- Email: [email protected]
Platelet release of β-thromboglobulin and platelet factor 4 and serotonin in plasma samples
Clinical Biochemistry 38 (2005) 1023 – 1026
Platelet release of h-thromboglobulin and platelet factor 4 and serotonin in plasma samples Ryunosuke Ohkawaa, Yuji Hirowatarib, Kazuhiro Nakamuraa, Shigeo Ohkuboa, Hitoshi Ikedaa, Mitsumasa Okadac, Minoru Tozukaa, Kazuhiko Nakaharad, Yutaka Yatomid,* a
Department of Clinical Laboratory, The University of Tokyo Hospital, Tokyo, Japan b Scientific Instruments Division, Tosoh Corporation, Kanagawa, Japan c Department of Biomolecular Science, Toho University, Chiba, Japan d Department of Laboratory Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan Received 8 February 2005; received in revised form 29 June 2005; accepted 4 July 2005 Available online 10 August 2005
Abstract Objectives: Platelet release of a granule-derived CXC chemokines and dense granule-derived serotonin in plasma samples was evaluated. Methods: Concentrations of the CXC chemokines h-TG and PF4 were assayed by an enzyme immunoassay; serotonin was measured by an HPLC method. Results: h-TG and PF4 were more easily released than serotonin by in vitro procedures. Use of the anti-platelet cocktail CTAD and preservation of the samples at 4-C were necessary to accurately measure h-TG and PF4, but not serotonin. Conclusions: Assaying serotonin may be useful for assessing platelet activation in vivo as a laboratory test because of facile preparation of plasma samples. D 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Plasma; Platelet factor 4 (PF4); Serotonin; h-thromboglobulin (h-TG)
Introduction Platelets play important roles in physiological hemostasis and pathological thrombosis. They can be activated by various stimuli, and detection of platelet activation in vivo could be useful for identifying patients at risk of thrombosis and for assessing the effect of anti-platelet therapy [1]. For this purpose, plasma levels of the substances released from activated platelets have been measured, including h-thromboglobulin (h-TG) and platelet factor 4 (PF4) [2]. These platelet-specific CXC chemokines Abbreviations: h-TG, h-thromboglobulin; PF4, platelet factor 4; TPA, 12-O-tetradecanoylphorbol 13-acetate; CTAD, citrate – theophylline – adenosine – dipyridamole. * Corresponding author. Fax: +81 3 5689 0495. E-mail address: [email protected] (Y. Yatomi).
are stored in platelet a-granules, released extracellularly upon activation, and elevated in the plasma of prethrombotic and thrombotic patients, reflecting in vivo platelet activation [2]. Serotonin (5-hydroxytryptamine) is also a bioactive substance released from activated platelets; its chemical structure and storage site in platelets are completely different from the CXC chemokines [3]. Circulating plasma serotonin is taken up by platelets mainly by an active transport mechanism and is stored in dense granules [3]. Serotonin is secreted from platelets at sites of endothelial injury, where it promotes thrombogenic reactions through interactions with platelets themselves and smooth muscle cells [4]. A sensitive HPLC method of assaying serotonin showed that plasma and whole blood concentration increases and decreases with age, respectively, and that the plasma/whole blood serotonin concentration ratios
0009-9120/$ - see front matter D 2005 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2005.07.008
1024
R. Ohkawa et al. / Clinical Biochemistry 38 (2005) 1023 – 1026
were significantly higher in patients with ischemic heart diseases than in healthy controls [5]. Accordingly, it seems likely that serotonin in the plasma can be a marker reflecting platelet activation in vivo, as is the case with CXC chemokines. In this report, we measured plasma h-TG, PF4, and serotonin under various conditions and evaluated and compared the measurement of these platelet-derived substances as laboratory tests.
