- Email: [email protected]
A simple and reproducible method to reliably assess platelet activation
Thrombosis Research 110 (2003) 53 – 56
Brief Communication
A simple and reproducible method to reliably assess platelet activation R.M. Nickels a, U.T. Seyfert b, E. Wenzel b, M.D. Menger a, B. Vollmar c,* a
b
Institute for Clinical and Experimental Surgery, University of Saarland, Homburg/Saar, Germany Department of Clinical Hemostaseology and Transfusion Medicine, University of Saarland, Homburg/Saar, Germany c Department of Experimental Surgery, University of Rostock, Schillingalle 70, 18055 Rostock, Germany Received 9 January 2003; received in revised form 23 April 2003; accepted 23 April 2003
Keywords: Platelet activation; Hemostasis; Thrombosis
1. Introduction
2. Materials and methods
Activation of platelets plays a crucial role in the pathophysiology of thrombosis and hemostasis [1 –3]. The series of events triggered after platelet activation depends on the strength of the applied stimulus. Resting platelets have a discoid shape that is rapidly transformed to a spherical shape upon activation. Weak stimulation leads to shape change and aggregation, while more intense stimulation is followed by arachidonate liberation and secretion of a-granules [4]. During the release reaction, granules fuse with the plasma membrane. Thereby, the transmembrane glycoprotein Pselectin, which resides within the a-granule membrane of unstimulated platelets, is phosphorylated and translocated to the plasma membrane, serving as a cell adhesion receptor [5]. Moreover, P-selectin has been demonstrated to be associated with the limiting membrane of dense granules, i.e. another type of secretory organelles, which are formed by endogeneous synthesis and endocytosis from the plasma membrane, as shown for a-granules [6]. The development of a simple and reproducible technology to assess the different degrees of platelet activation has long been a prime concern in research focusing on the evaluation of platelet responses both in vitro and in vivo. The objective of this communication is to present a simple, economical and reproducible method to assess platelet activation which comprises the adhesion and retention of platelets passing an athrombogenic filter via centrifugal force (retention test Homburg (RT-H)). RT-H was compared with conventional measures of platelet activation, such as flow cytometric analysis of P-selectin expression and assessment of platelet shape upon spreading.
2.1. Blood collection and PRP preparation
* Corresponding author. Tel.: +49-381-494-6220; fax: +49-381-4946222. E-mail address: [email protected] (B. Vollmar).
Blood from healthy volunteers was drawn from the left cubital vein with a 21-gauge needle into 5 ml S-Monovettes 9NC (Sarstedt, Nu¨mbrecht, Germany) (1:10 citrate v/v). After centrifugation for 15 min at 110 g and room temperature, platelet-rich plasma was transferred in a separate tube and platelet count was assessed with a Coulter AC.T diff Analyzer (Coulter Electronics, Luton, UK). Aliquots of platelet-rich plasma either served as unstimulated negative controls or were activated to serve as positive controls. 2.2. Flow cytometric analysis of platelets Flow cytometric (FACS) analysis was performed to assess surface expression of P-selectin, which reliably indicates cell activation [7]. Expression of P-selectin on platelets was investigated by direct immunofluorescence using a monoclonal antihuman fluorescein-isothiocyanate (FITC)-coupled P-selectin antibody (Ancell, Bayport, MN, USA), diluted 1:50 (v/v) with staining medium (0.1% sodium azide and 2% fetal calf serum in PBS). An FITCcoupled IgG1 isotype-matched control antibody (Ancell) was used to exclude unspecific binding. Platelet suspensions (50 Al) each were incubated with saturating amounts (80 Al) of diluted FITC-labelled monoclonal antihuman P-selectin antibody or isotype control antibody for 40 min at room temperature in the dark. After incubation, the cells were washed twice with 0.1% sodium in PBS (Cell Wash, Becton Dickinson) to remove excess of antibody and fixed with 1% paraformaldehyde in PBS (Cell Fix, Becton Dickinson). Flow cytometry was performed within the next 3 h. FACScan flow cytometer (Becton Dickinson) was calibrated with fluorescent standard microbeads (CaliBRITE
