Effects of platelets activated by different agonists on fibrin formation and thrombin generation

Figures & data

Figure 1. Platelets immobilized on plastic surface in 96-well plates. Washed platelets (0.5 x 10⁸/ml) were not activated (a) or activated by 0.2 mM arachidonic acid (b) or 10 µm A23187 (C) without stirring for 5 min at room temperature and spun down on the bottom of 96-well plates (100 µl/well) at 1500 g, 5 min. (see “Materials and methods”). The supernatant was removed; platelets were fixed with 1% paraformaldehyde and analyzed in a phase-contrast microscope. Scale bar −10 µm. Close pictures were obtained after platelet activation by arachidonic acid (b) and ADP and collagen (not shown). After activation by A23187 most platelets acquire a rounded shape.

Figure 2. Effects of platelets on fibrin formation in modified PRA. Platelets were not activated by exogenous agonists (“No exo agonists”) or activated by 20 µM TRAP (“TRAP”), 20 µM ADP (“ADP”), 0.2 mM arachidonic acid (“Arach acid”), 10 µg/ml collagen (“Collagen”) or 10 µM A23187 (“A23187”) and spun down in 96-well plates. Platelets were not added to the control samples (“No platelets”). After plasma addition, PRA was performed as described in “Materials and methods.” Representative curves out of ≥ 8 experiments. Statistical data are given in Table I.

Table I. Platelet activation and fibrin formation in a PRA.

	Lag phase, min	V max, %A450/min
No platelets (1) (n = 26)	19.1 ± 3.1	5.7 ± 1.4
No exogenous agonists (2) (n = 26)	10.9 ± 2.4 p(1) < 0.001	8.5 ± 3.6 p(1) = 0.008
TRAP, 20 µM (n = 18)	10.5 ± 2.1 p(1) < 0.001 p(2) = 0.569	8.6 ± 3.2 p(1) = 0.013 p(2) = 0.908
ADP, 20 µM (n = 8)	8.1 ± 2.9 p(1) < 0.001 p(2)<0.001	8.8 ± 3.0 p(1) = 0.061 p(2) = 0.870
Arachidonic acid, 0.2 mM (n = 8)	7.5 ± 1.9 p(1) < 0.001 p(2) < 0.001	11.0 ± 4.3 p(1) < 0.001 p(2) = 0.111
Collagen, 10 µg/ml (n = 18)	6.8 ± 1.6 p(1) < 0.001 p(2) < 0.001	12.6 ± 3.7 p(1) < 0.001 p(2) = 0.001
A23187, 10 µM (n = 19)	5.8 ± 0.9 p(1) < 0.001 p(2) < 0.001	17.8 ± 6.3 p(1) < 0.001 p(2) < 0.001

Means ± SD are presented. (n) – Number of experiments. Difference between multiple groups was highly significant for both “Lag phase” and “V max” – p < 0.001 (one way ANOVA). p(1), p(2) – Significance of differences from “No platelets” (1) and “No exogenous agonists” (2) (post-hoc analysis, Fisher test). Significant differences are highlighted in italics.

Figure 3. Effects of platelets on thrombin generation in TGT. Platelets were not activated by exogenous agonists (“No exo agonists”), or activated by 20 µM TRAP (“TRAP”), 20 µM ADP (“ADP”), 0.2 mM arachidonic acid (“Arach acid”), 10 µg/ml collagen (“Collagen”) or 10 µM A23187 (“A23187”) and spun down in 96-well plates. Platelets were not added to the control samples (“No platelets”). TGT was performed as described in “Materials and methods.” Representative curves out of ≥ 7 experiments. Statistical data are given in Table II.

Table II. Platelet activation and thrombin generation.

	Lag phase, min	ETP, nM x min	Peak, nM	V max, nM/min
No platelets (1) (n = 8)	4,5 ± 0,5	281 ± 45	18 ± 3	3,8 ± 0,7
No exogenous agonists (2) (n = 8)	3,9 ± 0,4 p(1) = 0.003	1222 ± 158 p(1) < 0.001	76 ± 12 p(1) <0.001	13,0 ± 2,9 p(1) = 0.092
TRAP, 20 µM (n = 7)	4,0 ± 0,3 p(1) = 0.006 p(2) = 0.754	1249 ± 211 p(1) < 0.001 p(2) = 0.724	97 ± 15 p(1) < 0.001 p(2) = 0.102	18,9 ± 3,5 p(1) = 0.010 p(2) = 0.303
ADP, 20 µM (n = 8)	3,9 ± 0,3 p(1) = 0.005 p(2) = 0.881	1180 ± 101 p(1) < 0.001 p(2) = 0.611	82 ± 15 p(1) < 0.001 p(2) = 0.666	15,1 ± 3,5 p(1) = 0.049 p(2) = 0.724
Arachidonic acid, 0.2 mM (n = 7)	4,1 ± 0,4 p(1) 0.048 p(2) = 0.307	1143 ± 109 p(1) < 0.001 p(2) = 0.334	101 ± 12 p(1) < 0.001 p(2) = 0.102	21,4 ± 2,7 p(1) = 0.004 p(2) = 0.162
Collagen, 10 µg/ml (n = 7)	4,0 ± 0,3 p(1) 0.009 p(2) = 0.665	1263 ± 175 p(1) < 0.001 p(2) = 0.600	131 ± 33 p(1) < 0.001 p(2) < 0.001	30,4 ± 12,0 p(1) < 0.001 p(2) = 0.003
A23187, 10 µM (n = 8)	3,5 ± 0,3 p(1) < 0.001 p(2) = 0.025	1307 ± 204 p(1) < 0.001 p(2) = 0.277	227 ± 44 p(1) < 0.001 p(2) < 0.001	81,1 ± 24,0 p(1) < 0.001 p(2) < 0.001

Means ± SD are presented. (n) – Number of experiments. Difference between multiple groups was highly significant for all variables (“Lag phase,” “ETP,” “Peak,” “V max”) – p < .001 (one way ANOVA). p(1), p(2) – Significance of differences from “No platelets” (1) and “No exogenous agonists” (2) (post-hoc analysis, Fisher test). Significant differences are highlighted in italics.

Figure 4. Platelet activation and PS expression. Platelets were not activated (No agonists) or activated by indicated agonists and PS was detected by staining with annexin V-FITC using flow cytometry. Thrombin was used in a concentration of 10 U/ml, and other agonists (A23187, TRAP, collagen, arachidonic acid and ADP) in the same concentrations as in recalcification assay (Figure 1 and Table I) and TGT (Figure 2 and Table II). (A) Examples of flow cytometry analysis. Fluorescence regions for PS-, PS+, and PS++ platelets are shown. Vertical line separating PS- and PS+ regions corresponds to 95% of the fluorescent peak of the negative control (platelets without annexin V-FITC, not shown). Vertical line separating PS+ and PS++ regions corresponds to the 95% of the fluorescent peak of A23187 activated platelets (to the right). (B) Amounts of PS+ (black bars) and PS++ (gray bars) platelets after their activation with the agonists. Means ± SD (n ≥ 9) are shown; *p < 0.05, ***p < 0.001, significance of differences from “No agonists” group (t-test for means). (C) Amounts of PS+ (black bars) and PS++ (grey bars) platelets after their activation by thrombin alone and by thrombin in combination (+) with ADP, arachidonic acid and collagen. Means ± SD (n ≥ 11) are shown; *p < 0.05, **p < 0.001, significance of differences from “Thrombin” group (paired t-test).