A role for the thiol isomerase protein ERP5 in platelet function by Peter A. Jordan, Joanne M. Stevens, Gary P. Hubbard, Natasha E. Barrett, Tanya Sage, Kalwant S. Authi, and Jonathan M. Gibbins Blood Volume 105(4):1500-1507 February 15, 2005 ©2005 by American Society of Hematology

Localization of ERP5 and specificity of anti-ERP5 antibodies. Localization of ERP5 and specificity of anti-ERP5 antibodies. (A) Localization of ERP5 to intracellular and plasma membranes of human platelets. Human (IM 40 μg) and PM (80 μg) membranes from resting platelets prepared by high-voltage free-flow electrophoresis were separated by SDS-PAGE and immunoblotted. ERP5 protein was detected by means of specific polyclonal antibodies. (B) Specificity of anti-ERP5 antibodies. Recombinant human PDI (4 μg) and human platelet lysates (4 μg and 20 μg) were Western blotted and probed for ERP5 (i) and PDI (ii) by means of sheep polyclonal anti-ERP5 and mouse monoclonal anti-PDI, respectively. Peter A. Jordan et al. Blood 2005;105:1500-1507 ©2005 by American Society of Hematology

Increase in ERP5 cell-surface expression in a concentration- and time-dependent manner in response to platelet stimulation by convulxin, collagen, or thrombin. Increase in ERP5 cell-surface expression in a concentration- and time-dependent manner in response to platelet stimulation by convulxin, collagen, or thrombin. (A) Concentration dependence with 90-second stimulation. (i) Stimulation by convulxin (Cvx). Basal level is indicated by filled histogram; stimulation with 10 ng/mL Cvx, by an empty histogram; and stimulation with 40 ng/mL Cvx, by an empty dotted-line histogram. (ii) Stimulation with collagen (Coll). Basal level is indicated by a filled histogram; stimulation with 25 μg/mL Coll, by an empty histogram; and stimulation with 100 μg/mL Coll, by an empty dotted-line histogram. (iii) Stimulation with thrombin (Thr). Basal level is indicated by filled histogram; stimulation with 0.2 U/mL Thr, by an empty histogram; and stimulation with 1.0 U/mL Thr, by an empty dotted-line histogram. (B) Time dependence. (i) Basal level is indicated by filled histogram; 45-second stimulation with 40 ng/mL Cvx by an empty histogram; and 300-second stimulation with 40 ng/mL Cvx, by an empty dotted-line histogram. (ii) Stimulation by Coll. Basal level is indicated by a filled histogram; 45-second stimulation with 25 μg/mL Coll by an empty histogram; and 300-second stimulation with 25 μg/mL Coll, by an empty dotted-line histogram. (iii) Stimulation by Thr. Basal level is indicated by a filled histogram; 45-second stimulation with 1.0 U/mL Thr, by an empty histogram; and 300-second stimulation with 1.0 U/mL Thr, by an empty dotted-line histogram. (C) Normalized plots for the increase in cell-surface exposure observed for ERP5 over time are given for convulxin (i), collagen (ii), and thrombin (iii). Data are presented as mean ± standard error (SE) (n = 3), where 100% represents the maximal response detected. Peter A. Jordan et al. Blood 2005;105:1500-1507 ©2005 by American Society of Hematology

Thiol isomerase activity of human ERP5. Thiol isomerase activity of human ERP5. Thiol isomerase activity was assessed as the ability to refold denatured, scrambled RNAse and enhance the degradation of cyclic 2′,3′-cytidine monophosphate, as followed by ultraviolet/visible (UV/vis) spectroscopy. (A) Activity was observed for a recombinant ERP5 fusion protein (21 μg/mL) and a recombinant PDI fusion protein (11 μg/mL) relative to samples containing only cyclic 2′,3′-cytidine monophosphate (blank) or only fusion protein. (B) Anti-ERP5 polyclonal antibodies (24 μg/mL) raised in sheep were able to partially inhibit the thiol isomerase activity of a recombinant ERP5 fusion protein (30 μg/mL). Preimmune IgG (24 μg/mL) and monoclonal anti-PDI antibodies (28 μg/mL) were found to possess no such inhibitory activity. Data are presented as mean ± SE from 3 different determinations. *P < .05. Peter A. Jordan et al. Blood 2005;105:1500-1507 ©2005 by American Society of Hematology

Inhibition of platelet aggregation by an inhibitory antibody for ERP5. Inhibition of platelet aggregation by an inhibitory antibody for ERP5. Platelets (4 × 108/mL) were incubated with anti-ERP5 IgG or control IgG at the given concentrations for 2.5 minutes prior to addition of agonist. Prior to the addition of antibodies, platelets were preincubated with a saturating concentration of an F(ab′) fragment of the IV.3 protein to prevent signaling through the FcγRIIa receptor. Control IgG was purified from the preimmune serum of the animal in which antibodies were raised. (A) Concentration effect of inhibitory antibodies. (i) Concentration effect of 2.5 μg/mL collagen incubated with preimmune IgG (36 μg/mL); anti-ERP5 antibody (6 μg/mL); or anti-ERP5 antibody (36 μg/mL). (ii) Concentration effect of 30 ng/mL convulxin incubated with preimmune IgG (24 μg/mL); anti-ERP5 antibody (12 μg/mL); or anti-ERP5 antibody (24 μg/mL). (B) Additive effect of inhibitory antibodies for ERP5 and PDI. (i) Additive effect of 4.0 μg/mL collagen incubated with preimmune IgG (36 μg/mL); anti-ERP5 (12 μg/mL) plus preimmune IgG (19 μg/mL); anti-PDI (6 μg/mL) plus preimmune IgG (19 μg/mL); or anti-ERP5 (12 μg/mL) plus anti-PDI (6 μg/mL). (ii) Additive effect of 150 ng/mL convulxin incubated with preimmune IgG (36 μg/mL); anti-ERP5 (12 μg/mL) plus preimmune IgG (19 μg/mL); anti-PDI (6 μg/mL) plus preimmune IgG (19 μg/mL); or anti-ERP5 (12 μg/mL) plus anti-PDI (6 μg/mL). Traces shown are representative of those observed for at least 3 different donors. (C) Mobilization of calcium from intracellular stores. Ca2+ release was measured by Fura-2 fluorescence in platelets stimulated with convulxin (300 ng/mL, ▪) or collagen (4 μg/mL, ▦). Prior to stimulation, platelets were incubated with anti-ERP5 antibodies or control antibodies (24 μg/mL) from preimmune sera. Data are presented as mean ± SE for 3 separate experiments. Peter A. Jordan et al. Blood 2005;105:1500-1507 ©2005 by American Society of Hematology