0049-3848/03/$ - see front matter D 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0049-3848(03)00256-1
54
R.M. Nickels et al. / Thrombosis Research 110 (2003) 53–56
Beads, Becton Dickinson) for accurate instrument setting. Platelets were identified by their characteristic forward and sideward scatter lights and selectively analysed for their fluorescence properties using the CellQuest data handling program (Becton Dickinson) with assessment of 30,000 events per sample. The relative fluorescence intensity of a given sample was calculated by substracting the signal obtained when cells were incubated with the isotype-specific control antibody from the signal generated by cells incubated with the test antibody (Fig. 1). 2.3. Retention test Homburg (RT-H) The retention test Homburg (RT-H) is based on the exposure of platelets to a standardized exogenic surface (retention tube) under defined conditions of flow and calculates the difference of platelet count before and after the retention tube passage. To perform the RT-H, 450 Al of platelet suspension was pipetted into the filter of special retention tubes (Sysmex, Norderstedt, Germany), and 50 Al of 0.3% sodium citrate (Braun) was added. After incubation for 5 min at room temperature, the tubes were gently vortexed for 1 min and the platelets were counted with a Coulter AC.T diff Analyzer (Coulter Electronics). Tubes were then centrifuged at 110 g for 5 min to allow the platelets to pass the filter. The filter was removed and the remaining platelets in the retention tube were mixed well and again counted. The difference value of the count before and after passing the filter indicates the percentage of retained platelets, i.e. the retention index (RI), calculated as RI (%)=[(platelet count before centrifugation platelet count after centrifugation)/platelet count before centrifugation] 100.
2.4. Spreading analysis test Platelet counts were assessed with a Coulter AC.T diff Analyzer (Coulter Electronics) and adjusted to 0.3 108/ml with 0.3% sodium citrate. About 2 – 4 ml of each platelet suspension were poured on plastic slides and incubated at room temperature for 30 min allowing platelets to adhere and to spread out on the slide. The slides were rinsed with 0.3% sodium citrate, fixed in 11% formaldehyde (Merck, Darmstadt, Germany) and dried at room temperature. Then the slides were oxidized for 5 min in 0.1 N potassium permanganate (Merck), rinsed with water and stained for 60 min in Giemsa (Merck). The shapes of spread platelets were differentiated using light microscopy (Model BX60F; Olympus Optical, Tokyo, Japan) at a magnification of 400. Hundred platelets were counted and the percentage of (i) small and large forms as well as (ii) spider forms was calculated. Moreover, grade of aggregation and agglutination was assessed semiquantitatively by no agglutination or aggregation = 0, minor agglutination or aggregation = 1, moderate agglutination or aggregation = 2, marked agglutination or aggregation = 3, and extensive agglutination or aggregation = 4, according to a modified method initially described by Breddin and Bauke [8]. 2.5. Activation of platelets Platelet activation was performed using adenosine diphosphate (ADP; Sigma, Deisenhofen, Germany) and thrombin receptor activating peptide (TRAP; Bachem Biochemica, Heidelberg, Germany) as agonists. Platelet-rich plasma was diluted to 1 108 platelets/ml with Ca2 +- and Mg2 +-free PBS. Platelet solution was incubated for 2 min at room temperature with 10, 50 or 100 AM ADP or for 5 min with 20 AM TRAP, and then further processed for analysis of both flow cytometry and spreading behaviour. 2.6. Statistics All values are given as means F S.E.M. Statistical analysis was performed using the unpaired Student’s t-test for differences of platelet preparations in P-selectin expression, retention index and spreading test (SigmaStat, California, USA). A p value < 0.05 was considered to be significant.
3. Results
Fig. 1. Flow cytometric analysis of CD62P expression on human platelets using an FITC-labelled antihuman CD62P antibody (solid line). Platelets were assessed from platelet-rich plasma and activated by exposure to TRAP (20 AM). To exclude unspecific binding (M1), an FITC-coupled IgG1 isotype-matched control antibody was used (dashed line).
