Apoptotic Vascular Endothelial Cells Become Procoagulant by Thomas Bombeli, Aly Karsan, Jonathan F. Tait, and John M. Harlan Blood Volume 89(7):2429-2442 April 1, 1997 ©1997 by American Society of Hematology

Flow cytometric cell cycle histogram. Flow cytometric cell cycle histogram. HUVECs treated with staurosporine (left panels) or kept in suspension with serum deprivation (right panels) for 4, 8, and 16 hours were permeabilized, fixed, treated with RNAse, and stained with PI. The region below the Go /G1 peak (designated Ao region) represents cells undergoing apoptosis-associated DNA degradation (hypodiploid DNA) and is expressed as a percentage of events with respect to the entire cell cycle. Thomas Bombeli et al. Blood 1997;89:2429-2442 ©1997 by American Society of Hematology

Internucleosomal DNA fragmentation. Internucleosomal DNA fragmentation. HUVECs treated with staurosporine (lane 2) or kept in suspension with serum deprivation (lane 3) for 6 hours were lysed and total cellular DNA was separated on a 2% agarose gel. HUVECs in lanes 2 and 3 show typical oligonucleosomal banding. Lanes M and 1 represent molecular weight markers (in kilobases) and normal HUVECs, respectively. Thomas Bombeli et al. Blood 1997;89:2429-2442 ©1997 by American Society of Hematology

Annexin V binding. Annexin V binding. HUVECs treated with staurosporine (left panels) or kept in suspension with serum deprivation (right panels) for 4, 8, and 16 hours were stained with FITC-conjugated recombinant annexin V in a buffer containing 2.5 mmol/L CaCl2 and subsequently analyzed using flow cytometry. Almost 100% of normal cells (upper panel, right peak) show an increased fluorescence compared with cells stained in EDTA. The marker M1 represents the percentage of cells exhibiting a higher fluorescence than the control (lower 6 panels, left peak). The results of one representative experiment of three performed are shown. Thomas Bombeli et al. Blood 1997;89:2429-2442 ©1997 by American Society of Hematology

Antigenic TF. (A) HUVECs were treated with LPS (▨) or staurosporine (▪) or were kept in suspension with serum deprivation () for 2, 4, 8, and 16 hours. Antigenic TF. (A) HUVECs were treated with LPS (▨) or staurosporine (▪) or were kept in suspension with serum deprivation () for 2, 4, 8, and 16 hours. Cells were then lysed and prepared for quantitative determination of TF by ELISA. Functional TF in adherent (B) and suspended cells (C). HUVECs either untreated or activated with LPS for 6 hours were treated with staurosporine (B; solid triangles) or kept in suspension with serum deprivation (C; shaded triangles) for 8 hours. After incubation with factor VIIa and factor X, the formation of factor Xa was measured using a chromogenic substrate. Controls were untreated cells (B and C; ○) and cells treated with LPS (B and C; □). Results are expressed as means ± SD (n = 4). Thomas Bombeli et al. Blood 1997;89:2429-2442 ©1997 by American Society of Hematology

Activity and inhibition of the intrinsic tenase complex. Activity and inhibition of the intrinsic tenase complex. Assays were performed using either adherent (left panels) or suspended cells (right panels). Apoptosis was induced by treatment with staurosporine (left panels, solid triangles) or culture in suspension with serum deprivation (right panels, shaded triangles) for 4 and 16 hours, respectively. The activity of the intrinsic tenase complex was determined by measuring the amidolytic activity of factor Xa after the addition of factor IXa, factor VIII, and factor X (A and B). The effect of the exposure of PS on the activity of the intrinsic tenase complex was assessed by the ability of annexin V to inhibit factor Xa formation (C and D). After preincubation of HUVECs with various concentrations of annexin V for 5 minutes, the activity of the intrinsic tenase complex was measured as described above. Controls were untreated cells (all panels; ○). Results are expressed as means ± SD (n = 4). Thomas Bombeli et al. Blood 1997;89:2429-2442 ©1997 by American Society of Hematology

