Inhibition of collagen-induced platelet aggregation by anopheline antiplatelet protein, a saliva protein from a malaria vector mosquito by Shigeto Yoshida, Toshiki Sudo, Masashi Niimi, Lian Tao, Bing Sun, Junichi Kambayashi, Hiroyuki Watanabe, Enjie Luo, and Hiroyuki Matsuoka Blood Volume 111(4):2007-2014 February 15, 2008 ©2008 by American Society of Hematology

Female salivary gland–specific expression of AAPP Female salivary gland–specific expression of AAPP. (A) Western blotting for AAPP in the salivary glands of female mosquitoes. Female salivary gland–specific expression of AAPP. (A) Western blotting for AAPP in the salivary glands of female mosquitoes. A homogenate of 2 pairs of female salivary glands was subjected to 10% SDS-PAGE. Lane 1, silver staining; lane 2, Western blotting using a mouse anti-rAAPP immune serum. (B) RT-qPCR of aapp mRNA. Total RNA was isolated from salivary glands (SG) and carcasses (Ca) of male and female mosquitoes. The expression of aapp mRNA was examined for its tissue- and sex-specificity using RT-qPCR. The fold induction was determined from threshold cycle values normalized for ubiquitin mRNA expression and then normalized to the values of each gene from the male salivary gland mRNA. (C) Immunostaining of salivary glands with the anti-rAAPP immune serum. The paired salivary glands of mosquitoes are present in the thorax flanking the esophagus. Each gland consists of 3 lobes: a distal lateral lobe (DL), proximal lateral lobe (PL), and medial lobe (M). (i) phase-contrast image of the salivary glands of a wild-type mosquito; (ii) immunostaining of the salivary glands of a wild-type mosquito with the anti-rAAPP immune serum; (iii) phase-contrast image of the salivary glands of a DsRed transgenic mosquito; and (iv) DsRed expression in the salivary glands of a DsRed transgenic mosquito. Scale bars: 100 μm. Shigeto Yoshida et al. Blood 2008;111:2007-2014 ©2008 by American Society of Hematology

AAPP specifically inhibits collagen-induced platelet aggregation. AAPP specifically inhibits collagen-induced platelet aggregation. PRP were incubated with 1000 nM rAAPP for 2 minutes followed by the addition of various platelet aggregation agonists, collagen (1.5 μg/mL), ADP (8 μM), TRAP (30 μM), epinephrine (0.5 μg/mL), PAF (0.125 μg/mL), U46619 (1 μM), and A23187 (20 μM). Tracings are representative results of a typical experiment. Shigeto Yoshida et al. Blood 2008;111:2007-2014 ©2008 by American Society of Hematology

AAPP specifically inhibits collagen-induced platelet aggregation by binding to collagen. AAPP specifically inhibits collagen-induced platelet aggregation by binding to collagen. (A) AAPP does not inhibit GPVI agonist-mediated platelet aggregation. PRP was incubated with the indicated concentrations of AAPP for 2 minutes followed by the addition of collagen (2 μg/mL). In the presence of 1000 nM rAAPP, PRPs were stimulated by CRP (0.5 μg/mL) or convulxin (10 ng/mL). As a control, the indicated concentrations of rAAPP were incubated in the absence of CRP or convulxin. Tracings are representative results of a typical experiment. (B) Dose-dependent inhibition of collagen-induced platelet aggregation by AAPP. PRPs were incubated with various amounts of rAAPP for 2 minutes followed by the addition of collagen. Results are means plus or minus SEM from 3 independent experiments. (C) Binding of AAPP to immobilized collagen. Various concentrations of rAAPP or rTrx were incubated in 96-well collagen-coated plates for 1 hour. After washing, bound proteins were detected using the Nickel-HRP. (D) Effect of AAPP on platelet adhesion to collagen. Washed platelet suspension and the indicated amounts of rAAPP were added to 96-well collagen-coated plates and incubated for 1 hour. After washing, adherent platelets were detected by the Dc protein assay kit. Results are means plus or minus SEM from 3 independent experiments. (E) GPVI binds to collagen but not AAPP. The indicated amounts of collagen or rAAPP were coated in 96-well plates. GPVI-expressing Jurkat cells were added to each well and incubated for 1 hour. After washing, adhesion cells were measured by the BCA protein assay. Results are representative of 3 independent experiments and expressed as the mean of tripli-cate reading plus or minus SEM for the indicated concentrations. Shigeto Yoshida et al. Blood 2008;111:2007-2014 ©2008 by American Society of Hematology

AAPP inhibits both GPVI- and integrin α2β1-mediated platelet adhesion to collagen. AAPP inhibits both GPVI- and integrin α2β1-mediated platelet adhesion to collagen. The indicated concentrations of rAAPP were incubated in 96-well plates coated with fibrillar type I collagen in the absence of Mg2+ (A) or with nonfibrillar type I collagen in the presence of Mg2+ (B) for 1 hour. After washing, washed platelet suspensions were allowed to adhere under static conditions to each well for 1 hour. After washing, adhesion platelets were quantified fluorometrically. As positive controls, washed platelet suspension were preincubated with antihuman GPVI mAb, OM-2 (1 μg/mL) (A) or anti-(human integrin α2-subunit) mAb, 6F1 (5 μg/mL) (B). Results are representative of 3 independent experiments and expressed as the mean of triplicate reading plus or minus SEM for the indicated concentrations. Shigeto Yoshida et al. Blood 2008;111:2007-2014 ©2008 by American Society of Hematology

AAPP inhibits GPVI-mediated platelet adhesion to collagen types I, III, but not type IV. Different types of collagen (types I, III, and IV) were coated in 96-well plates. AAPP inhibits GPVI-mediated platelet adhesion to collagen types I, III, but not type IV. Different types of collagen (types I, III, and IV) were coated in 96-well plates. The indicated concentrations of rAAPP were added to each well and incubated for 1 hour. After washing, GPVI-expressing Jurkat cells were allowed to adhere to each well for 1 hour. After washing, adhesion cells were quantified fluorometrically. The top box indicates the concentration of rAAPP (μg/mL). Results are representative of 3 independent experiments and expressed as the mean of triplicate reading plus or minus SEM for the indicated concentrations. Shigeto Yoshida et al. Blood 2008;111:2007-2014 ©2008 by American Society of Hematology

Effect of AAPP on [Ca2+]i in the early phase of collagen stimulation. Effect of AAPP on [Ca2+]i in the early phase of collagen stimulation. Fura-2 AM-loaded platelets were incubated with 1 mM CaCl2 for 5 minutes. Collagen (0.5 μg/mL) was added after a 60-second incubation with rAAPP (30, 100 or 300 nM), and the fura-2 fluorescence was determined. Tracings are representative results of a typical experiment. Shigeto Yoshida et al. Blood 2008;111:2007-2014 ©2008 by American Society of Hematology

Inhibition of ex vivo collagen-induced aggregation of rat platelets after injection of AAPP. SD rats were administered rAAPP (0.1, 0.3, or 1.0 mg/kg) intravenously, and blood was drawn 10 minutes after the administration. Inhibition of ex vivo collagen-induced aggregation of rat platelets after injection of AAPP. SD rats were administered rAAPP (0.1, 0.3, or 1.0 mg/kg) intravenously, and blood was drawn 10 minutes after the administration. Treatment with rAAPP results in inhibition of platelet aggregation in response to collagen, compared with treatment with PBS and rTrx, but has no effect on the ADP response. Results are means plus or minus SEM for 3 animals in each group. Shigeto Yoshida et al. Blood 2008;111:2007-2014 ©2008 by American Society of Hematology