LAIR-2/Fc inhibits adhesion of platelets to collagen under flow conditions.

<p><i>Panel</i> A: Collagen type III-coated coverslips were perfused with whole blood in the absence or presence of 100 µg/ml soluble LAIR-1/Fc, LAIR-2/Fc or SIRL-1/Fc. Representative pictures are shown. Perfusion was performed at a shear rate of 300 s⁻¹ (upper panels) or 1500 s⁻¹ (lower panels). Percentages below figures indicate percentage surface-coverage for each individual photo. <i>Panels</i> B & C: Quantitative representation of surface coverage of collagen type III-coated coverslips that were perfused in the absence or presence of 100 µg/ml soluble LAIR-1/Fc, LAIR-2/Fc or SIRL-1/Fc at 300 s⁻¹ (<i>panel</i> B) or 1500 s⁻¹ (<i>panel</i> C). Data represent mean±SD of three independent perfusions. <i>Panel</i> D: Dose dependent inhibition of surface platelet coverage in the presence of LAIR-2/Fc at shear rates of 300 s⁻¹ (open symbols) or 1500 s⁻¹ (closed symbols). *: <i>p</i><0.005.</p

LAIR-2/Fc interferes with VWF binding to collagen.

<p>Microtiter wells were coated with 50 µg/ml collagen I (<i>panel</i> A) or collagen III (<i>panel</i> B) and subsequently incubated with 0.1 µg/ml purified plasma-derived VWF in the presence of increasing concentrations of LAIR-1/Fc (open circles), LAIR-2/Fc (closed circles) or SIRL-1/Fc (squares). VWF binding was detected with horseradish-conjugated polyclonal anti-human VWF antibodies and 3-3′-5-5′-tetramethylbenzidine. Data are representative of 3 independent experiments.</p

LAIR-2/Fc inhibits collagen but not TRAP-induced platelet aggregation.

<p>Aggregation of platelet rich plasma (PRP) in response to collagen was measured using an optical aggregometer. <i>Panel</i> A: Platelet aggregation in response to 50 µM TRAP alone (PBS) or in the presence of 100 µg/ml LAIR-1/Fc, LAIR-2/Fc or SIRL-1/Fc. <i>Panel</i> B: Platelet aggregation in response to collagen (1 µg/ml) alone (PBS) or in the presence of 100 µg/ml LAIR-1/F, LAIR-2/Fc or SIRL-1/Fc. <i>Panel</i> C: Platelet aggregation in response to collagen (1 µg/ml) alone (PBS) or in the presence of 0.01 µg/ml, 0.1 µg/ml or 1.0 µg/ml LAIR-2/Fc. <i>Panel</i> D: Platelet aggregation in response to 0.5 µg/ml, 1 µg/ml, 2 µg/ml or 4 µg/ml collagen in the presence of 1.0 µg/ml LAIR-2/Fc.</p

Platelets do not express LAIR-1 or LAIR-2.

<p><i>Panel</i> A: Flowcytometric analysis of washed platelets (2×10E5/µl), unstimulated or stimulated for 5 min at RT with 10 µM TRAP or 5000 nM PMA. Upper panels represent staining for CD62L, lower panels represent LAIR-1 staining. <i>Panel</i> B: Western blots containing whole platelet lysates and controls were incubated with antibodies against LAIR-1 (<i>lanes 1–3</i>), LAIR-2 (<i>lanes 4–8</i>) or GpVI (<i>lane 9</i>). <i>Lane 1:</i> purified LAIR-1/Fc; <i>lane 2:</i> purified LAIR-2/Fc; <i>lane 3:</i> whole platelet lysate; <i>lane 4:</i> purified LAIR-1/Fc; <i>lane 5:</i> purified LAIR-2/Fc; <i>lane 6:</i> conditioned medium of non-transfected human 293T cells; <i>lane 7:</i> conditioned medium of human 293T cells secreting LAIR-2; <i>lane 8:</i> whole platelet lysate; <i>lane 9:</i> whole platelet lysate.</p