The Inhibitory Effect of Protamine on Platelets is Attenuated by Heparin without Inducing Thrombocytopenia in Rodents

Figures

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  Figure 1. Platelet number in (a) mice (n = 5–8) and (b) rats (n = 5–10) at 3, 15, 30, and 60 minutes, and on the 35th day after the administration of unfractionated heparin (UFH) and protamine sulfate (PS). The results are expressed as a percentage of the control samples, and are shown as a median (line) with the interquartile range (box), and maximum and minimum values (whiskers). The number of platelets in the vehicle-treated groups was 617 (565–675) at 3 and 15 minutes, 705 (670–752) at 60 minutes, and 427 (326–467) on the 35th day in the mice. In the rats, the control values were 446 (176–754), 725 (372–746), 668 (196–781), and 637 (494–660) at 15, 30, and 60 minutes, and on the 35th day, respectively. * p < 0.05; ** p < 0.01 vs. vehicle group; Kruskal–Wallis analysis of variance (ANOVA) with Dunn’s post-hoc test.

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  Figure 2. Platelet aggregation results in mice at 3, 15, and 60 minutes (n = 4–7), and on the 35th day (n = 5–7) after unfractionated heparin (UFH) and protamine sulfate (PS) administration. (a) Collagen-induced platelet aggregation expressed as the maximal extension (MaxA), (b) the slope of platelet aggregation (Slp), (c) lag time, and (d) the area under the curve (AUC). The results are expressed as a percentage of the control samples, and are shown as the median (line) with the interquartile range (box), and maximum and minimum values (whiskers). The control values at 3 and 15 minutes were 11.0 (10.0–13.0), 9.0 (6.0–10.0), 115.0 (104.0–174.0), and 31.3 (21.1–40.4); at 60 minutes they were 10.5 (10.0–11.0), 4.0 (4.0–5.0), 137.0 (112.0–151.0), and 25.5 (21.4–30.6); and on the 35th day, they were 6.0 (4.0–8.0), 3.0 (3.0–4.0), 190.0 (150.0–270.0), and 11.6 (3.8–15.7), for MaxA, Slp, lag time, and AUC, respectively. * p < 0.05; ** p < 0.01 vs. vehicle; ^ p < 0.05; ^^ p < 0.01 vs. PS group; Kruskal–Wallis ANOVA with Dunn’s post-hoc test.

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  Figure 3. Platelet aggregation results in rats at 60 min (n = 5–7) and on the 35th day (n = 9–10) after unfractionated heparin (UFH) and protamine sulfate (PS) administration. (a) Collagen-induced platelet aggregation expressed as the maximal extension (MaxA), (b) the slope of platelet aggregation (Slp), (c) lag time, and (d) area under the curve (AUC). The results are expressed as a percentage of the control samples and are shown as the median (line) with the interquartile range (box), and maximum and minimum values (whiskers). The control values at 60 min were 8.5 (6.0–10.5), 4.0 (3.0–4.5), 156.0 (102.5–199.0), and 20.7 (9.7–25.2), and on the 35th day were 7.0 (3.5–9.0), 4.0 (2.0–5.0), 133.5 (85.0–220.0), and 17.4 (5.4–27.3) for MaxA, Slp, lag time, and AUC, respectively. * p < 0.05 vs. vehicle group; Kruskal–Wallis ANOVA with Dunn’s post-hoc test.

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  Figure 4. Effects of unfractionated heparin (UFH) and protamine sulfate (PS) on (a) P-selectin, (b) platelet factor 4 (PF4), and (c) β-thromboglobulin (βTG) concentration in mice (n = 6–8) on the 35th day of the experiment. The results are shown as the median (line) with the interquartile range (box), and maximum and minimum values (whiskers). * p < 0.05; ** p < 0.01 vs. vehicle group; Kruskal–Wallis ANOVA with Dunn’s post-hoc test. VEH—vehicle.

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  Figure 5. Effects of unfractionated heparin (UFH) and protamine sulfate (PS) on the (a) activated partial thromboplastin time (aPTT) and (b) D-dimer concentration in mice (n = 3–7), and (c) thrombus weight, (d) prothrombin time (PT), (e) activated partial thromboplastin time (aPTT), (f) D-dimer concentration, (g) fibrinogen concentration, (h) anti-factor Xa (anti-fXa) activity, and (i) 6-keto PGF1α concentration in rats (n = 9–10) on the 35th day of the experiment. The results are shown as the median (line) with the interquartile range (box), and maximum and minimum values (whiskers). * p < 0.05; ** p < 0.01; Kruskal–Wallis ANOVA with Dunn’s post-hoc test.

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  Figure 6. The thrombopoietin level (TPO) during the repeated administration of unfractionated heparin (UFH) and protamine sulfate (PS) in mice (n = 6–8). The serum TPO concentration was measured by specific enzyme-linked immunosorbent assay (ELISA). The results are shown as the median (line) with the interquartile range (box), and maximum and minimum values (whiskers). * p < 0.05 vs. the first week within the same group; Friedman ANOVA with Dunn’s post-hoc test.

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  Figure 7. The morphology of megakaryocytes (MK), and their number per 100-cell count for bone marrow (%), shown as the median with range. (a) The arrows in the top panel indicate MK, and (b) the bottom panel represent the remaining bone marrow cell lines at a 1000-fold magnification in bone marrow smears from mice (n = 8) treated repeatedly with unfractionated heparin (UFH) and protamine sulfate (PS). May–Grünwald–Giemsa stain (Merck) following the standard protocol. Microscope: Olympus CH 30; objectives: DPlanC 100, 1.25 oil, 160/0.17; DPlanC 40, 0.65, 160/0.17; camera: Digital Sight DS.-Fi1 Nikon.

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  Figure 8. Schematic representation of (a) the study protocol, and (b) the mean body weight gain expressed as a percentage of the body weight before treatment with unfractionated heparin (UFH) and protamine sulfate (PS) in mice (n = 8) during the second experiment. The results are shown as mean ± standard deviation (SD).* p < 0.05 vs. vehicle, ANOVA with Fisher’s LSD post hoc test.

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  Figure 9. Schematic representation of (a) the study protocol and (b) the mean body weight gain expressed as a percentage of the body weight before treatment with unfractionated heparin (UFH) and protamine sulfate (PS) in rats (n = 9–10) during the fourth experiment. The results are shown as mean ± SD. ANOVA with Fisher’s LSD post-hoc test.