Isolation of mouse monocytes: Mouse monocytes were isolated using a modified

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1 Supplemental Material Extended Methods Isolation of mouse monocytes: Mouse monocytes were isolated using a modified method described (1). Citrated mouse whole blood was mixed with equal volume of PBS, and was laid over Histopaque -183 (Sigma-Aldrich). After centrifugation at 4g for 3 minutes, the cells from interface containing mononuclear cells were collected. After washing with PBS, the cell suspension was resuspended with PBS containing 2% BSA and 2 mm EDTA, followed by incubation with PE-conjugated antibodies against B22, CD49b, NK1.1, Ly6G, CD9, CD45R, and Ter-119 for removal of non-monocyte leukocytes. After washing with PBS to remove unlabeled antibodies, the cells were mixed with anti-pe antibody magnetic beads (Miltenyi). After the labeled cells were removed using a MACS Cell Separation column in the magnetic field of a MACS Separator (Miltenyi Biotec), and the unlabeled cells (monocytes) were collected. Isolation of mouse neutrophils: Mouse neutrophils were isolated from peripheral blood using a commercial kit (Miltenyi, Cat# ) by following the manufacturer s instruction. In brief, after removal of red blood cell using lysis buffer, the blood cells were incubated with biotin-labeled antibody cocktail negative for neutrophils. The biotin-labeled cells were removed by anti-biotin MicroBeads (Miltenyi Biotec) on a MS column in the magnetic field of a MACS Separator (Miltenyi Biotec), and the unlabeled neutrophils were collected. 1

2 Generation of rabbit anti-pdi antibody: PDI was purified from platelets as previously reported (2). A rabbit anti-pdi antibody was commercially generated against PDI in white New Zealand rabbits (Harlan) and purified on a protein A column. Measurement of plasma PDI levels: Plasma PDI was measured using the IMMUNOSET PDI ELISA development kit (Enzo) according to the manufacturer s instructions. Visualization of leukocyte accumulation at the site of vessel injury: Leukocytes at the site of laser-induced injury were visualized using two methods. Firstly, FITC anti-cd11b (.2 µg/g BW) (clone M1/7, BD Bioscience) was administered intravenously 1 minutes prior to injury (3). Secondly, to stain the nucleus of the leukocytes, Hoechst (2.5mg/kg, Invitrogen) was administered as described (4). Visualization of labeled leukocytes in non-injured venules was used to ensure adequate labeling of leukocytes for both techniques. Leukocyte rolling and adhesion was monitored for at least 4 minutes post injury using confocal imaging microscopy, as previously described (5). Confocal imaging studies of PDI at the site of injury: rpdi(oo-ss) was labeled with Alexa 488. The labeling did not affect the enzymatic activity of rpdi(oo-ss) in the Di-E-GSSG PDI assay (data not shown). Alexa-rPDI(oo-ss) (.3 µg/g BW) was administered intravenously 1 minutes prior to the laser-induced arteriole injury. 2

3 Platelets were detected using the anti-cd41 F(ab) 2 fragments conjugated to Alexa 647. rpdi(oo-ss) and platelet accumulation were monitored for at least 3 minutes using confocal imaging microscopy (5). 3

4 Supplemental Figures A 1 Thrombin (.3 U/ml) RL9 (3 µg/ml) RL9 (1 µg/ml) IgG (3 µg/ml) B IgG (3 µg/ml) RL9 (1 µg/ml) RL9 (3 µg/ml) ** Time (min) C EGSH (nm) PDI Time (min) (µm) 3 9 D EGSH (nm) ERp Time (min) (µm) 3 9 Supplemental Figure 1. The monoclonal anti-pdi antibody RL9 inhibits aggregation of PDI-null platelets. (A) Representative tracings show RL9 at the concentrations shown but not the isotype specific control IgG inhibits aggregation of PDI-null platelets; (B) Cumulative data; n = 3, mean ± SE, **P <.1, P <.1, ANOVA. The antibodies were incubated with the platelets for 2 minutes prior to addition of thrombin. (C and D) Quercetin-3-rutinoside in the concentrations shown inhibits ERp57 in the Di-E-GSSG assay. Inhibition of PDI (2 nm) (C) and ERp57 (2 nm) (D). The curves are composites of 3 separate experiments. 4

5 A Convulxin (6 ng/ml) Pf4-Cre/! 1! 1! 2! 3! 4! 5! Time (min)!! 12 P = ! Pf4-Cre/! B Thrombin (.8 U/ml) 1! 1! 2! 3! 4! 5! Time (min)! Pf4-Cre/!! !! Pf4-Cre/! C Convulxin (1ng/ml) 1! 1! 2! 3! 4! 5! Time (min)! Pf4-Cre/!! !! Pf4-Cre/! Supplemental Figure 2. Aggregation of PDI-null platelets. Platelet activation was induced by (A) convulxin, n = 5, (B) Thrombin, n = 3, and (C) convulxin, n = 3, at the indicated doses and compared to aggregation of platelets from Crenegative ( ) littermate mice. Representative tracings (left) and combined aggregation data (right); mean ± SE, t-test. 5