Materials and methods
Serotonin measurement by HPLC Serotonin in the plasma samples was separated by HPLC with a column-switching system and was specifically converted into a fluorescent derivative with benzylamine for convenient detection, as described previously [5]. The lower detection limit for serotonin is 0.13 nM, while linear relationship exists between the peak fluorescence intensity and the concentration in the range of 0 –25 nM. The assay CVs are within 10%. The Q1, median, and Q3 plasma serotonin concentrations were 2.95, 4.40, and 7.10 nM, respectively; the IQR was calculated as 2.075 (n = 75).
Plasma sample preparation Statistics Blood was collected from the antecubital vein of healthy adult volunteers who had not received any medication for the last 7 days and had given the informed consent. Whole blood samples were taken into vacuum tubes containing 3 mg/mL of ethylenediamine tetraacetic acid dipotassium salt 2H2O (EDTA), mixed with 10% vol of citrate – theophylline – adenosine– dipyridamole (CTAD) [6] (BD Biosciences, Tokyo, Japan), or mixed with 3 mg/ mL of EDTA plus 10% vol of CTAD. The anti-coagulated samples were centrifuged at 2500 g for 30 min to obtain plasma. b-TG and PF4 assays Antigen levels of h-TG and PF4 in the plasma samples were determined in accordance with manufacturer’s instructions by an enzyme immunoassay using an Asserachrom h-TG and an Asserachrom PF4 (Roche Diagnostics, Tokyo, Japan), respectively. The measuring limits for h-TG were 5– 200 ng/mL and for PF4 1 – 100 ng/mL; assay CVs were within 15%. When concentrations exceeded the upper measuring limits, samples were diluted with supplied diluents. Respective reference intervals for the plasma h-TG and PF4 were 10– 40 ng/mL and 0 –5 ng/mL.
When indicated, the statistical significance of the difference between the two groups (n = 3 or 4) was determined by means of paired Student’s t test. P < 0.05 was considered significant.
Results Platelet activation markers using plasma samples prepared by centrifugation at 4-C were measured. In the samples from blood treated with EDTA, h-TG and PF4 levels were much higher than those of samples prepared from blood treated with CTAD, a mixture of citrate – theophylline – adenosine – dipyridamole [6] (Fig. 1). Further addition of EDTA to CTAD did not affect the results (Fig. 1), indicating that anti-coagulation and anti-platelet effects by CTAD are necessary and enough to block in vitro release of these CXC chemokines. In contrast, the plasma level of serotonin was minimally affected by the choice of agents, i.e., EDTA vs. CTAD ( P value = 0.252, Fig. 1). Combined treatment with EDTA and CTAD resulted in a synergistic regulatory effect on in vitro platelet serotonin release (Fig. 1), although its mechanism remains to be elucidated.
Fig. 1. h-TG, PF4, and serotonin concentrations in plasma samples: effects of anti-platelet agents. Whole blood was mixed with EDTA (E), CTAD (C), or EDTA plus CTAD (E + C) immediately after venipuncture. Then, plasma samples were prepared by centrifugation at 4-C. h-TG (left panel), PF4 (middle panel), and serotonin (right panel) in these samples were then assayed. Results are means T SD of the data obtained from 4 healthy donors. Reference values for h-TG (below 50 ng/mL) and PF4 (below 20 ng/mL) were indicated by hatched lines. The mean T SD of plasma serotonin concentrations in healthy subjects was 5.7 T 3.0 nM [5]. *Statistically significant.
R. Ohkawa et al. / Clinical Biochemistry 38 (2005) 1023 – 1026
Concentrations of platelet releasates were measured in plasma samples prepared by centrifugation at 4-C and room temperature; all samples were treated with EDTA plus CTAD (Fig. 2A). The plasma concentrations of h-TG tended to increase when the samples were centrifuged at room temperature, although the difference was not significant: 13.0 T 3.3 ng/mL and 27.2 T 14.8 ng/mL under 4-C and room temperature centrifugation, respectively ( P value = 0.156). Similar results were obtained when the levels of PF4 were examined: 0.1 T 0.1 ng/mL and 3.6 T 3.2 ng/mL under 4-C and room temperature centrifugation, respectively ( P value = 0.113, Fig. 2A). In contrast, the plasma serotonin concentrations were significantly lower when the samples were centrifuged at room temperature (Fig. 2A): 2.9 T 0.3 nM and 1.8 T 0.6 nM under 4-C and room temperature centrifugation, respectively ( P value = 0.047). Cooling of platelets may inhibit reuptake of serotonin released by in vitro procedures. The effects of incubation at 4-C or room temperature were assessed before centrifugation for plasma sample preparation. Whole blood samples maintained at room temperature before centrifugation resulted in much higher levels of plasma h-TG and PF4, while keeping at 4-C did not (Fig. 2B). In contrast, the plasma serotonin levels were not affected by maintenance of the whole blood sample before centrifugation even at room temperature (Fig. 2B).
