Impaired activation of platelets lacking protein kinase C-θ isoform by Bela Nagy, Kamala Bhavaraju, Todd Getz, Yamini S. Bynagari, Soochong Kim, and Satya P. Kunapuli Blood Volume 113(11):2557-2567 March 12, 2009 ©2009 by American Society of Hematology

Activation of PKC-θ by different platelet agonists. Activation of PKC-θ by different platelet agonists. (A) Washed and aspirin-treated human platelets were stimulated with 2MeSADP, collagen-related peptide (CRP), and PAR agonists SFLLRN (PAR1) and AYPGKF (PAR4) peptides with various times as indicated at 37°C. (B) Aspirin-treated, washed human platelets were stimulated with SFLLRN (10 μM) and AYPGKF (500 μM) peptides and CRP (10 μg/mL) in the presence or absence of Gq selective inhibitor YM-254890 (50 nm) at 37°C. The stimulation times for all agonists were 60 seconds. The samples were analyzed for threonine phosphorylation of PKC-θ by Western blotting using monoclonal phospho(Thr538)-specific PKC-θ antibody. Equal lane loading was assured by probing the samples with total PKC-θ in the same blot. The Western blot shown is representative of 3 experiments done from 3 different donors. Data were quantified by densitometry and analyzed the fold increase over control. *P < .05. Bela Nagy Jr et al. Blood 2009;113:2557-2567 ©2009 by American Society of Hematology

Role of PKC-θ in GPVI- and PAR-mediated platelet aggregation and dense granule secretion. Role of PKC-θ in GPVI- and PAR-mediated platelet aggregation and dense granule secretion. Aspirin-treated washed human platelets in the presence or absence of PKC-θ antagonistic RACK peptide and isolated mouse platelets (as indicated) from PKC-θ−/− mice and WT littermates were stimulated with GPVI agonists (A) CRP (10 and 20 μg/mL) and collagen (10 and 20 μg/mL); and (B) PAR4 agonist AYPGKF (100 and 200 μM), PAR1 and PAR4 agonist thrombin (0.1 and 0.2 U/mL), and PAR1 agonist SFLLRN (5 and 10 μM) for 3.5 minutes at 37°C in stirring condition and their aggregation and simultaneous dense granule secretion were measured and compared. Dense-granule secretion is expressed as ATP released (nmol/108 platelets). The tracings are representative of results from at least 3 different donors. Bela Nagy Jr et al. Blood 2009;113:2557-2567 ©2009 by American Society of Hematology

Role of PKC-θ in αIIbβ3 activation and α-granule secretion downstream of GPVI and PARs. (A) Isolated platelets from PKC-θ−/− mice (■) and WT littermates (□) were stimulated with GPVI agonist CRP (10 and 20 μg/mL) and PAR4 agonsit AYPGKF (100 and 200 μM) to ... Role of PKC-θ in αIIbβ3 activation and α-granule secretion downstream of GPVI and PARs. (A) Isolated platelets from PKC-θ−/− mice (■) and WT littermates (□) were stimulated with GPVI agonist CRP (10 and 20 μg/mL) and PAR4 agonsit AYPGKF (100 and 200 μM) to test the JON/A binding to activated αIIbβ3 receptors. (B) WT and PKC-θ−/− murine platelets and (C) washed human platelets with or without PKC-θ RACK peptide were stimulated with CRP (10 and 20 μg/mL) as well as AYPGKF (100 and 200 μM) and PAR1 agonist SFLLRN (5 μM; only in human platelets) for 15 minutes at 37°C in nonstirring condition in the presence of FITC-labeled anti–P-selectin (CD62)-antibody. Reactions were terminated by fixing the platelets with PFA and then analyzed by flow cytometry. Each bar is the average of 3 experiments plus or minus SD from 3 different donors. (D) Dot plots of dual-color labeling for JON/A binding and CD62 expression on murine platelets are representative data of panels A and B. Bela Nagy Jr et al. Blood 2009;113:2557-2567 ©2009 by American Society of Hematology

PKC-θ-mediated syntaxin-4 phosphorylation. PKC-θ-mediated syntaxin-4 phosphorylation. Washed human platelets were stimulated with CRP (10 μg/mL) and AYPGKF (100 μM) at 37°C in the presence of PKC-θ RACK peptide or control peptide and then immunoprecipitated for syntaxin-4, and immunoblotted for phosphorylation on threonine residues of syntaxin-4. Normal mouse IgG served as a negative control. Phosphorylation data were quantified and analyzed in fold increase over control. Bela Nagy Jr et al. Blood 2009;113:2557-2567 ©2009 by American Society of Hematology

Role of PKC-θ on thromboxane generation and ERK phosphorylation. Role of PKC-θ on thromboxane generation and ERK phosphorylation. (A) Non–aspirin-treated mouse platelets separated from PKC-θ−/− mice (■) and WT littermates (□) were stimulated with collagen (10 μg/mL) and CRP (10 μg/mL) as well as AYPGKF (100 μM) and thrombin (0.05 U/mL) for 3.5 minutes at 37°C in stirring condition. Reactions were terminated and the generated TXB2 levels were measured. Data are presented as maximal percentage of TXB2 generated in the WT controls. Each bar is the average ( ± SD) of 3 experiments from 3 different donors. (B) Stimulated washed mouse samples were also analyzed by Western blotting using antibodies against antiphospho-ERK and total ERK as lane loading control. The phenotype of the mice was assured by probing the mice samples with total PKC-θ antibody. The Western blot shown is representative of results from 3 different donors. Phosphorylation data were quantified and analyzed in fold increase over control. Bela Nagy Jr et al. Blood 2009;113:2557-2567 ©2009 by American Society of Hematology

Role of PKC-θ in thrombus formation in vivo. Role of PKC-θ in thrombus formation in vivo. Using the FeCl3 carotid artery injury model of in vivo thrombosis (2 minutes of exposure to 10% FeCl3), we analyzed the percentage of mice forming stable thrombi (A) and the average time of occlusion (B) in PKC-θ−/− and WT mice. PKC-θ−/− mice demonstrated prolonged average occlusion time (mean ± SEM; 15.7 ± 3.3 minutes) and a failure to form a stable thrombus versus littermates (7.6 ± 1.1 minutes of occlusion time; P < .05 by unpaired Student t test). Fisher exact probability test was used for analyzing data shown in panel A. Bela Nagy Jr et al. Blood 2009;113:2557-2567 ©2009 by American Society of Hematology

Model depicting the functional role of PKC-θ downstream of GPVI and PAR activation in platelets. Model depicting the functional role of PKC-θ downstream of GPVI and PAR activation in platelets. Collagen and CRP act through GPVI, whereas thrombin, SFLLRN (PAR1), and AYPGKF (PAR4) act via PARs and cause activation of the Gq/PLC pathways. PLC activation leads to generation of IP3, which mobilizes calcium from intracellular stores. Increased DAG leads to the translocation to the membrane and subsequent phosphorylation of the threonine 538 residues on PKC-θ. Activated PKC-θ has a significant role in granule secretion via mediating syntaxin-4 phosphorylation and thromboxane generation via regulating ERK phosphorylation. Furthermore, PKC-θ is involved in both inside-out and outside-in αIIbβ3 signaling pathways. Bela Nagy Jr et al. Blood 2009;113:2557-2567 ©2009 by American Society of Hematology