TI - Results . AB - The inhibition , by PECAM-1 , of platelet response to GPVI stimulation , has been demonstrated by us and others [13,14] , although some debate surrounds the issue of whether PECAM-1 modulates signaling stimulated by other platelet agonists . We therefore explored this in more detail by measuring platelet reactivity to pathway specific activation by CRP-XL , thrombin and ADP in the presence of cross-linked anti-PECAM-1 antibody which we have previously shown to cause PECAM-1 PHOSphorylation [13] , or isotype control . Following PECAM-1 cross-linking total platelet [Ca2+] i reduced by 7.5 + - 2.0% in response to CRP-XL , and 11.2 + - 3.3% in response to thrombi . Similarly , peak [Ca2+] i was reduced by 6.2 + - 2.2% and 10.0 + - 2.8% , and final [Ca2+] i reduced by 8.8 + - 1.3% and 14.9 + - 4.0% , respectively ( P lt 005 , in all cases ) . Neither total nor peak [Ca2+] i in response to ADP were affected by PECAM-1 cross-linking ( Fig 1a ) . PECAM-1 cross-linking did not initiate [Ca2+] i in the absence of agonist ( data not shown ) . Furthermore PECAM-1 cross-linking , across a range of CRP-XL concentrations inhibited P-selectin exposure by 11.2% ( range : . . 2.7-20.8% , P = 0.036 ) and fibrinogen binding to alphaIIbbeta3 by 9.9% ( range : . . 3.3-17.4% , P = 0.046 ) ( Fig 1B and C ) . Similar magnitudes of inhibition were seen in response to both thrombin and ADP ( Fig 1B and C ) . The mean inhibition of P-selectin exposure in response to thrombin was 8.1% ( range : . . 0.8-14.9% , P = 0.021 ) and in response to ADP was 14.9% ( range : . . 3.9-22.7% , P = 0.013 ) . Fibrinogen binding was inhibited by 9.3% ( range : . . 6.1-16.1% , P = 0.024 ) in response to thrombin and 10.7% ( range : . . 2.8-24.4% , P = 0.035 ) in response to ADP . The inhibitory effect seen here was specific for cross-linked anti-PECAM-1 antibody . The level of stimulation achieved in the presence of the cross-linked isotype control was not significantly different to that seen in the absence of antibodies ( data not shown ) and no other combination of antibodies , apart from cross-linked anti-PECAM-1 antibody , caused any significant change to platelet activation ( Fig 2 ) . Platelet response was not inhibited if incubated with the PECAM-1 antibody and F ( ab' ) fragments of the cross-linking antibody ( Fig 2 ) . Furthermore , pre-incubation with IV.3 F ( ab' ) fragments , to block the low-affinity receptor for IgG FcgammaRIIA , did not alter the inhibitory effect of PECAM-1 cross-linking ( Fig 2 ) . Therefore the inhibition of platelet reactivity by PECAM-1 is not a generic antibody effect . As with [Ca2+] i no combination of antibodies caused any platelet activation in the absence of agonists ( data not shown ) . If PECAM-1 function is important for the regulation of platelet function , variation in the surface expression of the protein should impact on platelet reactivity . To test this we examined the relationship between surface expression of PECAM-1 on unactivated platelets and its association with P-selectin exposure and fibrinogen binding to platelets in response to CRP-XL , ADP and TRAP , in 89 healthy subjects [20] . Surface expression of PECAM-1 was negatively correlated with P-selectin expression and fibrinogen binding in response to CRP-XL ( P = 0009 and 0003 , respectively ) and ADP ( P = 0017 and 0003 , respectively ) although not to TRAP ( Table 1 ) . While the strength of this association is modest , accounting for between 6% and 10% of the variation in platelet response to CRP-XL and ADP , it was of a similar magnitude to the positive association seen between platelet reactivity and GPVI or alphaIIbbeta3 surface expression . Associations of this magnitude are entirely expected in this context given that platelet activation is a multi-factorial process requiring the integration of multiple signaling steps . Variation in any of these steps will have a moderate impact on the final level of response . It is important here to separate out the information being given by the r2 value and the P-value .r2 is the proportion of variation in platelet function explained by the regression model and gives information about the strength of the association , while the P-value gives information on whether that association is likely to have occurred by chance . Therefore given an appropriate sample size it is entirely possible , as is the case here , to identify weak associations that are statistically significant . The negative association identified here confirms our findings of an inhibitory role for PECAM-1 in CRP-XL and ADP induced platelet activation . There is a clear dichotomy in these results . While PECAM-1 cross-linking inhibited platelet response to thrombin ( Fig 1 ) , PECAM-1 expression showed no relationship with platelet activation following stimulation by TRAP in the association studies ( Table 1 ) . Subsequent investigation established that PECAM-1 cross-linking did not inhibit either fibrinogen binding or P-selectin expression in response to TRAP ( Fig 3 ) . These results support the findings of the association study , however , a discrepancy remains between the inhibitory effect of PECAM-1 on thrombin activation of platelets and its lack of effect on TRAP stimulation . This may stem from the involvement of glycoprotein ( GP ) Ibalpha in thrombin [23] ( which is PAR-1 specific ) induced platelet activation and may indicate a role for PECAM-1 in mediating the action of GPIbalpha during thrombin stimulation of platelets . PECAM-1 has previously been shown to be involved in GPIbalpha mediated activation of platelets , becoming tyrosine PHOSphorylated upon vWF binding to GPIbalpha and PECAM-1 deficient mice show enhanced aggregation in response to vWF [24] . Thus it is not inconceivable that stimulation of PECAM-1 inhibits the action of GPIbalpha causing reduced thrombin induced platelet activation . During thrombus formation platelets are exposed to a variety of stimuli simultaneously , including collagen , thrombin and ADP . It is , therefore , possible that the small inhibitory effects in multiple pathways , that we have described so far , interact resulting in a greater inhibition of platelet response and thrombus formation . This could be seen in the flow cytometry assay when comparing the response of platelets to CRP-XL in the presence and absence of inhibitors of endogenous thrombin , ADP and thromboxane . PECAM-1 cross-linking had a greater inhibitory effect on CRP-XL (01 mug/mL) stimulation in the absence of hirudin , apyrase and indomethacin , thereby allowing secondary activation pathways to play a role , than in the presence of these inhibitors ; fibrinogen binding was reduced by 15.5 + - 2.7% compared to 4.9 + - 2.8% , P = 0.002 , and P-selectin expression was reduced by 15.5 + - 1.8% compared to 6.2 + - 4.9% , P = 0.01 , respectively ( Fig 4 ) . A similar increase in inhibition was seen when comparing the effect of PECAM-1 cross-linking on platelets stimulated with CRP-XL (1 mug/mL) in the presence of inhibitors or CRP-XL (01 mug/mL) in their absence . While both treatments gave comparable levels of platelet activation with the isotype control , the level of inhibition resulting from PECAM-1 cross-linking was significantly greater in the absence of inhibitors ( Fig 4 ) . To identify the magnitude of the inhibitory effect of PECAM-1 in conditions that are more reflective of in vivo processes , thrombi were formed under arterial flow conditions in collagen coated micro-capillary slides . Following PECAM-1 cross-linking total thrombus volume was reduced by 49.8 + - 9.9% ( P = 0002 ) compared to treatment with the isotype control , and the area of the glass capillary covered by thrombi was similarly reduced by 54.1 + - 7.9% ( P = 00005 ) ( Fig 5 ) . The total amount of protein eluted from the slides following thrombus formation also decreased in the presence of cross-linked PECAM-1 by 16.9 + - 4.3% ( Fig 5D ) .