1025
Discussion The present study confirmed that the usage of CTAD is required to measure plasma h-TG and PF4; platelets must be activated at least slightly during centrifugation. This is in agreement with the previous report on the superiority of CTAD as an anti-platelet agent [6]. In contrast, the choice of EDTA or CTAD hardly affected the plasma level of serotonin. Furthermore, centrifugation (to isolate plasma) at room temperature resulted in undesired in vitro h-TG and PF4 release into plasma, which is not the case with serotonin. Finally, keeping the whole blood sample at room temperature (before centrifugation) resulted in in vitro release of h-TG and PF4, but not serotonin. All these results indicate that strict sampling procedures to obtain suitable plasma samples are needed for the measurement of h-TG and PF4, compared with serotonin. Platelets contain a variety of bioactive substances in their granules including a granules and dense granules and release the contents upon activation [7,8]. Our results are consistent with the idea that CXC chemokines in a granule are more easily released than serotonin in dense granules [9]. It is established that the detection of platelet activation in vivo is clinically useful (see Introduction). Although changes in h-TG and PF4 plasma levels have been used for such platelet activation markers, strict and careful blood
Fig. 2. h-TG, PF4, and serotonin concentrations in plasma samples. (A) Effects of cooling or not during centrifugation for plasma preparation. Whole blood was mixed with EDTA plus CTAD immediately after venipuncture. Then, plasma samples were prepared by centrifugation at 4-C (4-C) or room temperature (R). h-TG (left panel), PF4 (middle panel), and serotonin (right panel) in these samples were then assayed. Results are means T SD of the data obtained from 4 healthy donors (open circle, solid circle, open square, and solid square). (B) Effects of incubation at 4-C or room temperature before centrifugation for plasma preparation. Whole blood was mixed with EDTA plus CTAD (h-TG and PF4) or EDTA (serotonin) immediately after venipuncture. Then, the whole blood samples were kept at room temperature for 2 h (R2) or 4 h (R4) or at 4-C for 4 h (4-C). Then, plasma samples were prepared by centrifugation at 4-C. h-TG, PF4, and serotonin in these samples were then assayed. The results are expressed as the percentages of the values for plasma prepared immediately after whole blood collection (C), which were 37.5 T 12.4 ng/mL, 17.1 T 8.22 ng/mL, and 3.8 T 1.9 nM for h-TG, PF4, and serotonin, respectively, and are means T SD of the data obtained from 4 healthy donors.
1026
R. Ohkawa et al. / Clinical Biochemistry 38 (2005) 1023 – 1026
processing in order to avoid the in vitro platelet activation is required due to the low threshold of platelet activation for release of these a granule contents [2]. Very recently, a sensitive method of assaying serotonin, which is stored in platelet dense granules and can be released upon activation, was developed, and it is likely that serotonin in the plasma can be a marker reflecting platelet activation in vivo, as is the case with CXC chemokines [5,10]. As we have revealed in this study, serotonin may be more useful to detect platelet activation in vivo as laboratory tests than CXC chemokines because of facile preparation of plasma samples.
