Impaired “outside-in” integrin αIIbβ3 signaling and thrombus stability in TSSC6-deficient mice by Matt W. Goschnick, Lai-Man Lau, Janet L. Wee, Yong S. Liu, P. Mark Hogarth, Lorraine M. Robb, Michael J. Hickey, Mark D. Wright, and Denise E. Jackson Blood Volume 108(6):1911-1918 September 15, 2006 ©2006 by American Society of Hematology

TSSC6 is expressed on the surface and in an intracellular pool of platelets in complex with integrin αIIbβ3. TSSC6 is expressed on the surface and in an intracellular pool of platelets in complex with integrin αIIbβ3. (A-B) Flow cytometric analysis of TSSC6 surface expression on resting and thrombin-stimulated (0-5 U/mL) wild-type murine platelets. P-selectin exposure on thrombin-stimulated (1 U/mL) wild-type murine platelets in the absence and presence of FITC-antimouse P-selectin mAb is included as a positive control marker of alpha granule release. Platelets were stained with the indicated anti–murine 14A6 TSSC6 monoclonal antibody followed by a secondary FITC-conjugated antirat antibody. Data were collected through a live platelet gate based on forward versus side scatter profiles on a FACS Calibur flow cytometer. Results are cumulative data derived from 3 independent experiments and presented as mean fluorescence intensity (MFI) ± SEM (n = 3). (C) 1.5 mg of human platelet lysates solubilized with indicated detergents were immunoprecipitated with antibodies directed against normal mouse IgG1, PECAM-1, GPIb-IX-V complex, and integrin complex-specific αIIbβ3 Ab, P2. Samples were resolved on 12.5% SDS-PAGE under reducing conditions and Western blotted for TSSC6 with a polyclonal anti–human TSSC6 antibody. Matt W. Goschnick et al. Blood 2006;108:1911-1918 ©2006 by American Society of Hematology

TSSC6–/– mice display features of unstable hemostasis in vivo. TSSC6–/– mice display features of unstable hemostasis in vivo. Hemostasis in 6- to 8-week-old mice was assessed using an in vivo tail-bleeding assay. (A) Time taken for cessation of bleeding for wild-type and TSSC6–/– mice (n = 20). P < .05 (Student t test). (B) Volume of blood lost during tail-bleeding time for wild-type and TSSC6–/– mice (n = 20). P < .05 (Student t test). (C) Rebleeding occurrences in wild-type and TSSC6–/– mice (n = 20). P < .005 (Student t test). Errors bars indicate SEM. Matt W. Goschnick et al. Blood 2006;108:1911-1918 ©2006 by American Society of Hematology

Absence of platelet TSSC6 results in secondary instability in platelet thrombi formed in vivo. Absence of platelet TSSC6 results in secondary instability in platelet thrombi formed in vivo. (A) Thrombus formation in vivo was monitored on mesenteric arterioles after topical application in 7.5% FeCl3. Time to artery congestion and primary clot formation in seconds after injury. Each symbol represents 1 monitored arteriole for each wild-type versus TSSC6–/– mouse. Wild-type: 773.4 ± 40.2 seconds; TSSC6–/–: 885.7 ± 36.3 seconds. P < .05; n = 30. (B) Time of clot stability in seconds after injury and occlusion of vessel. Each symbol represents 1 monitored arteriole for each wild-type versus TSSC6–/– mouse. Wild type: 314.4 ± 47.9 seconds; TSSC6–/–: 186.7 ± 38.4 seconds. P < .05; n = 11. (C) Number of thrombi embolizing more than 20 μm in diameter formed during the 30- to 45-minute observation period. Each symbol represents 1 monitored arteriole for each wild-type versus TSSC6–/– mouse. Wild-type: 4.0 ± 1.3 emboli; TSSC6–/–: 9.0 ± 1.9 emboli. P < .05; n = 23. Matt W. Goschnick et al. Blood 2006;108:1911-1918 ©2006 by American Society of Hematology