6 A EC! Monocytes Neutrophils kda 148 B kda 25 C PLCγ2 PDI ERp ERp WT WT WT Platelets PDI ERp57 ERp72 ERp5 MFI D non-activated ** E MFI ** ** MFI WT WT WT FITC-IgG FITC-anti PDI NA Act - Thrombin NA Act - Convulxin F Plasma PDI (ng/ml) WT G PT (s) WT!! APTT (s) WT!! Supplemental Figure 3. Characterization of endothelial, blood cells and plasma of the transgenic mice. (A) Western blots of isolated endothelial cells (EC), neutrophil, and monocytes for PDI, ERp57, and ERp72. Shown are the PLCγ2 loading controls for PDI. Separate loading controls were run for ERp57 and ERp72 with similar amounts of protein found in each sample (not shown). Western blots using rabbit anti-pdi antibody of (B) platelets and (C) purified PDI, ERp57, ERp72 and ERp5. Blots are representative of 3 separate experiments. (D) Binding of FITC-labeled rabbit anti-pdi antibody and FITC-labeled control rabbit antibody (IgG) to non-activated 6

7 platelets; mean ± SE, n = 4, **P <.1, P <.1, ANOVA. (E) Binding of FITC-rabbit anti-pdi antibody to non-activated (NA) and activated (Act) platelets: Left panel: thrombin (2 U/ml) (n=3), and right panel: convulxin (5 ng/ml) (n = 3); mean ± SE, **P <.1, P <.1, ANOVA. (F) Plasma PDI levels in WT and transgenic mice; mean ± SE, n = 5, t-test. (G) PT (left) and APTT (right) on plasma from wild type (WT) and transgenic mice []. The PT and APTT were performed as described in methods on plasma obtained from the transgenic mice and WT control mice; mean ± SE, n = 1, t-test. 7

8 A! 1 B!! 1 C!! 1! Convulxin (4ng/ml) 1! 2! 3! 4! 5! Time (min)! Thrombin (.8U/ml) 1! 2! 3! 4! 5! Time (min)! Convulxin (8ng/ml) 1! 2! 3! 4! 5! Time (min)!! WT!! WT!! WT! P =.6! WT!!! WT!!! WT!! Supplemental Figure 4. Aggregation of platelets from transgenic and wild type (WT) mice. Platelet activation was induced by (A) convulxin (n = 5). (B) Thrombin (n = 3), and (C) convulxin (n = 3) at the indicated doses. Representative tracings (left) and combined aggregation data (right); mean ± SE, t-test. 8

9 Count! A NA! PAC-1! Activated! B NA! P-selectin! Activated! C P-selectin! Activated + r Activated 15 1 MFI! 5 P= ! 1 1! 1 2! 1 3! 1 4! 1! 1 1! 1 2! 1 3! 1 4! FITC-PAC1! PE-CD62p! D Non-activated (NA) Activated Activated + Eptifibatide 1! 1 1! 1 2! 1 3! 1 4! PE-CD62p! PBS rpdi (ss-oo)! Activated! Time (min)! PBS r 1 nm ATP % ATP secretion 1 5 PBS P=.1 r 1 5 PBS r Supplemental Figure 5. P-selectin expression in human platelets is independent of αiibβ3 and dependent on the second active site of PDI. (A and B) Platelets were pretreated with or without 4 mm eptifibatide, followed by activation with.1 U/ml thrombin. PAC1 binding (A) and P-selectin expression (B) were measured by flow cytometry. Histograms in A and B are representative of 3 separate experiments. (C) Platelets were pretreated with 2 mm r and activated with.1 U/ml thrombin. Representative histogram (left) and combined data (right); mean ± SE, n = 3, t-test. (D) Platelets were pretreated with or without 1 mm r for 5 minutes prior to activation with thrombin (.2 U/ml). Aggregation and ATP secretion were monitored in the lumi-aggregometer. Representative aggregation tracings with ATP release (left) and combined data of ATP secretion and aggregation (right, n = 3); mean ± SE, t-test. 9