[3]
[4]
[5]
[6]
Acknowledgment [7]
This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
[8] [9]
References [10] [1] Wagner DD, Burger PC. Platelets in inflammation and thrombosis. Arterioscler Thromb Vasc Biol 2003;23:2131 – 7. [2] Kaplan KL, Owen J. Plasma levels of beta-thromboglobulin and
platelet factor 4 as indices of platelet activation in vivo. Blood 1981;57:199 – 202. Shirakawa R, Higashi T, Tabuchi A, Yoshioka A, Nishioka H, Fukuda M, et al. Munc13-4 is a GTP-Rab27-binding protein regulating dense core granule secretion in platelets. J Biol Chem 2004;279:10730 – 7. Crowley ST, Dempsey EC, Horwitz KB, Horwitz LD. Platelet-induced vascular smooth muscle cell proliferation is modulated by the growth amplification factors serotonin and adenosine diphosphate. Circulation 1994;90:1908 – 18. Hirowatari Y, Hara K, Kamihata H, Iwasaka T, Takahashi H. Highperformance liquid chromatographic method with column-switching and post-column reaction for determination of serotonin levels in platelet-poor plasma. Clin Biochem 2004;37:191 – 7. Macey M, Azam U, McCarthy D, Webb L, Chapman ES, Okrongly D, et al. Evaluation of the anticoagulants EDTA and citrate, theophylline, adenosine, and dipyridamole (CTAD) for assessing platelet activation on the ADVIA 120 hematology system. Clin Chem 2002;48:891 – 9. Blockmans D, Deckmyn H, Vermylen J. Platelet activation. Blood Rev 1995;9:143 – 56. Flaumenhaft R. Molecular basis of platelet granule secretion. Arterioscler Thromb Vasc Biol 2003;23:1152 – 60. Krishnamurthi S, Joseph S, Kakkar VV. Synergistic potentiation of 5-hydroxytryptamine secretion by platelet agonists and phorbol myristate acetate despite inhibition of agonist-induced arachidonate/thromboxane and b-thromboglobulin release and Ca2+ mobilization by phorbol myristate acetate. Biochem J 1986;238:193 – 9. Hara K, Hirowatari Y, Yoshika M, Komiyama Y, Tsuka Y, Takahashi H. The ratio of plasma to whole-blood serotonin may be a novel marker of atherosclerotic cardiovascular disease. J Lab Clin Med 2004;144:31 – 7.
Platelet release of h-thromboglobulin and platelet factor 4 and serotonin in plasma samples Ryunosuke Ohkawaa, Yuji Hirowatarib, Kazuhiro Nakamuraa, Shigeo Ohkuboa, Hitoshi Ikedaa, Mitsumasa Okadac, Minoru Tozukaa, Kazuhiko Nakaharad, Yutaka Yatomid,* a
Department of Clinical Laboratory, The University of Tokyo Hospital, Tokyo, Japan b Scientific Instruments Division, Tosoh Corporation, Kanagawa, Japan c Department of Biomolecular Science, Toho University, Chiba, Japan d Department of Laboratory Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan Received 8 February 2005; received in revised form 29 June 2005; accepted 4 July 2005 Available online 10 August 2005
Abstract Objectives: Platelet release of a granule-derived CXC chemokines and dense granule-derived serotonin in plasma samples was evaluated. Methods: Concentrations of the CXC chemokines h-TG and PF4 were assayed by an enzyme immunoassay; serotonin was measured by an HPLC method. Results: h-TG and PF4 were more easily released than serotonin by in vitro procedures. Use of the anti-platelet cocktail CTAD and preservation of the samples at 4-C were necessary to accurately measure h-TG and PF4, but not serotonin. Conclusions: Assaying serotonin may be useful for assessing platelet activation in vivo as a laboratory test because of facile preparation of plasma samples. D 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Plasma; Platelet factor 4 (PF4); Serotonin; h-thromboglobulin (h-TG)
Introduction Platelets play important roles in physiological hemostasis and pathological thrombosis. They can be activated by various stimuli, and detection of platelet activation in vivo could be useful for identifying patients at risk of thrombosis and for assessing the effect of anti-platelet therapy [1]. For this purpose, plasma levels of the substances released from activated platelets have been measured, including h-thromboglobulin (h-TG) and platelet factor 4 (PF4) [2]. These platelet-specific CXC chemokines Abbreviations: h-TG, h-thromboglobulin; PF4, platelet factor 4; TPA, 12-O-tetradecanoylphorbol 13-acetate; CTAD, citrate – theophylline – adenosine – dipyridamole. * Corresponding author. Fax: +81 3 5689 0495. E-mail address: [email protected] (Y. Yatomi).