Flow cytometry revealed that a fraction of less than 5% of platelets from PRP expressed P-selectin (Fig. 2A). Upon ADP exposure, the fraction of P-selectin expressing platelets increased by five- to sixfold to approximately 17%, regardless of the dose of ADP used (10 – 100 AM; Fig. 2A). Thrombin receptor-activating peptide (TRAP) proved to be a stronger platelet agonist causing membrane translocation of P-selectin in almost half of the PRP platelets (Fig. 2A).
R.M. Nickels et al. / Thrombosis Research 110 (2003) 53–56
55
Table 1 Spreading test of unstimulated, ADP- and TRAP-stimulated platelets from platelet-rich plasma Sample
Large/ small form
Transition/ spider form
Agglutination
Aggregation
Platelets, no activation Platelets, ADP activation Platelets, TRAP activation
69 F 4
31 F 4
0.2 F 0.2
1.0 F 0.3
86 F 4*
14 F 4*
1.3 F 0.3*
2.5 F 0.3*
83 F 2*
17 F 2*
3.2 F 0.6*
3.6 F 0.2*
Values are given as means F S.E.M. * p < 0.05 vs. platelets, no activation. Fig. 2. P-selectin expression (A) and retention index (B) of platelets isolated from platelet-rich plasma and activated by either ADP (10 – 100 AM) or TRAP (20 AM). Unstimulated platelets served as controls (C). Values are given as means F S.E.M.; *p < 0.05 vs. untreated control platelets; #p < 0.05 vs. ADP-stimulated platelets.
Unexposed control platelets from PRP revealed a retention index of 14.4 F 1.8% (Fig. 2B). Activation with ADP resulted in a four- to fivefold increase of platelet retention index. In parallel to P-selectin expression, retention index did not differ between platelets stimulated by either low or high ADP concentration (Fig. 2B). TRAP activation caused maximal platelet retention with an average value of 86.6 F 2.0% (Fig. 2B). Regression analysis between retention index and P-selectin expression of activated and nonactivated PRP platelets revealed a significant ( p < 0.01) linear correlation between both measures of platelet activation such that the higher the P-selectin expression of platelets, the more pronounced the retention index (Fig. 3). Quantitative analysis of spreading of platelets revealed that 60– 70% of platelets from platelet-rich plasma were found as small and large forms on the slide, while 30 – 40%
Fig. 3. Regression analysis between retention index and P-selectin expression of unstimulated (circles), ADP-stimulated (triangles) and TRAP-stimulated (squares) platelets from platelet-rich plasma. r = regression coefficient.
were presented as transition or spider forms (Table 1). Platelet agglutination and aggregation were found to be almost absent. Stimulation with ADP and TRAP caused about 85% of all types of platelets to spread to small, but mostly to large forms, while the fraction of transition or spider forms decreased to 15% (Table 1). Marked activation of ADP- and TRAP-exposed platelets was further indicated by pronounced agglutination and aggregation (Table 1).