Delayed kinetics of clot retraction for TSSC6–/– platelets in the presence of normal integrin αIIbβ3 expression. Delayed kinetics of clot retraction for TSSC6–/– platelets in the presence of normal integrin αIIbβ3 expression. (A) Photographs showing in vitro kinetics of clot retraction over a 21-hour time frame using platelet rich plasma (PRP) (normalized platelet counts) from wild-type and TSSC6-deficient mice. Samples were treated with 2.5 units thrombin. Each photograph is representative of at least 3 experiments. (B) The expression of surface markers on platelets was determined by staining with an isotype control (FITC-CD3), positive control FITC-CD44 mAb, FITC-CD9, and FITC-integrin β3 mAb for both wild-type and TSSC6–/– platelets. FITC-labeled samples were analyzed on a FACS Calibur analyzer. Results are cumulative data derived from 3 independent experiments and presented as MFI ± SEM. Matt W. Goschnick et al. Blood 2006;108:1911-1918 ©2006 by American Society of Hematology

TSSC6–/– platelets display restricted cytoskeletal reorganization upon spreading on immobilized fibrinogen. TSSC6–/– platelets display restricted cytoskeletal reorganization upon spreading on immobilized fibrinogen. (A) Washed wild-type and TSSC6–/– platelets were allowed to adhere for 0 to 40 minutes at 37°C to a fibrinogen matrix. Adherent platelets were fixed and imaged by DIC microscopy. Images were captured with an Axiovert 135 microscope (Zeiss, Oberkochen, Germany) with a 63×/1.25 oil immersion lens and a PixeLINK megapixel firewire camera (model PL-A661; PixeLINK, Ottawa, ON, Canada) and PixeLINK software version 3.2. (B) The percentage of spread platelets for each genotype were quantitated per high-powered field (*P < .05, n = 3). DIC images shown are a representative of 3 independent experiments. (C) The number of adherent platelets for each genotype was quantitated per high-powered field (P > .05; n = 3). Results are representative of 3 independent experiments. Matt W. Goschnick et al. Blood 2006;108:1911-1918 ©2006 by American Society of Hematology

TSSC6–/– platelets display normal platelet aggregation responses to PAR-4, ADP, type 1 collagen, and calcium ionophore. TSSC6–/– platelets display normal platelet aggregation responses to PAR-4, ADP, type 1 collagen, and calcium ionophore. Aggregation responses of PRP (platelet count adjusted to 100 × 109/L) for wild-type and TSSC6–/– mice were determined following activation with different concentrations of various agonists: PAR-4 agonist peptide (125-250 μM), ADP (5-20 μM), type 1 collagen (5-20 μg/mL), and calcium ionophore (2.5-10 μg/mL), respectively. Matt W. Goschnick et al. Blood 2006;108:1911-1918 ©2006 by American Society of Hematology

TSSC6–/– platelets display normal soluble FITC-fibrinogen binding, JON/A mAb binding, and alpha granule release following treatment with thrombin, PAR-4 peptide, ADP, and ADP in synergy with epinephrine. TSSC6–/– platelets display normal soluble FITC-fibrinogen binding, JON/A mAb binding, and alpha granule release following treatment with thrombin, PAR-4 peptide, ADP, and ADP in synergy with epinephrine. (A) FACS analysis of FITC-conjugated fibrinogen binding to platelets stimulated with thrombin (1 U/mL), PAR-4 (250 μM), PMA (20 μM), ADP (10 μM), ADP (10 μM) + epinephrine (20 μM), or unstimulated (control). Results are cumulative data from 3 independent assays and are presented as MFI ± SEM. (B) Flow cytometric analysis of JON/A-PE mAb binding to platelets stimulated with 0.5 to 1.0 U/mL thrombin, 125 to 250 μM PAR-4, 10 to 20 μM ADP, 10 to 20 μM ADP + 20 μM epinephrine, 2.5 to 5 μg/mL CRP, or unstimulated (control). Results are cumulative data from 3 independent experiments and are presented as MFI ± SEM. (C) Surface expression of P-selectin was determined for washed platelets stimulated by 0.5 to 5 U/mL thrombin or 100 to 400 μM PAR-4 agonist peptide and then stained with either a buffer control and FITC–P-selectin mAb for wild-type and TSSC6–/– platelets. FITC-labeled samples were analyzed on a FACS Calibur analysis. Results are cumulative data from 3 independent experiments and are presented as MFI ± SEM. Matt W. Goschnick et al. Blood 2006;108:1911-1918 ©2006 by American Society of Hematology