The inhibitory monoclonal anti-ERp57 Ab inhibits platelet aggregation, activation of αIIbβ3, P-selectin expression, and prolongs the tail bleeding times and the time to thrombosis in mice. (A) Representative tracings show Mab1 but not Mab2 inhibits aggregation of human platelets stimulated with collagen (i), the peptide SFLLRN (ii), or calcium ionophore (ii; A23187). (B) The combined data ± SE for at least 3 separate experiments (for collagen (i) and SFLLRN (ii), Mab1 at 10 μg/mL also inhibited aggregation to a P < .05 compared with either the IgG2a or Mab2 controls.) Calcium ionophore (iii) was used with platelets preincubated with apyrase (10 U/mL) and MeSAMP (100μM). (Ci) Mab1 inhibits PAC1 binding to SFLLRN (1μM)–activated platelets. PAC1 binding to nonactivated platelets (NA) is seen on the left. (ii) The results of inhibition of PAC1 binding as the percentage of fluorescent intensity relative to the normal IgG2a control ± SE (n = 3). (Di) A representative histogram and (ii) the combined data show the inhibitory monoclonal anti-ERp57 Ab Mab1 inhibits P-selectin binding to convulxin (10 ng/mL)–activated platelets relative to normal IgG2a ± SE (n = 7). (A-D) The platelets were preincubated with the inhibitory mAb (Mab1), noninhibitory Ab (Mab2), or control mouse IgG2a at 30 μg/mL for 10 minutes before the addition of the agonist. (E) A total of 200 μg of each Ab was infused into mice and the tail bleeding time recorded up to 15 minutes. Horizontal bars represent mean bleeding times. (Fi) The systolic and diastolic mouse carotid artery blood velocity monitored in mm of blood per second (mm/s) using the small animal Doppler probe with the Visual Sonics Vevo2100 flowmeter. (ii) Complete occlusion of blood flow after treatment with FeCl₃. (iii) Mab1 inhibits FeCl₃-induced occlusion relative to the normal mouse IgG2a control (n = 8). In these experiments, the carotid artery was treated with 5% FeCl₃ for 2 minutes as described in “Studies using FaCl₂-induced thrombosis of the carotid artery. A total of 450 μg of the control IgG2a or Mab1 were infused immediately before the filter paper soaked in 5% FeCl₃ was applied to the carotid artery.”