are stored in platelet a-granules, released extracellularly upon activation, and elevated in the plasma of prethrombotic and thrombotic patients, reflecting in vivo platelet activation [2]. Serotonin (5-hydroxytryptamine) is also a bioactive substance released from activated platelets; its chemical structure and storage site in platelets are completely different from the CXC chemokines [3]. Circulating plasma serotonin is taken up by platelets mainly by an active transport mechanism and is stored in dense granules [3]. Serotonin is secreted from platelets at sites of endothelial injury, where it promotes thrombogenic reactions through interactions with platelets themselves and smooth muscle cells [4]. A sensitive HPLC method of assaying serotonin showed that plasma and whole blood concentration increases and decreases with age, respectively, and that the plasma/whole blood serotonin concentration ratios
0009-9120/$ - see front matter D 2005 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2005.07.008
1024
R. Ohkawa et al. / Clinical Biochemistry 38 (2005) 1023 – 1026
were significantly higher in patients with ischemic heart diseases than in healthy controls [5]. Accordingly, it seems likely that serotonin in the plasma can be a marker reflecting platelet activation in vivo, as is the case with CXC chemokines. In this report, we measured plasma h-TG, PF4, and serotonin under various conditions and evaluated and compared the measurement of these platelet-derived substances as laboratory tests.
Materials and methods
Serotonin measurement by HPLC Serotonin in the plasma samples was separated by HPLC with a column-switching system and was specifically converted into a fluorescent derivative with benzylamine for convenient detection, as described previously [5]. The lower detection limit for serotonin is 0.13 nM, while linear relationship exists between the peak fluorescence intensity and the concentration in the range of 0 –25 nM. The assay CVs are within 10%. The Q1, median, and Q3 plasma serotonin concentrations were 2.95, 4.40, and 7.10 nM, respectively; the IQR was calculated as 2.075 (n = 75).
Plasma sample preparation Statistics Blood was collected from the antecubital vein of healthy adult volunteers who had not received any medication for the last 7 days and had given the informed consent. Whole blood samples were taken into vacuum tubes containing 3 mg/mL of ethylenediamine tetraacetic acid dipotassium salt 2H2O (EDTA), mixed with 10% vol of citrate – theophylline – adenosine– dipyridamole (CTAD) [6] (BD Biosciences, Tokyo, Japan), or mixed with 3 mg/ mL of EDTA plus 10% vol of CTAD. The anti-coagulated samples were centrifuged at 2500 g for 30 min to obtain plasma. b-TG and PF4 assays Antigen levels of h-TG and PF4 in the plasma samples were determined in accordance with manufacturer’s instructions by an enzyme immunoassay using an Asserachrom h-TG and an Asserachrom PF4 (Roche Diagnostics, Tokyo, Japan), respectively. The measuring limits for h-TG were 5– 200 ng/mL and for PF4 1 – 100 ng/mL; assay CVs were within 15%. When concentrations exceeded the upper measuring limits, samples were diluted with supplied diluents. Respective reference intervals for the plasma h-TG and PF4 were 10– 40 ng/mL and 0 –5 ng/mL.
When indicated, the statistical significance of the difference between the two groups (n = 3 or 4) was determined by means of paired Student’s t test. P < 0.05 was considered significant.
Results Platelet activation markers using plasma samples prepared by centrifugation at 4-C were measured. In the samples from blood treated with EDTA, h-TG and PF4 levels were much higher than those of samples prepared from blood treated with CTAD, a mixture of citrate – theophylline – adenosine – dipyridamole [6] (Fig. 1). Further addition of EDTA to CTAD did not affect the results (Fig. 1), indicating that anti-coagulation and anti-platelet effects by CTAD are necessary and enough to block in vitro release of these CXC chemokines. In contrast, the plasma level of serotonin was minimally affected by the choice of agents, i.e., EDTA vs. CTAD ( P value = 0.252, Fig. 1). Combined treatment with EDTA and CTAD resulted in a synergistic regulatory effect on in vitro platelet serotonin release (Fig. 1), although its mechanism remains to be elucidated.