10 A β-actin PDI! ERp72! ERp57 Platelets B Band density of PDI 15 P< PDI ERp57 ERp72 β-actin C Count D EC 8 7 control 6 CD31! β-actin PDI! ERp72! ERp57 EC PE E Band density of PDI 5 Count EC VE-Cadherin! control APC 15 P<.1 1 PDI ERp57 ERp72 β-actin Count EC 8 7 control 6 Flk1! PE F PDI! ERp72! ERp57 Fibroblasts G Band density of PDI P<.1 PDI ERp57 ERp72 H PLCγ2 PDI ERp72 ERp57 Neutrophils! Monocytes! I Plasma PDI (ng/ml)! P=.2 Supplemental Figure 6. Characterization of PDI deficiency in vascular wall, blood cells and plasma of mice. (A) Reprehensive Western blot of platelet lysates using the polyclonal anti-pdi antibody (with β-actin control), and antibodies against ERp72 and ERp57. (B) Quantitative analysis of protein level by densitometry band density of PDI (% of WT control); mean ± SE, n = 4, t-test. (C) Flow cytometry analysis of isolated endothelial cells for expression of CD31, VE-cadherin, Flk1, compared to isotype specific control antibodies. Histograms are representative of 3 separate experiments. (D) Western blot for PDI (with β-actin control), ERp72 and ERp57 in isolated mouse endothelial cells. (E) Quantitative analysis of EC protein level by 1

11 densitometry; mean ± SE, n = 5, t-test. (F) Western blot for PDI, ERp72 and ERp57 in isolated fibroblasts. (G) Quantitative analysis; mean ± SE, n = 3, t-test. (H) Western blot of neutrophils and monocytes for PDI (with PLCγ2 control), ERp72 and ERp57. Blots are representative of 3 separate experiments. Separate β-actin or PLCγ2 loading controls were run for ERp57 and ERp72 in A, D and H with similar amounts of protein found in each sample (not shown). (I) Plasma PDI levels; mean ± SE, n = 4, t-test. 11

12 A Platelet Count 1 9 /L C B MFI αiibβ GpIb GpVI PT (s) 1 5 APTT (s) Supplemental Figure 7. Platelet counts, platelet glycoprotein expression and coagulation times in mice. (A) Platelet counts in Mx1- Cre/ mice and littermate control mice treated identically were measured a week after the last treatment with poly(i:c); mean ± SE, n = 1, t-test. (B) Glycoprotein (αiibβ3, GPIb or GPVI) expression on platelets from and littermate controls was analyzed by flow cytometry; mean ± SE, n = 1, t-test. (C) PT and PTT on plasma obtained from Mx1- Cre/ mice and littermate controls; mean ± SE, n = 1, t-test. 12

13 A! WT 12 + rerp57(ss-oo) 2µg B! WT 3 + rerp57(ss-oo) 2µg Fl platelet (x1 4 ) 8 4 ** Fl Fibrin(x1 4 ) 2 1 * * C! Fl platelet (x1 4 ) Time (seconds) + rerp57(ss-oo) 2µg * D! Fl Fibrin(x1 4 ) Time (seconds) + rerp57(ss-oo) 2µg * Time (seconds) Time (seconds) E! ERp57 fl/fl Tie2-Cre/ERp57 fl/fl F! ERp57 fl/fl Tie2-Cre/ERp57 fl/fl 12 Tie2-Cre/ERp57 fl/fl + r 2µg 3 Tie2-Cre/ERp57 fl/fl + r 2µg Fl platelet (x1 4 ) 8 4 * * Time (seconds) Time (seconds) Fl Fibrin(x1 4 ) 2 1 Supplemental Figure 8. Distinct roles for the C-terminal active site of PDI and ERp57 in platelet accumulation and fibrin deposition. Infusion of rerp57(ss-oo) into transgenic mice (A and B) or mice (C and D) results in further inhibition of platelet accumulation and fibrin deposition. Infusion of r into Tie2-Cre/ERp57 fl/fl mice further inhibits platelet accumulation (E) and fibrin deposition (F). Shown are the median integrated fluorescence intensities (FI) of anti-cd41 (platelets) and anti-fibrin (fibrin) antibodies. Data were analyzed as described for Figure 9 with only significant differences shown; *P <.5; **P <.1; P <.1, Mann-Whitney rank sum test. The data were obtained from 3 thrombi in 3 mice for each experimental condition. 13

14 A! 12 + rerp57(oo-ss) 2 µg B! 3 + rerp57(oo-ss) 2 µg Fl platelet (x1 4 ) 8 4 P=.17 Fl Fibrin(x1 4 ) 2 1 P= Time (seconds) Time (seconds) C! 12 Tie2-Cre/ERp57 fl/fl Tie2-Cre/ERp57 fl/fl + rpdi(oo-ss) 2 µg D! Tie2-Cre/ERp57 fl/fl 3 Tie2-Cre/ERp57 fl/fl + rpdi(oo-ss) 2 µg Fl platelet (x1 4 ) Time (seconds) P=.35 Fl Fibrin(x1 4 ) Time (seconds) P=.21 Supplemental Figure 9. Lack of significant recovery of thrombosis by infusion of rerp57(oo-ss) into mice and infusion of rpdi(oo-ss) into Tie2-Cre/ERp57 fl/fl mice. Infusion of rerp57(oo-ss) into Mx1- Cre/ mice did not result in significant recovery of platelet accumulation (A) or fibrin deposition (B). Infusion of rpdi(oo-ss) into Tie2-Cre/ERp57 fl/fl mice did not result in significant recovery of platelet accumulation (C) and fibrin deposition (D). Shown are the median integrated fluorescence intensities (FI) of anti-cd41 (platelets) and anti-fibrin (fibrin) antibodies. Data were analyzed as described for Figure 9 without significant differences for any of the comparisons, Mann- Whitney rank sum test. The data were obtained from 3 thrombi in 3 mice for each experimental condition. 14

