Figure 1
Figure 1. PECAM-1 and Lyn depend on one another to inhibit platelet responses to GPVI-specific agonists. (A) To control for genetic differences other than PECAM-1 or Lyn, platelets were obtained from age- and sex-matched PECAM-1–positive/Lyn-positive (P+/+/L+/+) and PECAM-1–negative/Lyn-positive (P−/−/L+/+) offspring of pecam-1+/−/lyn+/+ breeding pairs (i), Lyn-positive/PECAM-1–positive (L+/+/P+/+) and Lyn-negative/PECAM-1–positive (L−/−/P+/+) offspring of lyn+/−/pecam-1+/+ breeding pairs (ii), PECAM-1–positive/Lyn-negative (P+/+/L−/−) and PECAM-1-negative/Lyn-negative (P−/−/L−/−/) offspring of pecam-1+/−/lyn−/− breeding pairs (iii), and Lyn-positive/PECAM-1–negative (L+/+/P−/−) and Lyn-negative/PECAM-1–negative (L−/−/P−/−) offspring of lyn+/−/pecam-1−/− breeding pairs (iv). Pairwise combinations of platelets were stimulated in an aggregometer with various doses of CRP as described in “Isolation and stimulation of platelets” under constant stirring conditions. Low, intermediate, and high doses of CRP were empirically determined for each pairwise comparison. A low dose of CRP (range, 0.025-0.1 μg/mL) was defined as the highest dose of CRP that failed to induce aggregation of double-positive platelets for the data shown in subpanels i-ii, or of single-positive platelets for the data shown in subpanels iii-iv. A high dose of CRP (range, 0.2-0.6 μg/mL) was defined as the lowest dose of CRP that induced 100% aggregation of double-positive platelets for the data shown in subpanels i-ii, or of single-positive platelets for the data shown in subpanels iii-iv. An intermediate dose of CRP (range, 0.05-0.3 μg/mL) was between the high and low doses for each experiment. The percentage of aggregation was quantified in multiple pairwise comparisons (n = 3), and results are presented as mean percentage (± SEM) of aggregation. An asterisk identifies a statistically significant difference in platelet aggregation (P < .05). “NS” identifies differences that are not statistically significant. Note that PECAM-1 inhibits platelet responses to intermediate doses of CRP in the presence (i) but not in the absence (iii) of Lyn, and that Lyn is inhibitory in the presence (ii) but not in the absence (iv) of PECAM-1. (B) CRP-induced aggregation responses of platelets from age- and sex-matched mice of all 4 genotypes were compared in a single experiment using a dose of CRP (0.3 μg/mL) that induced an intermediate level of responsiveness from P+/+/L+/+ platelets. Note that PECAM-1–deficient, Lyn-deficient, and PECAM-1/Lyn double-deficient platelets are equally hyperresponsive, relative to wild-type platelets, to an intermediate dose of the GPVI-specific agonist CRP.

PECAM-1 and Lyn depend on one another to inhibit platelet responses to GPVI-specific agonists. (A) To control for genetic differences other than PECAM-1 or Lyn, platelets were obtained from age- and sex-matched PECAM-1–positive/Lyn-positive (P+/+/L+/+) and PECAM-1–negative/Lyn-positive (P−/−/L+/+) offspring of pecam-1+/−/lyn+/+ breeding pairs (i), Lyn-positive/PECAM-1–positive (L+/+/P+/+) and Lyn-negative/PECAM-1–positive (L−/−/P+/+) offspring of lyn+/−/pecam-1+/+ breeding pairs (ii), PECAM-1–positive/Lyn-negative (P+/+/L−/−) and PECAM-1-negative/Lyn-negative (P−/−/L−/−/) offspring of pecam-1+/−/lyn−/− breeding pairs (iii), and Lyn-positive/PECAM-1–negative (L+/+/P−/−) and Lyn-negative/PECAM-1–negative (L−/−/P−/−) offspring of lyn+/−/pecam-1−/− breeding pairs (iv). Pairwise combinations of platelets were stimulated in an aggregometer with various doses of CRP as described in “Isolation and stimulation of platelets” under constant stirring conditions. Low, intermediate, and high doses of CRP were empirically determined for each pairwise comparison. A low dose of CRP (range, 0.025-0.1 μg/mL) was defined as the highest dose of CRP that failed to induce aggregation of double-positive platelets for the data shown in subpanels i-ii, or of single-positive platelets for the data shown in subpanels iii-iv. A high dose of CRP (range, 0.2-0.6 μg/mL) was defined as the lowest dose of CRP that induced 100% aggregation of double-positive platelets for the data shown in subpanels i-ii, or of single-positive platelets for the data shown in subpanels iii-iv. An intermediate dose of CRP (range, 0.05-0.3 μg/mL) was between the high and low doses for each experiment. The percentage of aggregation was quantified in multiple pairwise comparisons (n = 3), and results are presented as mean percentage (± SEM) of aggregation. An asterisk identifies a statistically significant difference in platelet aggregation (P < .05). “NS” identifies differences that are not statistically significant. Note that PECAM-1 inhibits platelet responses to intermediate doses of CRP in the presence (i) but not in the absence (iii) of Lyn, and that Lyn is inhibitory in the presence (ii) but not in the absence (iv) of PECAM-1. (B) CRP-induced aggregation responses of platelets from age- and sex-matched mice of all 4 genotypes were compared in a single experiment using a dose of CRP (0.3 μg/mL) that induced an intermediate level of responsiveness from P+/+/L+/+ platelets. Note that PECAM-1–deficient, Lyn-deficient, and PECAM-1/Lyn double-deficient platelets are equally hyperresponsive, relative to wild-type platelets, to an intermediate dose of the GPVI-specific agonist CRP.

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