High fragmentation in platelet concentrates impacts the activation, procoagulant, and aggregatory capacity of platelets

Figures & data

Figure 1. Illustration of gating strategies to detect platelet subpopulations. The dot plots depict the formation of platelet subpopulations, exemplified by a whole blood sample from a healthy individual, before and after stimulation with same agonists as used for the platelet concentrates. a: Spontaneously activated platelets (with HEPES instead of agonist). b: Platelets activated with a combination of CRP-XL (1.33 µg/mL) + PAR1-AP (30 µm) + PAR4-AP (300 µm).

Figure 2. Examples of flow cytometry dot plots from platelet units with high and low level of platelet fragmentation at day 1. a, c: Spontaneously activated (with HEPES instead of agonist). b, d: platelets activated with a combination of CRP-XL (1.33 µg/mL) + PAR1-AP (30 µm) + PAR4-AP (300 µm).

Table I. Platelet subpopulations in PCs on day 1 of storage.

	Normal-sized platelets		Small platelets		Platelet fragments
	HEPES	Agonist	HEPES	Agonist	HEPES	Agonist
All PCs (n = 10)	82 (66–91)	86 (71–91)	10 (5–19)	8 (5–14)	8 (3–17)	7 (3–15)
PCs with low fragmentation (n = 5)	91 (89–93)	90 (88–96)	6 (4–7)	6 (2–8)	3 (2–4)	4 (2–5)
PCs with high fragmentation (n = 5)	72 (46–75)^##	73 (60–80)^##	18 (13–27)^##	13 (8–18)	14 (10–27)^##	11 (10–26)^##

Note: Platelet subpopulations based on FSC/CD41+ properties. The results are shown as percentage of platelets in each population. Platelet subpopulations in PCs formed spontaneously (HEPES) or after stimulation with agonist combination of CRP-XL (1.33 µg/mL) + PAR1-AP (30 µM) + PAR4-AP (300 µM). Median and 25^th-75^th percentiles. ^#/## = p < .05/0.01 as compared to PCs with low fragmentation.

Table II. Expression of activation markers on all platelets and platelet subpopulations on day 1 of storage.

	All platelets		Normal-sized platelets		Small platelets		Platelet fragments
	HEPES	Agonist	HEPES	Agonist	HEPES	Agonist	HEPES	Agonist
PAC-1+	0.8 (0.6–1.1)	85 (70–93)**	0.8 (0.5–1.3)	96 (86–98)**	1.5 (1.2–1.8)^nn	46 (17–58)^**nn	0.3 (0.2–0.4)^n	0.8 (0.1–1.0)^nn
LAMP-1+	1.8 (1.3–3.9)	65 (49–68)**	1.6 (1.2–3.6)	70 (63–71)**	4.3 (2.8–8.0)^nn	35 (16–47)^**nn	0.8 (0.3–1.4)^nn	0.6 (0.2–1.3)^nn
Annexin V+	4.2 (2.7–8.1)	7.6 (4.3–10.6)**	1.5 (1.3–2.1)	2.4 (1.4–5.7)*	22 (13–31)^nn	48 (40–52)^**nn	11 (7–17)^nn	19 (7–34)^*n
P-selectin+	39 (31–46)	94 (89–98)**	36 (28–40)	99 (96–99)**	58 (49–78)^nn	88 (80–91)^**nn	43 (35–48)	49 (42–57)^**nn
DiIC1(5)+	86 (70–94)	70 (59–77)**	98 (98–98)	81(78–87)*	48 (22–67)^nn	4.3 (4.0–12.0)^**nn	5.4 (4.1–10.0)^nn	0.7 (0.2–1.3)^**nn

Note: Expression of markers on all platelets and on the different platelet subpopulations spontaneously (HEPES) or after stimulation with agonist combination of CRP-XL (1.33 µg/ml) + PAR1-AP (30 µM) + PAR4-AP (300 µM). The expression is shown as percentage positive platelets. Median and 25th–75th percentiles for n = 10. ^*/** = p < .05/0.01 as compared to HEPES for each subpopulation. Statistical comparison between the subpopulations with ^n/nn = p < .05/0.01 as compared to normal-sized platelets for HEPES and agonist combination, respectively.

Table III. Aggregation and coagulation response of platelets in PCs with high and low fragmentation.

	Day 1	Day 5	Day 7
TRAP-AUC (AU x min)
PCs with low fragmentation (n = 5)	649 (565–704)	612 (600–677)	622 (544–687)
PCs with high fragmentation (n = 5)	557 (553–661)	576 (536–664)	609 (538–821)
COL-AUC (AU x min)
PCs with low fragmentation (n = 5)	105 (94–168)	70 (63–96)	41 (29–70)**
PCs with high fragmentation (n = 5)	77 (36–97)	25 (19–46)^*,#	31 (29–46)
Clotting time (s)
PCs with low fragmentation (n = 5)	830 (635–922)	777 (739–807)	739 (678–914)
PCs with high fragmentation (n = 5)	728 (676–796)	753 (745–779)	751 (710–822)
G’max (Pa)
PCs with low fragmentation (n = 5)	1041 (971–1145)	1293 (1163–1431)	1340 (1189–1366)*
PCs with high fragmentation (n = 5)	976 (629–1092)	1162 (807–1313)*	1076 (771–1262)

Note: Aggregation response to PAR-1 AP (TRAP) and collagen (COL) determined with impedance aggregometry as well as clotting time and maximum elasticity (G’max) determined by a viscoelastic device. Median and 25th-75th percentiles. ^*/** = p < .05/0.01 as compared to day 1 for PCs with low and high fragmentation, respectively, ^#/## = p < .05/0.01 as compared to PCs with low fragmentation.

Figure 3. Spontaneous exposure of platelet activation markers during storage in PCs with initially high (n = 5) and low (n = 5) platelet fragmentation. The result is for CD41+ events of normal- and small size. The bars show median values and error bars the interquartile ranges. ^*/** = p <.05/0.01 compared to day 1 for low- and high fragmentation, respectively. ^#/## = p <.05/0.01 as compared to PCs with low fragmentation.

Figure 4. Activation-induced response in PCs with initially high (n = 5) and low (n = 5) platelet fragmentation. The result is for CD41+ events of normal- and small size. The bars show median values and error bars the interquartile ranges. ^*/** = p <.05/0.01 compared to day 1 for low- and high fragmentation, respectively. # = p <.05 as compared to PCs with low fragmentation. The activation-induced response was calculated where the percentage for the non-agonist stimulated samples (i.e., with HEPES) was subtracted from the percentage of the agonist-stimulated samples.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.