15 Supplemental Videos Supplemental Video 1. Visualization of leukocytes in venules using anti- CD11b The FITC-anti-CD11b antibody was administered intravenously to detect circulating leukocytes. The microvenule circulation of the cremaster muscle was visualized. Endogenous leukocytes were seen moving through the venule. Confocal images were acquired using a 6X objective at 1 frame/second. Signal of CD11b labeling is shown in green. Supplemental Video 2. Visualization of leukocytes in arterioles using anti- CD11b To detect leukocytes at the site of laser-induced injury, the anti-cd11b antibody was administered intravenously 1 minutes prior to vessel wall injury. The arteriole circulation of the cremaster muscle was visualized at the site of the laser pulse. Confocal images were acquired using a 6X objective at 1 frame/second after laser injury. A representative video from 1 separate injuries is shown. Platelets labeled with Alexa 647 anti-cd41fab are shown in red and leukocytes labeled with FITC-anti-CD11b antibody are shown in green. Leukocyte rolling and adhesion was monitored for 8 minutes post injury. Supplemental Video 3. Visualization of leukocytes in venules using staining of leukocyte nuclei Hoescht was infused intravenously 1 minutes prior to laser injury. The microvenule circulation of the cremaster muscle was visualized. Endogenous leukocytes were seen moving through the venule. Confocal images were acquired using a 6X objective at 1 frame/second. Hoescht signal is shown in blue. Supplemental Video 4. Visualization of leukocytes in arterioles using staining of leukocyte nuclei Hoescht was infused intravenously 1 minutes prior to laser injury. The arteriole circulation of the cremaster muscle was visualized at the site of the laser pulse. Confocal images were acquired using a 6X objective at 1 frame/second after laser injury. Shown is a representative video from 1 separate injuries. Platelets are shown in red and Hoescht signal is shown in blue. Leukocyte rolling and adhesion was monitored for 1 minutes post injury. Supplemental Video 5. Visualization of rpdi(oo-ss) accumulation at the site of injury in wild type mice 15 µg of Alexa 488 rpdi(oo-ss) together with 185 µg of unlabeled rpdi(oo-ss) was infused into C57Bl/6 mice 5 minutes prior to laser injury of the cremaster arteriole. Alexa 647 anti-cd41 F(ab)2 was used to visualize platelets. Confocal images were acquired using a 6X objective at 1 frame/second after laser injury. Shown is a representative video from 1 separate injuries. Platelets are shown in red and rpdi(oo-ss) deposition is shown in green. rpdi(oo-ss) and platelet accumulation were monitored for 3 minutes. 15

16 Supplemental Video 6. Visualization of rpdi(oo-ss) accumulation at the site of injury in Pf4-Cre/ mice 15 µg of Alexa 488 rpdi(oo-ss) together with 185 µg of unlabeled rpdi(oo-ss) was infused into Pf4-Cre/ mice 5 minutes prior to laser injury of the cremaster arteriole. Alexa 647 anti-cd41 F(ab)2 was used to visualize platelets. Confocal images were acquired using a 6X objective at 1 frame/second after laser injury. Shown is a representative video from 9 separate injuries. Platelets are shown in red and rpdi(oo-ss) deposition is shown in green. rpdi(oo-ss) and platelet accumulation were monitored for 3 minutes. 16

17 Supplemental References 1. Swirski, FK, et al. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science. 29;325(594): Chen, K, Detwiler, TC, and Essex, DW. Characterization of protein disulphide isomerase released from activated platelets. Br J Haematol. 1995;9(2): Darbousset, R, et al. Tissue factor-positive neutrophils bind to injured endothelial wall and initiate thrombus formation. Blood. 212;12(1): Nishimura, S, et al. In vivo imaging visualizes discoid platelet aggregations without endothelium disruption and implicates contribution of inflammatory cytokine and integrin signaling. Blood. 212;119(8):e Rauova, L, et al. Monocyte-bound PF4 in the pathogenesis of heparininduced thrombocytopenia. Blood. 21;116(23):