4. Discussion Platelet activation is characterized by an ordered sequence of events that includes shape change, increase in cytoplasmic Ca2 +, induction of fibrinogen receptor expression, release of granular contents with P-selectin translocation, aggregation, and formation of a stable hemostatic plug [9]. While this response is essential for hemostasis, it is also important in the pathogenesis of a broad spectrum of diseases, including atherosclerosis, myocardial infarction, unstable angina, stroke and thrombosis. Specialised glycoprotein receptors enable platelets to adhere to proteins that are exposed in areas of vascular damage. The process of adhesion and the interaction of soluble agonists with platelet receptors activate the platelets, which are then able to aggregate. This aggregation creates a platelet plug that seals the breach in the vessel wall and prevents excess blood loss. Activated platelets then facilitate secondary hemostasis, the formation of a fibrin clot, by carrying coagulation factors and providing a catalytic surface for the major interactions of the coagulation cascade. Thus, for evaluating the state of platelet activation in vitro, it is mandatory to combine specific techniques, e.g. flow cytometry determining specific platelet membrane structures with methods assessing the ability of platelets to adhere and to form aggregates. The RT-H is based on the exposure of platelets to a standardized exogenic surface (retention tube) under defined conditions of flow and calculates the difference of platelet count before and after the retention tube passage. Morphological evaluation using scanning as well as transmission electron microscopy of the polyurethane filter in the retention tube has demonstrated that platelets adhere and spread out at the surface and form aggregates [10]. The spreading
56
R.M. Nickels et al. / Thrombosis Research 110 (2003) 53–56
analysis test, as used herein, determines adhesion and spreading as well as spontaneous aggregation and agglutination of platelets from a morphological point of view without application of shear forces. In the present study, regression analysis revealed a significant linear correlation between retention index and P-selectin expression of resting, ADP- and TRAP-activated PRP platelets. Stimulation of PRP platelets with TRAP led to an enormous increase of P-selectin-positive platelets in contrast to the stimulation with ADP causing only approximately 17% of platelets to express P-selectin. While TRAP, a strong platelet agonist, was able to overcome platelet desensitization and caused extreme a-degranulation, ADP as weaker agonist failed to induce sustained release of agranules. However, ADP seemed to induce a change in both platelet shape and adhesion behaviour, as mirrored by markedly increased retention indices found in the present study. In support of this, spreading analysis test revealed increased agglutination and aggregation.
5. Summary RT-H proved to directly reflect activation of platelets from platelet-rich plasma, inasmuch as there was a positive linear correlation between retention index and surface Pselectin expression of nonstimulated and agonist-stimulated platelets. Thus, the RT-H might be an ideal adjunctive to refine the tools for assessment platelet response in physiology and pathophysiology.
Acknowledgements B.V. is recipient of a Heisenberg Stipendium (Vo 450/6-1 and 6-2). The study is supported by a grant from the
Deutsche Forschungsgemeinschaft, Bonn Bad-Godesberg (Vo 450/7-1).
References [1] Subramaniam M, Frenette PS, Saffaripour S, Johnson RC, Hynes RO, Wagner DD. Defects in hemostasis in P-selectin-deficient mice. Blood 1996;87:1238 – 42. [2] Denis C, Methia N, Frenette PS, Rayburn H, Ullman-Cullere M, Hynes RO, et al. A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis. Proc Natl Acad Sci U S A 1998;95:9524 – 9. [3] Vollmar B, Schmits R, Kunz D, Menger MD. Lack of in vivo function of CD31 in vascular thrombosis. Thromb Haemost 2001;85:160 – 4. [4] Holmsen H. Platelet secretion. In: Colman RW, Hirsh I, Marder VJ, Salzman EW, editors. Hemostasis and thrombosis. Basic principles and clinical practice, 2nd ed. Philadelphia, PA: JB Lippincott; 1987. p. 606 – 17. [5] Furie B, Furie BC, Flaumenhaft R. A journey with platelet P-selectin: the molecular basis of granule secretion, signalling and cell adhesion. Thromb Haemost 2001;86:214 – 21. [6] Youssefian T, Masse JM, Rendu F, Guichard J, Cramer EM. Platelet and megakaryocyte dense granules contain glycoproteins Ib and IIb – IIIa. Blood 1997;89:4047 – 57. [7] Jy W, Mao WW, Horstman LL, Valant PA, Ahn YS. A flow cytometric assay of platelet activation marker P-selectin (CD62P) distinguishes heparin-induced thrombocytopenia (HIT) from HIT with thrombosis (HITT). Thromb Haemost 1999;82:1255 – 9. [8] Breddin HK, Bauke J. Thrombozytenagglutination und Gefa¨ßkrankheiten. Blut 1965;11:144 – 64. [9] Blockmans D, Deckmyn H, Vermylen J. Platelet activation. Blood Rev 1995;9:143 – 56. [10] Mestres P, Pu¨tz N, Zhu H, Wenzel E. Description of a method for detection of adherent platelets in scanning electron microscopy. Eur J Cell Biol 1999;78(Suppl 50):32.
