SUPPLEMENTARY FIGURE 1

Transcription

1 SUPPLEMENTRY FIGURE 1 C D E F G * Supplementary Fig. 1: Platelets from Pik3c2b mice exhibit normal function. () Expression of platelet-specific glycoproteins on the surface of platelets in whole blood taken from littermate wild-type (C2 +/+) and Pik3c2b (C2 ) mice, determined via flow cytometry. n = 3. () ggregation of platelets isolated from C2 +/+ and C2 mice in response to a PR4- activating peptide (PR4-P; YPGKF), collagen-related peptide (CRP), or DP. Each agonist was used at two concentrations one known to induce near maximal aggregation and one known to

2 cause a sub-maximal response. Shown are the maximum level of aggregation induced within the 10 min observation period (Max) and the minimum level observed after the maximum had been reached (Min), as a measure of aggregate stability. n = 3 8. (C) inding of Oregon Greenlabelled fibrinogen and (D) surface P-selectin expression on platelets from C2 +/+ and C2 mice following stimulation with either PR4-P (100 M) or CRP (10 g/ml), as determined via flow cytometry. n = 5 each. (E) Release of 14 C-serotonin from C2 +/+ or C2 platelets in response to PR4-P (100 μm). n = 3. (F) Tail bleeding times in untreated C2 +/+ and C2 mice. Data points represent time to cessation of bleeding in an individual mouse following a defined incision in the tail. Red bars are mean ± sem. (G) In vivo thrombosis in C2 +/+ and C2 mice. Electrolytic injury of carotid arteries was induced under stasis by a current of 18 m for 2 min (injury). Shown are body weight adjusted blood flow rates from 5 min before to 30 min after injury. n = 5. (-G) ll data are presented as mean ± sem and were analyzed using unpaired, two-tailed, Student s t-tests. *, P < 0.05.

3 SUPPLEMENTRY FIGURE 2 Supplementary Fig. 2: Platelet counts and volumes are normal in PI3KC2-deficient mice. () Peripheral blood platelet counts and () mean platelet volumes in an allelic series of PI3KC2- deficient mice. Shown are measurements taken in whole blood from wild-type (+/+), Pik3c2a +/gt1 (C2 +/-), Pik3c2b (C2 ), and Pik3c2a +/gt1 ; Pik3c2b (C2 +/-; ) mice on a Hemavet automated blood cell analyser. No significant differences observed (one-way NOV, with onferroni correction for multiple comparisons vs +/+).

4 SUPPLEMENTRY FIGURE 3 C D Supplementary Fig. 3: Platelets from C2 +/-; mice exhibit normal function in vitro. () Expression of platelet-specific glycoproteins on the surface of platelets in whole blood taken from wild-type (+/+) and Pik3c2a +/gt1 ; Pik3c2b (C2 +/-; ) mice, determined via flow cytometry. n = 3. () ggregation of platelets isolated from +/+ and C2 +/-; mice in response to a PR4- activating peptide (PR4-P; YPGKF), collagen-related peptide (CRP), or DP. n = (C) inding of Oregon Green-labelled fibrinogen and (D) surface P-selectin expression. n = 3 5 each. (-D) ll data are presented as mean ± sem and were analyzed using unpaired, two-tailed, Student s t-tests. No significant differences were observed by genotype at any point.

5 SUPPLEMENTRY FIGURE 4 Supplementary Fig. 4: Inducible GFP expression in platelets from shpi3kc2 mice. CMV-rtT; TRE-GFP-shPI3KC2 bi-transgenic mice (shpi3kc2 ) were fed doxycyclinecontaining food (dox; 600 mg/kg) for the indicated times and GFP expression was analysed in isolated platelets via flow cytometry. Note that GFP expression plateaued beyond 9 days at > 95%. ll experimental mice were examined after 10 days on doxycycline-containing food. Data are mean ± sem. n = 7 10.

6 SUPPLEMENTRY FIGURE 5 C D E Supplementary Fig. 5: Platelets from PI3KC2 mice exhibit normal function. () Expression of platelet-specific glycoproteins on the surface of platelets in whole blood taken from littermate wild-type (C2 +/+) and Pik3c2a gt1 (C2 ) mice, determined via flow cytometry. n = 5 7. () ggregation of platelets isolated from C2 +/+ and C2 mice in response to a PR4-activating peptide (PR4-P; YPGKF), collagen-related peptide (CRP), or DP. n = (C) inding of Oregon Green-labelled fibrinogen and (D) surface P-selectin expression. n = (E) Release of 14 C-serotonin from C2 +/+ or C2 platelets in response to PR4-P (100 μm). n = 3. (-E) ll data are presented as mean ± sem and were analyzed using unpaired, two-tailed, Student s t-tests. No significant differences were detected by genotype at any point in any assay.

7 SUPPLEMENTRY FIGURE 6 C Supplementary Fig. 6: Platelets from shpi3kc2 mice exhibit normal function. () ggregation of platelets isolated from CMV-rtT; TRE-GFP-shPI3KC2 bi-transgenic mice (shpi3kc2 ) and controls (pooled wild-type and monotransgenic littermates) in response to a PR4-activating peptide (PR4-P; YPGKF), collagen-related peptide (CRP), or DP. () inding of the activation-specific IIb 3 antibody and (C) surface P-selectin expression on platelets from shpi3kc2 and littermate controls. ll data are presented as mean ± sem of n = 3 5 independent experiments (mice) and were analyzed using unpaired, two-tailed, Student s t-tests. No significant differences were detected by genotype at any point in any assay.

8 SUPPLEMENTRY FIGURE 7 shcontrol shpi3kc2 actin tubulin Supplementary Fig. 7: Normal cytoskeleton in platelets from shpi3kc2 mice. Representative images of platelets from shpi3kc2 and shcontrol mice adherent to fibrinogen following perfusion of anticoagulated whole blood for 4 min at a rate of 600 s -1 and then fixed and stained with () phalloidin or () an anti- -tubulin antibody. Scale bars are () 2 m and () 10 m.

9 SUPPLEMENTRY FIGURE 8 Figure 1 Figure 4 C2 C2g C2 -actin C2 Figure 6 -actin -actin Supplementary Fig. 8: Uncropped scans of immunoblots.