Increased thrombosis susceptibility and altered fibrin formation in STAT5-deficient mice by Sarah M. Nordstrom, Brian A. Holliday, Brandon C. Sos, James W. Smyth, Robert E. Levy, Jonathan W. Dukes, Susan T. Lord, and Ethan J. Weiss Blood Volume 116(25):5724-5733 December 16, 2010 ©2010 by American Society of Hematology

In vivo thrombosis and in vitro coagulation in STAT5-deficient mice. In vivo thrombosis and in vitro coagulation in STAT5-deficient mice. (A) STAT5g male (- - -), STAT5g female (gray - - -), control male (—), and control female (gray —) mice were injected with 2.0 μL/g 1:40 TF dilution in a PE model of thrombosis. STAT5g males and females had reduced survival and shortened median survival times compared with sex-matched controls. No differences were found between genotype-matched males and females. (B) Whole blood was isolated from STAT5g male (○; n = 10), STAT5g female (□; n = 8), control male (●; n = 9), and control female (■; n = 14) mice. STAT5g mice had shortened TF-triggered clotting times compared with sex-matched controls. **P < .01, ***P < .001; 1-way ANOVA with Bonferroni post test. (C) STAT5l male (- - -), STAT5l female (gray - - -), control male (—), and control female (gray —) mice were injected with TF as in panel A. STAT5l males and females had reduced survival and shortened median survival times compared with sex-matched controls. No differences were found between genotype-matched males and females. (D) Whole blood was isolated from STAT5l male (○; n = 9), STAT5l female (□; n = 7), control male (●; n = 39), and control female (■; n = 38) mice. STAT5l mice had shortened TF-triggered clotting times compared with sex-matched controls. ***P < .001; 1-way ANOVA with Bonferroni post test. Sarah M. Nordstrom et al. Blood 2010;116:5724-5733 ©2010 by American Society of Hematology

In vivo thrombosis and in vitro coagulation in STAT5-deficient and little intercrossed mice. In vivo thrombosis and in vitro coagulation in STAT5-deficient and little intercrossed mice. (A) Control male (—; control/control), Lit male (gray —; litm/m/control), ST5 male (- - -; control/STAT5−/−), and LitST5 male (gray - - -; litm/m/STAT5−/−;) mice were injected with 3.0 μL/g 1:40 TF dilution in a PE model of thrombosis. Survival of male LitST5 was reduced compared with Lit and control male mice, but was similar to ST5. (B) Control female (—), Lit female (gray —), ST5 female (- - -), and LitST5 female (gray - - -) mice were injected with TF as in panel A. Survival of LitST5 female mice was reduced compared with Lit and control female mice, but was similar to ST5. (C) Whole blood was isolated from control male (●; control/control; n = 25), Lit male (○; n = 18), ST5 male (■; n = 9), and LitST5 male (□; n = 7) mice. TF-triggered whole blood clotting times were significantly shorter in LitST5 mice versus Lit, but were comparable to ST5. ***P < .001; 1-way ANOVA with Bonferroni post test. (D) Whole blood was isolated from control female (●; n = 28), Lit female (○; n = 5), ST5 female (■; n = 11), and LitST5 female (□; n = 8) mice. TF-triggered whole blood clotting times were significantly shorter in LitST5 mice versus Lit and control, but were comparable to ST5. *P < .05, ***P < .001; 1-way ANOVA with Bonferroni post test. Sarah M. Nordstrom et al. Blood 2010;116:5724-5733 ©2010 by American Society of Hematology

Thrombin generation in STAT5-deficient plasma. Thrombin generation in STAT5-deficient plasma. (A) Expression of clotting factors: mRNA was isolated from control (open) and STAT5-deficient (filled) livers, and quantitative reverse transcription PCR was performed on cDNA. A raw GCN was calculated as described in the Methods, and each GCN was normalized to control mice. STAT5-deficient and control mice had comparable expression of coagulation factors, with the exception of F11 (n = 3, *P = .0069; Student t test). (B) Coagulation factor activity: plasma from control (open) and STAT5-deficient (filled) mice were mixed with the indicated human factor-deficient plasma; factor activities were measured as described in the Methods and normalized to control mice. STAT5-deficient mice had slightly increased FV, FIX, and FX activities, but were comparable to control for all other factor activities (n = 3; *P < .05; Student t test). (C) Thrombin generation: platelet-poor plasma was isolated from STAT5-deficient (- - -) and control mice (—). A fluorogenic thrombin substrate was added to plasma, and fluorescence was measured over time after the addition of TF. STAT5-deficient plasma had comparable endogenous thrombin potential to control mice. Sarah M. Nordstrom et al. Blood 2010;116:5724-5733 ©2010 by American Society of Hematology