Brief Communication
A simple and reproducible method to reliably assess platelet activation R.M. Nickels a, U.T. Seyfert b, E. Wenzel b, M.D. Menger a, B. Vollmar c,* a
b
Institute for Clinical and Experimental Surgery, University of Saarland, Homburg/Saar, Germany Department of Clinical Hemostaseology and Transfusion Medicine, University of Saarland, Homburg/Saar, Germany c Department of Experimental Surgery, University of Rostock, Schillingalle 70, 18055 Rostock, Germany Received 9 January 2003; received in revised form 23 April 2003; accepted 23 April 2003
Keywords: Platelet activation; Hemostasis; Thrombosis
1. Introduction
2. Materials and methods
Activation of platelets plays a crucial role in the pathophysiology of thrombosis and hemostasis [1 –3]. The series of events triggered after platelet activation depends on the strength of the applied stimulus. Resting platelets have a discoid shape that is rapidly transformed to a spherical shape upon activation. Weak stimulation leads to shape change and aggregation, while more intense stimulation is followed by arachidonate liberation and secretion of a-granules [4]. During the release reaction, granules fuse with the plasma membrane. Thereby, the transmembrane glycoprotein Pselectin, which resides within the a-granule membrane of unstimulated platelets, is phosphorylated and translocated to the plasma membrane, serving as a cell adhesion receptor [5]. Moreover, P-selectin has been demonstrated to be associated with the limiting membrane of dense granules, i.e. another type of secretory organelles, which are formed by endogeneous synthesis and endocytosis from the plasma membrane, as shown for a-granules [6]. The development of a simple and reproducible technology to assess the different degrees of platelet activation has long been a prime concern in research focusing on the evaluation of platelet responses both in vitro and in vivo. The objective of this communication is to present a simple, economical and reproducible method to assess platelet activation which comprises the adhesion and retention of platelets passing an athrombogenic filter via centrifugal force (retention test Homburg (RT-H)). RT-H was compared with conventional measures of platelet activation, such as flow cytometric analysis of P-selectin expression and assessment of platelet shape upon spreading.
2.1. Blood collection and PRP preparation
* Corresponding author. Tel.: +49-381-494-6220; fax: +49-381-4946222. E-mail address: [email protected] (B. Vollmar).
Blood from healthy volunteers was drawn from the left cubital vein with a 21-gauge needle into 5 ml S-Monovettes 9NC (Sarstedt, Nu¨mbrecht, Germany) (1:10 citrate v/v). After centrifugation for 15 min at 110 g and room temperature, platelet-rich plasma was transferred in a separate tube and platelet count was assessed with a Coulter AC.T diff Analyzer (Coulter Electronics, Luton, UK). Aliquots of platelet-rich plasma either served as unstimulated negative controls or were activated to serve as positive controls. 2.2. Flow cytometric analysis of platelets Flow cytometric (FACS) analysis was performed to assess surface expression of P-selectin, which reliably indicates cell activation [7]. Expression of P-selectin on platelets was investigated by direct immunofluorescence using a monoclonal antihuman fluorescein-isothiocyanate (FITC)-coupled P-selectin antibody (Ancell, Bayport, MN, USA), diluted 1:50 (v/v) with staining medium (0.1% sodium azide and 2% fetal calf serum in PBS). An FITCcoupled IgG1 isotype-matched control antibody (Ancell) was used to exclude unspecific binding. Platelet suspensions (50 Al) each were incubated with saturating amounts (80 Al) of diluted FITC-labelled monoclonal antihuman P-selectin antibody or isotype control antibody for 40 min at room temperature in the dark. After incubation, the cells were washed twice with 0.1% sodium in PBS (Cell Wash, Becton Dickinson) to remove excess of antibody and fixed with 1% paraformaldehyde in PBS (Cell Fix, Becton Dickinson). Flow cytometry was performed within the next 3 h. FACScan flow cytometer (Becton Dickinson) was calibrated with fluorescent standard microbeads (CaliBRITE
0049-3848/03/$ - see front matter D 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0049-3848(03)00256-1