Flow cytometric determination of TM Flow cytometric determination of TM. HUVECs treated with staurosporine (left panels) or kept in suspension with serum deprivation (right panels) for 2, 4, and 8 hours were harvested, washed, and stained with an antihuman TM MoAb, followed by an FITC-conjuga... Flow cytometric determination of TM. HUVECs treated with staurosporine (left panels) or kept in suspension with serum deprivation (right panels) for 2, 4, and 8 hours were harvested, washed, and stained with an antihuman TM MoAb, followed by an FITC-conjugated goat antimouse antibody. The M1-region is defined as a percentage of events below the control (all panels, right peak). The results of one representative experiment of three performed are shown. Thomas Bombeli et al. Blood 1997;89:2429-2442 ©1997 by American Society of Hematology

Functional activity of TM (A), HS (B), and TFPI (C). Functional activity of TM (A), HS (B), and TFPI (C). Assays were performed using either adherent (left panels) or suspended cells (right panels). Apoptosis in adherent and suspended HUVECs was induced by treatment with staurosporine (left panels, solid triangles) or culture in suspension with serum deprivation (right panels, shaded triangles) for 8 and 16 hours, respectively. All activities were determined by chromogenic substrate assays as follows: thrombin-dependent activation of protein C (TM activity), ATIII-dependent inhibition of thrombin (HS activity), and inhibition of factor Xa formation generated by the TF-factor VIIa complex (TFPI activity). Controls were untreated cells (all panels; ○). As negative controls (all panels; □), cells were pretreated with LPS (TM), heparinase III (HS), and rabbit antihuman TFPI antibody (TFPI), respectively. Results are expressed as mean ± SD (n = 4). Thomas Bombeli et al. Blood 1997;89:2429-2442 ©1997 by American Society of Hematology

Flow cytometric determination of HS Flow cytometric determination of HS. HUVECs treated with staurosporine (left panels) or kept in suspension with serum deprivation (right panels) for 2, 4, and 8 hours were harvested, washed, and incubated with ATIII for 30 minutes. Flow cytometric determination of HS. HUVECs treated with staurosporine (left panels) or kept in suspension with serum deprivation (right panels) for 2, 4, and 8 hours were harvested, washed, and incubated with ATIII for 30 minutes. Cells were then stained with an antihuman ATIII MoAb, followed by an FITC-conjugated goat antimouse antibody. The M1-region is defined as the percentage of events below the control (all panels, right peak). The results of one representative experiment of three performed are shown. Thomas Bombeli et al. Blood 1997;89:2429-2442 ©1997 by American Society of Hematology

Flow cytometric determination of TFPI Flow cytometric determination of TFPI. HUVECs treated with staurosporine (left panels) or kept in suspension with serum-deprivation (right panels) for 2, 4, and 8 hours were harvested, washed, and stained with a rabbit antihuman TFPI antibody, followed by a... Flow cytometric determination of TFPI. HUVECs treated with staurosporine (left panels) or kept in suspension with serum-deprivation (right panels) for 2, 4, and 8 hours were harvested, washed, and stained with a rabbit antihuman TFPI antibody, followed by an FITC-conjugated antirabbit MoAb. The M1-region is defined as the percentage of events below the control (all panels, right peak). The results of one representative experiment of three performed are shown. Thomas Bombeli et al. Blood 1997;89:2429-2442 ©1997 by American Society of Hematology

Thrombin generation in plasma with adherent (A) or suspended cells (B). Thrombin generation in plasma with adherent (A) or suspended cells (B). Apoptosis in adherent and suspended HUVECs was induced by treatment with staurosporine for 2 hours (A; solid triangles) and by culture in suspension with serum deprivation for 16 hours (B; shaded triangles), respectively. Cells were then incubated with 0.5 mL of citrated platelet-rich (adherent cells) and platelet-poor plasma (suspended cells). After starting the coagulation process with 0.5 mL of prewarmed CaCl2 , the formation of thrombin was monitored using a chromogenic substrate. Controls were untreated cells (A and B; ○). As negative controls (A and B; □), subendothelial matrix and no cells were used, respectively. Results are expressed as mean ± SD (n = 6). Thomas Bombeli et al. Blood 1997;89:2429-2442 ©1997 by American Society of Hematology