Inhibition of fibrinogen binding in platelets following blocking of cell-surface ERP5. Inhibition of fibrinogen binding in platelets following blocking of cell-surface ERP5. Binding of FITC-labeled fibrinogen was measured with the use of flow cytometry on platelets stimulated with the agonists convulxin or collagen. Prior to stimulation, platelets were incubated with anti-ERP5 antibodies, anti-PDI antibodies, or control antibodies from preimmune sera. (A) Histogram for fluorescence of FITC-fibrinogen labeled platelets in response to the agonist convulxin (100 ng/mL). Control IgG (12 μg/mL) is indicated by a filled histogram; anti-ERP5 antibody (12 μg/mL), by an empty histogram; and anti-PDI antibody (33 μg/mL), by a dotted-line empty histogram. (Bi) Residual binding of FITC-fibrinogen following incubation of platelets with preimmune IgG (12 μg/mL), anti-ERP5 antibody (12 μg/mL), or anti-PDI antibody (33 μg/mL). Agonists were 100 ng/mL convulxin. (Bii) Additive effect of inhibitory antibodies on residual binding of FITC-fibrinogen following incubation of platelets with preimmune IgG (36 μg/mL); anti-ERP5 (12 μg/mL) plus preimmune IgG (19 μg/mL); anti-PDI (6 μg/mL) plus preimmune IgG (19 μg/mL); or anti-ERP5 (12 μg/mL) plus anti-PDI (6 μg/mL). Agonists were 300 ng/mL convulxin. (Ci) Residual binding of FITC-fibrinogen following incubation of platelets with antibodies as described for panel Bi. Agonists were 10 μg/mL collagen. (Cii) Additive effect of inhibitory antibodies on residual binding of FITC-fibrinogen following incubation of platelets with antibodies as described for panel Bii. Agonists were 4 μg/mL collagen. Data are presented as mean ± SE for 4 separate experiments (*P < .05; **P < .005). Peter A. Jordan et al. Blood 2005;105:1500-1507 ©2005 by American Society of Hematology

Inhibition of P-selectin expression in platelets following blocking of cell-surface ERP5. Inhibition of P-selectin expression in platelets following blocking of cell-surface ERP5. Binding of PE-conjugated anti-CD62p was measured by means of flow cytometry on platelets stimulated with the agonists convulxin or collagen. Prior to stimulation, platelets were incubated with anti-ERP5 antibodies, anti-PDI antibodies, or control antibodies from preimmune sera. (A) Histogram for fluorescence of PE anti-CD62p–labeled platelets in response to the agonist convulxin (100 ng/mL). Control IgG (12 μg/mL) is indicated by a filled histogram; anti-ERP5 antibody (12 μg/mL), by an empty histogram; and anti-PDI antibody (33 μg/mL), by a dotted-line empty histogram. (B,C) Data are presented as mean ± SE for 4 separate experiments. *P < .05. **P < .005. (Bi,Ci) Residual binding of PE anti-CD62p following incubation of platelets with preimmune IgG (12 μg/mL), anti-ERP5 antibody (12 μg/mL), anti-PDI antibody (33 μg/mL). Agonists were 100 ng/mL convulxin (Bi); and 10 μg/mL collagen (Ci). (Bii,Cii) Additive effect for inhibitory antibodies on residual binding of PE anti-CD62p following incubation of platelets with preimmune IgG (36 μg/mL); anti-ERP5 (12 μg/mL) plus preimmune IgG (19 μg/mL); anti-PDI (6 μg/mL) plus preimmune IgG (19 μg/mL); or anti-ERP5 (12 μg/mL) plus anti-PDI (6 μg/mL). Agonists were 300 ng/mL convulxin (Bii) and 4 μg/mL collagen (Cii). Peter A. Jordan et al. Blood 2005;105:1500-1507 ©2005 by American Society of Hematology

Stimulation-dependent association of ERP5 with integrin β3. Stimulation-dependent association of ERP5 with integrin β3. Platelets (1 × 109/mL) were stimulated in the presence of EGTA, apyrase, and indomethacin at varying concentrations of convulxin (ng/mL) or thrombin (U/mL) for 90 seconds (panel A), or at fixed concentrations of agonist (100 ng Cvx, 1.0 U Thr) for increasing durations (panel B). Following sample lysis, proteins were precipitated and separated with specific antibodies and protein A–sepharose. Immunoblotting was used to show interacting proteins. Blots were stripped and reprobed to verify equivalent levels of target antigen in each sample lane. Peter A. Jordan et al. Blood 2005;105:1500-1507 ©2005 by American Society of Hematology