54
R.M. Nickels et al. / Thrombosis Research 110 (2003) 53–56
Beads, Becton Dickinson) for accurate instrument setting. Platelets were identified by their characteristic forward and sideward scatter lights and selectively analysed for their fluorescence properties using the CellQuest data handling program (Becton Dickinson) with assessment of 30,000 events per sample. The relative fluorescence intensity of a given sample was calculated by substracting the signal obtained when cells were incubated with the isotype-specific control antibody from the signal generated by cells incubated with the test antibody (Fig. 1). 2.3. Retention test Homburg (RT-H) The retention test Homburg (RT-H) is based on the exposure of platelets to a standardized exogenic surface (retention tube) under defined conditions of flow and calculates the difference of platelet count before and after the retention tube passage. To perform the RT-H, 450 Al of platelet suspension was pipetted into the filter of special retention tubes (Sysmex, Norderstedt, Germany), and 50 Al of 0.3% sodium citrate (Braun) was added. After incubation for 5 min at room temperature, the tubes were gently vortexed for 1 min and the platelets were counted with a Coulter AC.T diff Analyzer (Coulter Electronics). Tubes were then centrifuged at 110 g for 5 min to allow the platelets to pass the filter. The filter was removed and the remaining platelets in the retention tube were mixed well and again counted. The difference value of the count before and after passing the filter indicates the percentage of retained platelets, i.e. the retention index (RI), calculated as RI (%)=[(platelet count before centrifugation platelet count after centrifugation)/platelet count before centrifugation] 100.
2.4. Spreading analysis test Platelet counts were assessed with a Coulter AC.T diff Analyzer (Coulter Electronics) and adjusted to 0.3 108/ml with 0.3% sodium citrate. About 2 – 4 ml of each platelet suspension were poured on plastic slides and incubated at room temperature for 30 min allowing platelets to adhere and to spread out on the slide. The slides were rinsed with 0.3% sodium citrate, fixed in 11% formaldehyde (Merck, Darmstadt, Germany) and dried at room temperature. Then the slides were oxidized for 5 min in 0.1 N potassium permanganate (Merck), rinsed with water and stained for 60 min in Giemsa (Merck). The shapes of spread platelets were differentiated using light microscopy (Model BX60F; Olympus Optical, Tokyo, Japan) at a magnification of 400. Hundred platelets were counted and the percentage of (i) small and large forms as well as (ii) spider forms was calculated. Moreover, grade of aggregation and agglutination was assessed semiquantitatively by no agglutination or aggregation = 0, minor agglutination or aggregation = 1, moderate agglutination or aggregation = 2, marked agglutination or aggregation = 3, and extensive agglutination or aggregation = 4, according to a modified method initially described by Breddin and Bauke [8]. 2.5. Activation of platelets Platelet activation was performed using adenosine diphosphate (ADP; Sigma, Deisenhofen, Germany) and thrombin receptor activating peptide (TRAP; Bachem Biochemica, Heidelberg, Germany) as agonists. Platelet-rich plasma was diluted to 1 108 platelets/ml with Ca2 +- and Mg2 +-free PBS. Platelet solution was incubated for 2 min at room temperature with 10, 50 or 100 AM ADP or for 5 min with 20 AM TRAP, and then further processed for analysis of both flow cytometry and spreading behaviour. 2.6. Statistics All values are given as means F S.E.M. Statistical analysis was performed using the unpaired Student’s t-test for differences of platelet preparations in P-selectin expression, retention index and spreading test (SigmaStat, California, USA). A p value < 0.05 was considered to be significant.
3. Results
Fig. 1. Flow cytometric analysis of CD62P expression on human platelets using an FITC-labelled antihuman CD62P antibody (solid line). Platelets were assessed from platelet-rich plasma and activated by exposure to TRAP (20 AM). To exclude unspecific binding (M1), an FITC-coupled IgG1 isotype-matched control antibody was used (dashed line).
