PECAM-1 expression and activity negatively regulate multiple platelet signaling pathways

Abstract

Platelet endothelial cell adhesion molecule-1 (PECAM-1) inhibits platelet response to collagen and may also inhibit two other major platelet agonists ADP and thrombin although this has been less well explored. We hypothesized that the combined effect of inhibiting these three platelet activating pathways may act to significantly inhibit thrombus formation. We demonstrate a negative relationship between PECAM-1 surface expression and platelet response to cross-linked collagen related peptide (CRP-XL) and ADP, and an inhibitory effect of PECAM-1 clustering on platelet response to CRP-XL, ADP and thrombin. This combined inhibition of multiple signaling pathways results in a marked reduction in thrombus formation.

Supplementary Fig. S1

Association between PECAM-1 surface expression and platelet reactivity. A graphical representation of the linear regression analysis summarised in Table 1 showing the association between surface expression of four platelet surface molecules and plate reactivity (light gray – P < 0.05). In line with previous reports surface expression of GPVI is positively associated with platelet response to CRP-XL [25] accounting for approximately 20% of the variation in platelet response to this agonist. Surface expression of αIIbβ3 was also positively correlated with platelet fibrinogen binding in response to ADP and TRAP. Surface expression of PECAM-1 showed a negative association with both fibrinogen binding and P-selectin expression in response to CRP-XL and ADP. GPVI and αIIbβ3 expression, time of day, and mean platelet volume were includes as confounding variables, as indicated in Table 1.

Fig. 1

Inhibitory effect of PECAM-1 cross-linking on platelet function. (A) [Ca²⁺]_i in response to agonists following anti-PECAM-1 antibody (dotted line) and the isotype control (solid line) cross-linking (mean of four individuals). (B) P-selectin expression and (C) fibrinogen binding to platelets in response to stimulation following anti-PECAM-1 antibody (open circles) and the isotype control (full circles) cross-linking. All points are the mean ± S.E. of five individuals. P-values compare the difference between the two curves over the entire concentration range. (D) Representative flow cytometry histograms showing P-selectin expression and fibrinogen binding to platelets in response to stimulation following anti-PECAM-1 antibody (black line) and the isotype control (gray line) cross-linking.

Fig. 2

Inhibitory effect of PECAM-1 is not a generic antibody effect. (A and C) P-selectin expression and (B and D) fibrinogen binding to platelets in response to stimulation is only inhibited by cross-linking anti-PECAM-1 antibodies, and (A and B) not with any other combination of the anti-PECAM-1, isotype control, cross-linking antibodies or (C and D) F(ab′) of the cross-linking antibody. Nor (C and D) was this inhibition attenuated by pre-incubation with IV.3 F(ab′) fragments. (A and B) stimulation occurred in the presence of appropriate agonists. (C and D) No inhibitors were used. All bars are the mean ± S.E. of four individuals. ^∗P < 0.05, ^∗∗P < 0.01. P-values compare the difference between the samples incubated with cross-linked anti-PECAM-1 and all other points.

Fig. 3

Inhibitory effect of PECAM-1 cross-linking does not reduce TRAP induced platelet activation. P-selectin exposure and fibrinogen binding to platelets in response to TRAP (comparing the entire concentration range, P = 0.566 and 0.489, respectively). Cross-linked anti-PECAM-1 antibody (open circles) and the isotype control (full circles). All data are given as the mean ± S.E. of four individuals.

Fig. 4

The inhibitory effects of PECAM-1 on multiple activation pathways interact to enhance the inhibition of platelet activation. (A and B) P-selectin exposure and fibrinogen binding to platelets in response to CRP-XL in the presence or absence of inhibitors of endogenous thrombin, ADP, and thromboxane. P-values compare cross-linked anti-PECAM-1 antibody (gray bar) and the isotype control (black bar) treated samples. (C and D) Inhibition of platelet response following PECAM-1 cross-linking. P-values compare the level of inhibition in the absence of inhibitors with that seen in their presence. All data are given as the mean ± S.E. of four individuals. ^∗P < 0.05, ^∗∗P < 0.005, ^∗∗∗P < 0.0005.

Fig. 5

Inhibitory effect of PECAM-1 cross-linking on thrombus formation. (A and B) Representative images showing thrombus formation on collagen coated slides following cross-linking of the isotype control or anti-PECAM-1 antibodies, respectively. (C) Mean volume, (D) the total amount of protein eluted from the slides, and (E) coverage following thrombus formation following anti-PECAM-1 antibody or isotype control cross-linking. To correct for inter-individual variation in thrombus formation all data are expressed as a percentage of the isotype control. Data are the mean ± S.E. of seven individuals (C and E) and 3 individuals (D). ^∗P < 0.05, ^∗∗P < 0.005, ^∗∗∗P < 0.0005.