International units, PAI1 = plasminogen activator inhibitor-1, t-PA = tissue plasminogen activator. * Compared to age-matched control p < 0.01. ** Compared to age-matched control p < 0.001.
Fig. 2. Plasma activity of tissue plasminogen activator (t-PA) in patients during different stages of ischemic stroke. *Compared to aged or patient control, p < 0.001; ** Compared to age-matched or patient control, p < 0.01; ***Compared to aged or patient control, p < 0.05; ###Compared to acute stage, p < 0.05.
1126
P. Zhuang et al. / Journal of Clinical Neuroscience 22 (2015) 1123–1127
Fig. 3. Plasma activity of plasminogen activator inhibitor-1 (PAI-1) in patients during different stages of ischemic stroke. *Compared to age-matched or patient control (p < 0.001); **Compared to age-matched or patient control (p < 0.01); # Compared to acute stage (p < 0.001); ##Compared to acute stage (p < 0.01); D Compared to convalescent stage (p < 0.05).
Fig. 4. Bar graph showing the average fold changes of plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) activities in patients with ischemic stroke at each stage (acute, convalescent, chronic). (A) Average fold change of plasma t-PA; (B) Average fold change of plasma PAI-1. *Kruskal–Wallis H test (p < 0.001).
has very weak activity. However, once thrombosis occurs along with vascular endothelial cell activation, t-PA is abundantly secreted and has higher activity to dissolve the thrombus. As we observed, the plasma t-PA activity in patients with acute ischemic stroke was significantly higher than in the age-matched healthy controls. Essentially, it can be speculated that this was a result of a compensatory function and indicated a decrease in fibrinolytic activity.
PAI-1, a fast-acting inhibitor of t-PA, can rapidly combine with t-PA forming a 1:1 ratio compound which inactivates t-PA. When t-PA combines with fibrin it can activate plasminogen. PAI-1 has a strong inhibitive effect on free t-PA. However, it has a very weak effect on t-PA bound to fibrin [19–21]. We found that plasma PAI-1 activity in patients with acute ischemic stroke was significantly lower than that of age-matched healthy controls or non-ischemic stroke patient controls. Furthermore, there was a significant negative correlation between plasma t-PA and PAI-1 activities in patients with acute ischemic stroke suggesting that plasminogen activation is enhanced in patients with acute ischemic stroke. The abnormalities in plasma t-PA and PAI-1 activities in acute ischemic stroke patients may simply be the result of cerebral infarction and hence may not reflect the diseased state during the period of onset. For this reason, we studied the plasma t-PA and PAI-1 activities in patients during convalescent and chronic stages. The results showed that plasma t-PA activity decreased in the later stages together with the functional recovery of limbs whereas the plasma PAI-1 activity increased considerably suggesting plasminogen activation is weakened. Along with the reduction of t-PA activity, plasma PAI-1 activity increased considerably during convalescent and chronic stages. Hence, in the later stages following cerebral thrombosis the activity of plasma t-PA and PAI-1 changed inversely, especially plasma PAI-1 activity which increased significantly during the chronic stage and is therefore a potential cause of recurrent ischemic stroke. Previous research [18] confirmed that the development of thrombotic disease was not only correlated with decreased t-PA activity but also the secretory increase of PAI-1. This means that an increased circulatory activity of PAI-1 is associated with an increased risk of ischemic stroke. Therefore, in order to reduce the incidence of ischemic stroke greater attention should be paid to the circulating activity of PAI-1. t-PA is mainly found in the blood and acts as a central nervous system (CNS) vascular permeability regulator. It is also expressed in the CNS [22,23]. Although t-PA has proven benefits related to vessel recanalization and can be used for the treatment of thromboembolic stroke, in some patients there are reports of adverse reactions, especially