Flow cytometry revealed that a fraction of less than 5% of platelets from PRP expressed P-selectin (Fig. 2A). Upon ADP exposure, the fraction of P-selectin expressing platelets increased by five- to sixfold to approximately 17%, regardless of the dose of ADP used (10 – 100 AM; Fig. 2A). Thrombin receptor-activating peptide (TRAP) proved to be a stronger platelet agonist causing membrane translocation of P-selectin in almost half of the PRP platelets (Fig. 2A).
R.M. Nickels et al. / Thrombosis Research 110 (2003) 53–56
55
Table 1 Spreading test of unstimulated, ADP- and TRAP-stimulated platelets from platelet-rich plasma Sample
Large/ small form
Transition/ spider form
Agglutination
Aggregation
Platelets, no activation Platelets, ADP activation Platelets, TRAP activation
69 F 4
31 F 4
0.2 F 0.2
1.0 F 0.3
86 F 4*
14 F 4*
1.3 F 0.3*
2.5 F 0.3*
83 F 2*
17 F 2*
3.2 F 0.6*
3.6 F 0.2*
Values are given as means F S.E.M. * p < 0.05 vs. platelets, no activation. Fig. 2. P-selectin expression (A) and retention index (B) of platelets isolated from platelet-rich plasma and activated by either ADP (10 – 100 AM) or TRAP (20 AM). Unstimulated platelets served as controls (C). Values are given as means F S.E.M.; *p < 0.05 vs. untreated control platelets; #p < 0.05 vs. ADP-stimulated platelets.
Unexposed control platelets from PRP revealed a retention index of 14.4 F 1.8% (Fig. 2B). Activation with ADP resulted in a four- to fivefold increase of platelet retention index. In parallel to P-selectin expression, retention index did not differ between platelets stimulated by either low or high ADP concentration (Fig. 2B). TRAP activation caused maximal platelet retention with an average value of 86.6 F 2.0% (Fig. 2B). Regression analysis between retention index and P-selectin expression of activated and nonactivated PRP platelets revealed a significant ( p < 0.01) linear correlation between both measures of platelet activation such that the higher the P-selectin expression of platelets, the more pronounced the retention index (Fig. 3). Quantitative analysis of spreading of platelets revealed that 60– 70% of platelets from platelet-rich plasma were found as small and large forms on the slide, while 30 – 40%
Fig. 3. Regression analysis between retention index and P-selectin expression of unstimulated (circles), ADP-stimulated (triangles) and TRAP-stimulated (squares) platelets from platelet-rich plasma. r = regression coefficient.
were presented as transition or spider forms (Table 1). Platelet agglutination and aggregation were found to be almost absent. Stimulation with ADP and TRAP caused about 85% of all types of platelets to spread to small, but mostly to large forms, while the fraction of transition or spider forms decreased to 15% (Table 1). Marked activation of ADP- and TRAP-exposed platelets was further indicated by pronounced agglutination and aggregation (Table 1).