Thrombin-triggered clotting. Thrombin-triggered clotting. (A) Thrombin was added to platelet-poor plasma. Clotting times were 31.4% shorter in STAT5-deficient mice (○) compared with control (●; n = 8,6; *P < .0001; Student t test). (B) Thrombin-triggered fibrin clot formation and firmness were measured over time in whole blood isolated from STAT5-deficient (- - -) and control mice (—) using a thromboelastometer. This experiment was performed 3 times, and a representative curve is shown. The clotting times were shorter (P = .0002; n = 3; Student t test) and maximal clot firmness was greater (P = .0316; n = 3; Student t test) in STAT5-deficient plasma compared with control. (C) STAT5-deficient (○) and control (●) defibrinated plasma were supplemented with human fibrinogen to achieve a final concentration of 3.0 mg/mL. Thrombin-triggered clot times were 25.4% shorter in STAT5-deficient versus control samples (n = 3; *P = .0289; Student t test). (D) Thrombin-triggered fibrin clot formation and firmness were measured as in panel B using defibrinated pooled (n = 3) STAT5-deficient (- - -), defibrinated pooled control (n = 3; —), and defibrinated normal human plasma (gray —) mixed 1:1 with normal human plasma. This experiment was performed 2 times, and a representative curve is shown. The addition of STAT5-deficient plasma prolonged thrombin-triggered clot times by 185% compared with human, while control plasma prolonged clot times by 370% compared with human. Maximum clot firmness was increased by 20% with addition of control plasma and 60% with STAT5-deficient plasma. Sarah M. Nordstrom et al. Blood 2010;116:5724-5733 ©2010 by American Society of Hematology

Confocal microscopy of fibrin polymerization. Confocal microscopy of fibrin polymerization. Platelet-poor plasma isolated from control (left panels) and STAT5-deficient (right panels) mice was supplemented with 80 μg/mL human fibrinogen labeled with AlexaFluor 488. Thrombin-triggered clotting was initiated in the center of a 35-mm glass-bottomed dish, and polymerization was recorded at 37°C using a Nikon Ti inverted microscope, 100×/1.49 Apo TIRF objective, Yokogowa CSU-X1 spinning disk confocal unit with 486 nm DPSS laser source, and a Photometrics Cascade II 512 camera. Images are maximum intensity projections of 5-μm stacks at the indicated time points. No discernable fibers were present at the beginning of capture (A-B). Fibrin fibrils first appeared at 120 seconds in STAT5-deficient plasma (C-D) and 180 seconds in control plasma (E-F). At 460 seconds, both control and STAT5-deficient plasma had established extensive fibrin networks (G-H). Sarah M. Nordstrom et al. Blood 2010;116:5724-5733 ©2010 by American Society of Hematology

Kinetics of FpA and FpB release. Kinetics of FpA and FpB release. Defibrinated control (●) and STAT5-deficient (○) plasma were supplemented with 1.0 mg/mL human fibrinogen. Reactions were initiated with thrombin and quenched at the indicated time points. FpA and FpB were quantified by HPLC analysis and normalized to the calculated FpA max values for each sample. FpA: STAT5-deficient (□) and control (■) mice had comparable release of FpA over time (n = 4; P = .6731; P > .05 at all time points; 2-way ANOVA with matching and Bonferroni post test). The rate constant, k1, was derived by fitting FpA curves to a first-order reaction (black lines); the rate of FpA release was similar between genotypes (n = 4; P = .5705; F test). FpB: In contrast, STAT5-deficient mice (○) demonstrated greater release of FpB over time compared with controls (●; n = 4; P = .0119; P < .05 at 0.5-, 2-, and 5-minute time points; 2-way ANOVA with matching and Bonferroni post test). Data points were plotted with a simple connecting line (gray lines). Sarah M. Nordstrom et al. Blood 2010;116:5724-5733 ©2010 by American Society of Hematology