intracerebral hemorrhage [24]. Additionally, up to 13% of ischemic stroke patients suffer re-occlusions following treatment with t-PA [25]. Animal studies also suggest that t-PA deficiency increases the transient middle cerebral artery occlusion after vascular fibrous deposition with an increase in brain injury. However, most studies show that excessive t-PA in the CNS leads to neuronal death. The endogenous t-PA in the CNS directly increases vascular permeability causing loss of blood-brain barrier integrity. Thrombolysis with t-PA is associated with increased vascular permeability [23,24] and delayed reperfusion with recombinant t-PA can cause hemorrhagic transformation. In view of the above reasons, the use of t-PA is strictly regulated in a clinical setting [19,26]. Previous research has shown that t-PA plays an inhibitory role in platelet aggregation in vitro. It has been shown that platelets can release b-TG and other substances after aggregation [27]. Therefore, the increase in plasma t-PA activity could lead to the decrease of b-TG level. Similarly, we observed a negative correlation between plasma t-PA activity and b-TG content within the same patient. Regarding the relationship between platelet function and thrombotic disease, tests which merely assess platelet aggregation and adhesion cannot provide direct evidence of platelet activation. Therefore, determining plasma b-TG content as a specific marker is crucial in reflecting platelet activation and release in thrombotic disease [9,28]. In accordance with an earlier study [11], our results showed that plasma b-TG level in patients with ischemic stroke was significantly higher than healthy controls suggesting that
P. Zhuang et al. / Journal of Clinical Neuroscience 22 (2015) 1123–1127
platelet function was in an augmented state and hence its release was enhanced. This is probably the reason why impaired vascular endodermis is more likely to cause platelet adhesion and aggregation and thus induce excessive activation of platelets. Previous research confirmed that b-TG has an inhibitory role on the enzyme prostacyclin (PGI2) synthase, resulting in the decrease of PGI2 synthesis in vascular endothelial cells [28]. On the other hand, because PGI2 has an inhibitive role on platelet aggregation, an increase of plasma b-TG level enhances platelet aggregation and subsequently accelerates thrombosis. Hence, it can be speculated that an increase in plasma b-TG level not only implies the presence of ischemic stroke but is likely to be a cause of ischemic stroke. In summary, because the application of t-PA is strictly limited in a clinical setting, the addition of thrombolytic therapies which could improve platelet function and lower b-TG levels would be invaluable in order to reduce re-occlusions and facilitate the recovery of brain function. Conflicts of Interest/Disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication. Acknowledgments We thank the staff of the Departments of Health Examination and Neurology at Shanghai East Hospital, Tongji University School of Medicine for providing plasma samples. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jocn.2014.12.027. References [1] Lou M. Thrombolysis in acute ischemic stroke: issues and options. Zhejiang Da Xue Xue Bao Yi Xue Ban 2014;43:1–6. [2] Jung S, Stapf C, Arnold M. Stroke unit management and revascularisation in acute ischemic stroke. Eur Neurol 2015;73:98–105. [3] Cesarman-Maus G, Hajjar KA. Molecular mechanisms of fibrinolysis. Brit J Haematol 2005;129:307–21. [4] Chudy´ P, Kotulicová D, Staško J, et al. The relationship among TAFI, t-PA, PAI-1 and F1 + 2 in type 2 diabetic patients with normoalbuminuria and microalbuminuria. Blood Coagul Fibrinol 2011;22:493–8. [5] Yoon BK, Kang YH, Oh WJ, et al. Impact of lysophosphatidylcholine on the plasminogen activator system in cultured vascular smooth muscle cells. J Korean Med Sci 2012;27:803–10. [6] Kario K, Yano Y, Matsuo T, et al. Additional impact of morning haemostatic risk factors and morning blood pressure surge on stroke risk in older Japanese hypertensive patients. Eur Heart J 2011;32:574–80.