4. Discussion Platelet activation is characterized by an ordered sequence of events that includes shape change, increase in cytoplasmic Ca2 +, induction of fibrinogen receptor expression, release of granular contents with P-selectin translocation, aggregation, and formation of a stable hemostatic plug [9]. While this response is essential for hemostasis, it is also important in the pathogenesis of a broad spectrum of diseases, including atherosclerosis, myocardial infarction, unstable angina, stroke and thrombosis. Specialised glycoprotein receptors enable platelets to adhere to proteins that are exposed in areas of vascular damage. The process of adhesion and the interaction of soluble agonists with platelet receptors activate the platelets, which are then able to aggregate. This aggregation creates a platelet plug that seals the breach in the vessel wall and prevents excess blood loss. Activated platelets then facilitate secondary hemostasis, the formation of a fibrin clot, by carrying coagulation factors and providing a catalytic surface for the major interactions of the coagulation cascade. Thus, for evaluating the state of platelet activation in vitro, it is mandatory to combine specific techniques, e.g. flow cytometry determining specific platelet membrane structures with methods assessing the ability of platelets to adhere and to form aggregates. The RT-H is based on the exposure of platelets to a standardized exogenic surface (retention tube) under defined conditions of flow and calculates the difference of platelet count before and after the retention tube passage. Morphological evaluation using scanning as well as transmission electron microscopy of the polyurethane filter in the retention tube has demonstrated that platelets adhere and spread out at the surface and form aggregates [10]. The spreading
56
R.M. Nickels et al. / Thrombosis Research 110 (2003) 53–56
analysis test, as used herein, determines adhesion and spreading as well as spontaneous aggregation and agglutination of platelets from a morphological point of view without application of shear forces. In the present study, regression analysis revealed a significant linear correlation between retention index and P-selectin expression of resting, ADP- and TRAP-activated PRP platelets. Stimulation of PRP platelets with TRAP led to an enormous increase of P-selectin-positive platelets in contrast to the stimulation with ADP causing only approximately 17% of platelets to express P-selectin. While TRAP, a strong platelet agonist, was able to overcome platelet desensitization and caused extreme a-degranulation, ADP as weaker agonist failed to induce sustained release of agranules. However, ADP seemed to induce a change in both platelet shape and adhesion behaviour, as mirrored by markedly increased retention indices found in the present study. In support of this, spreading analysis test revealed increased agglutination and aggregation.
5. Summary RT-H proved to directly reflect activation of platelets from platelet-rich plasma, inasmuch as there was a positive linear correlation between retention index and surface Pselectin expression of nonstimulated and agonist-stimulated platelets. Thus, the RT-H might be an ideal adjunctive to refine the tools for assessment platelet response in physiology and pathophysiology.
Acknowledgements B.V. is recipient of a Heisenberg Stipendium (Vo 450/6-1 and 6-2). The study is supported by a grant from the
Deutsche Forschungsgemeinschaft, Bonn Bad-Godesberg (Vo 450/7-1).
References [1] Subramaniam M, Frenette PS, Saffaripour S, Johnson RC, Hynes RO, Wagner DD. Defects in hemostasis in P-selectin-deficient mice. Blood 1996;87:1238 – 42. [2] Denis C, Methia N, Frenette PS, Rayburn H, Ullman-Cullere M, Hynes RO, et al. A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis. Proc Natl Acad Sci U S A 1998;95:9524 – 9. [3] Vollmar B, Schmits R, Kunz D, Menger MD. Lack of in vivo function of CD31 in vascular thrombosis. Thromb Haemost 2001;85:160 – 4. [4] Holmsen H. Platelet secretion. In: Colman RW, Hirsh I, Marder VJ, Salzman EW, editors. Hemostasis and thrombosis. Basic principles and clinical practice, 2nd ed. Philadelphia, PA: JB Lippincott; 1987. p. 606 – 17. [5] Furie B, Furie BC, Flaumenhaft R. A journey with platelet P-selectin: the molecular basis of granule secretion, signalling and cell adhesion. Thromb Haemost 2001;86:214 – 21. [6] Youssefian T, Masse JM, Rendu F, Guichard J, Cramer EM. Platelet and megakaryocyte dense granules contain glycoproteins Ib and IIb – IIIa. Blood 1997;89:4047 – 57. [7] Jy W, Mao WW, Horstman LL, Valant PA, Ahn YS. A flow cytometric assay of platelet activation marker P-selectin (CD62P) distinguishes heparin-induced thrombocytopenia (HIT) from HIT with thrombosis (HITT). Thromb Haemost 1999;82:1255 – 9. [8] Breddin HK, Bauke J. Thrombozytenagglutination und Gefa¨ßkrankheiten. Blut 1965;11:144 – 64. [9] Blockmans D, Deckmyn H, Vermylen J. Platelet activation. Blood Rev 1995;9:143 – 56. [10] Mestres P, Pu¨tz N, Zhu H, Wenzel E. Description of a method for detection of adherent platelets in scanning electron microscopy. Eur J Cell Biol 1999;78(Suppl 50):32.