Fig. 1. h-TG, PF4, and serotonin concentrations in plasma samples: effects of anti-platelet agents. Whole blood was mixed with EDTA (E), CTAD (C), or EDTA plus CTAD (E + C) immediately after venipuncture. Then, plasma samples were prepared by centrifugation at 4-C. h-TG (left panel), PF4 (middle panel), and serotonin (right panel) in these samples were then assayed. Results are means T SD of the data obtained from 4 healthy donors. Reference values for h-TG (below 50 ng/mL) and PF4 (below 20 ng/mL) were indicated by hatched lines. The mean T SD of plasma serotonin concentrations in healthy subjects was 5.7 T 3.0 nM [5]. *Statistically significant.
R. Ohkawa et al. / Clinical Biochemistry 38 (2005) 1023 – 1026
Concentrations of platelet releasates were measured in plasma samples prepared by centrifugation at 4-C and room temperature; all samples were treated with EDTA plus CTAD (Fig. 2A). The plasma concentrations of h-TG tended to increase when the samples were centrifuged at room temperature, although the difference was not significant: 13.0 T 3.3 ng/mL and 27.2 T 14.8 ng/mL under 4-C and room temperature centrifugation, respectively ( P value = 0.156). Similar results were obtained when the levels of PF4 were examined: 0.1 T 0.1 ng/mL and 3.6 T 3.2 ng/mL under 4-C and room temperature centrifugation, respectively ( P value = 0.113, Fig. 2A). In contrast, the plasma serotonin concentrations were significantly lower when the samples were centrifuged at room temperature (Fig. 2A): 2.9 T 0.3 nM and 1.8 T 0.6 nM under 4-C and room temperature centrifugation, respectively ( P value = 0.047). Cooling of platelets may inhibit reuptake of serotonin released by in vitro procedures. The effects of incubation at 4-C or room temperature were assessed before centrifugation for plasma sample preparation. Whole blood samples maintained at room temperature before centrifugation resulted in much higher levels of plasma h-TG and PF4, while keeping at 4-C did not (Fig. 2B). In contrast, the plasma serotonin levels were not affected by maintenance of the whole blood sample before centrifugation even at room temperature (Fig. 2B).
1025
Discussion The present study confirmed that the usage of CTAD is required to measure plasma h-TG and PF4; platelets must be activated at least slightly during centrifugation. This is in agreement with the previous report on the superiority of CTAD as an anti-platelet agent [6]. In contrast, the choice of EDTA or CTAD hardly affected the plasma level of serotonin. Furthermore, centrifugation (to isolate plasma) at room temperature resulted in undesired in vitro h-TG and PF4 release into plasma, which is not the case with serotonin. Finally, keeping the whole blood sample at room temperature (before centrifugation) resulted in in vitro release of h-TG and PF4, but not serotonin. All these results indicate that strict sampling procedures to obtain suitable plasma samples are needed for the measurement of h-TG and PF4, compared with serotonin. Platelets contain a variety of bioactive substances in their granules including a granules and dense granules and release the contents upon activation [7,8]. Our results are consistent with the idea that CXC chemokines in a granule are more easily released than serotonin in dense granules [9]. It is established that the detection of platelet activation in vivo is clinically useful (see Introduction). Although changes in h-TG and PF4 plasma levels have been used for such platelet activation markers, strict and careful blood
Fig. 2. h-TG, PF4, and serotonin concentrations in plasma samples. (A) Effects of cooling or not during centrifugation for plasma preparation. Whole blood was mixed with EDTA plus CTAD immediately after venipuncture. Then, plasma samples were prepared by centrifugation at 4-C (4-C) or room temperature (R). h-TG (left panel), PF4 (middle panel), and serotonin (right panel) in these samples were then assayed. Results are means T SD of the data obtained from 4 healthy donors (open circle, solid circle, open square, and solid square). (B) Effects of incubation at 4-C or room temperature before centrifugation for plasma preparation. Whole blood was mixed with EDTA plus CTAD (h-TG and PF4) or EDTA (serotonin) immediately after venipuncture. Then, the whole blood samples were kept at room temperature for 2 h (R2) or 4 h (R4) or at 4-C for 4 h (4-C). Then, plasma samples were prepared by centrifugation at 4-C. h-TG, PF4, and serotonin in these samples were then assayed. The results are expressed as the percentages of the values for plasma prepared immediately after whole blood collection (C), which were 37.5 T 12.4 ng/mL, 17.1 T 8.22 ng/mL, and 3.8 T 1.9 nM for h-TG, PF4, and serotonin, respectively, and are means T SD of the data obtained from 4 healthy donors.