1127
[7] Hosomi N, Lucero J, Heo JH, et al. Rapid differential endogenous plasminogen activator expression after acute middle cerebral artery occlusion. Stroke 2001;32:1341–8. [8] Niego B, Medcalf RL. Plasmin-dependent modulation of the blood–brain barrier: a major consideration during tPA-induced thrombolysis? J Cereb Blood Flow Metab 2014;34:1283–96. [9] Ravindran R, Krishnan LK. A biochemical study on the effect of proteolysis of beta-thromboglobulin proteins released from activated platelets on fibroblast proliferation. Pathophysiol Haemost Thromb 2007;36:285–9. [10] Taomoto K, Asada M, Kanazawa Y, et al. Usefulness of the measurement of plasma beta-thromboglobulin (beta-TG) in cerebrovascular disease. Stroke 1983;14:518–24. [11] Iskra T, Turaj W, Stowik A, et al. Hemostatic markers of endothelial injury in ischaemic stroke caused by large or small vessel disease. Pol Merkur Lekarski 2006;21:429–33. [12] Montaner J. Stroke biomarkers: can they help us to guide stroke thrombolysis? Timely Top Med Cardiovasc Dis 2007;11:E11. [13] Soeki T, Tamura Y, Fukuda N, et al. Plasma and platelet plasminogen activator inhibitor-1 in patients with acute myocardial infarction. Jpn Circ J 2000;64:547–53. [14] Vuckovic´ B, Deric´ M, Zarkov M. Influence of fibrinolitic potential reduction in ischemic brain disease development. Med Pregl 2008;61:247–51. [15] Wang JY, Ja HY, Song HY. Determination of plasma tissue type plasminogen activator and its inhibitor activity. Chin J Lab Med 1989;12:163–6. [16] Shi AL. b-TG RIA and clinical application. Chin J Nucl Med 1985;5:98. [17] Abanonu GB, Daskin A, Akdogan MF, et al. Mean platelet volume and bthromboglobulin levels in familial mediterranean fever: effect of colchicine use? Eur J Intern Med 2012;23:661–4. [18] Vaughan DE. PAI-1 antagonists: the promise and the peril. Trans Am Clin Climatol Assoc 2011;122:312–25. [19] Suzuki Y, Nagai N, Umemura K. Novel situations of endothelial injury in stroke – mechanisms of stroke and strategy of drug development: intracranial bleeding associated with the treatment of ischemic stroke: thrombolytic treatment of ischemia-affected endothelial cells with tissue-type plasminogen activator. J Pharmacol Sci 2011;116:25–9. [20] Lou M, Selim M. Does body weight influence the response to intravenous tissue plasminogen activator in stroke patients? Cerebrovasc Dis 2009;27:84–90. [21] Ekmekçi H, Güngör Öztürk Z, Ekmekçi OB, et al. Significance of vitronectin and PAI-1 activity levels in carotid artery disease: comparison of symptomatic and asymptomatic patients. Minerva Med 2013;104:215–23. [22] Samson AL, Medcalf RL. Tissue-type plasminogen activator: a multifaceted modulator of neurotransmission and synaptic plasticity. Neuron 2006;50: 673–8. [23] Yepes M, Sandkvist M, Moore EG, et al. Tissue-type plasminogen activator induces opening of the blood–brain barrier via the LDL receptor-related protein. J Clin Invest 2003;112:1533–40. [24] Nicole O, Docagne F, Ali C, et al. The proteolytic activity of tissue-plasminogen activator enhances NMDA receptor-mediated signaling. Nat Med 2001;7: 59–64. [25] Fernandez-Cadenas I, Del Rio-Espinola A, Rubiera M, et al. PAI-1 4G/5G polymorphism is associated with brain vessel reocclusion after successful fibrinolytic therapy in ischemic stroke patients. Int J Neurosci 2010;120: 245–51. [26] Yamashita T, Abe K. Therapeutic approaches to vascular protectionin ischemic stroke. Acta Med Okayama 2011;65:219–23. [27] Schoorl M, Schoorl M, Nubé MJ, et al. Platelet depletion, platelet activation and coagulation during treatment with hemodialysis. Scand J Clin Lab Invest 2011;71:240–7. [28] Sun LM, Ma ST, Geng M. Study on the relationship between plasmic b-TG, PF4 levels and hemorheology in gerontal patients with the insufficiency of Qi and stagnation of blood. J Pract Tradition Chin Int Med 2007;21:74–5.