1026
R. Ohkawa et al. / Clinical Biochemistry 38 (2005) 1023 – 1026
processing in order to avoid the in vitro platelet activation is required due to the low threshold of platelet activation for release of these a granule contents [2]. Very recently, a sensitive method of assaying serotonin, which is stored in platelet dense granules and can be released upon activation, was developed, and it is likely that serotonin in the plasma can be a marker reflecting platelet activation in vivo, as is the case with CXC chemokines [5,10]. As we have revealed in this study, serotonin may be more useful to detect platelet activation in vivo as laboratory tests than CXC chemokines because of facile preparation of plasma samples.
[3]
[4]
[5]
[6]
Acknowledgment [7]
This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
[8] [9]
References [10] [1] Wagner DD, Burger PC. Platelets in inflammation and thrombosis. Arterioscler Thromb Vasc Biol 2003;23:2131 – 7. [2] Kaplan KL, Owen J. Plasma levels of beta-thromboglobulin and
platelet factor 4 as indices of platelet activation in vivo. Blood 1981;57:199 – 202. Shirakawa R, Higashi T, Tabuchi A, Yoshioka A, Nishioka H, Fukuda M, et al. Munc13-4 is a GTP-Rab27-binding protein regulating dense core granule secretion in platelets. J Biol Chem 2004;279:10730 – 7. Crowley ST, Dempsey EC, Horwitz KB, Horwitz LD. Platelet-induced vascular smooth muscle cell proliferation is modulated by the growth amplification factors serotonin and adenosine diphosphate. Circulation 1994;90:1908 – 18. Hirowatari Y, Hara K, Kamihata H, Iwasaka T, Takahashi H. Highperformance liquid chromatographic method with column-switching and post-column reaction for determination of serotonin levels in platelet-poor plasma. Clin Biochem 2004;37:191 – 7. Macey M, Azam U, McCarthy D, Webb L, Chapman ES, Okrongly D, et al. Evaluation of the anticoagulants EDTA and citrate, theophylline, adenosine, and dipyridamole (CTAD) for assessing platelet activation on the ADVIA 120 hematology system. Clin Chem 2002;48:891 – 9. Blockmans D, Deckmyn H, Vermylen J. Platelet activation. Blood Rev 1995;9:143 – 56. Flaumenhaft R. Molecular basis of platelet granule secretion. Arterioscler Thromb Vasc Biol 2003;23:1152 – 60. Krishnamurthi S, Joseph S, Kakkar VV. Synergistic potentiation of 5-hydroxytryptamine secretion by platelet agonists and phorbol myristate acetate despite inhibition of agonist-induced arachidonate/thromboxane and b-thromboglobulin release and Ca2+ mobilization by phorbol myristate acetate. Biochem J 1986;238:193 – 9. Hara K, Hirowatari Y, Yoshika M, Komiyama Y, Tsuka Y, Takahashi H. The ratio of plasma to whole-blood serotonin may be a novel marker of atherosclerotic cardiovascular disease. J Lab Clin Med 2004;144:31 – 7.