Interspecies differences in protein expression do not impact the spatiotemporal regulation of glycoprotein VI mediated activation

Figure 1. Flow cytometry can be used to determine the copy number of mouse platelet receptors. Saturating concentrations of directly dye conjugated monoclonal antibodies against mouse GPVI (JAQ1) were used to label platelets (A) and beads of known antigen binding capacity (B). The geometric mean was used to construct a calibration graph, which can then be used to calculate the number of proteins exposed at the platelet cell surface (C). Comparison between mouse and human platelet copy numbers taken from this study, Zeiler et al (28) and Mazet et al (26) (D). Comparison between mouse and human protein densities, based on platelet volumes of 4.3 and 7.4 fL for mice and human, respectively (E)

Figure 2. Virtual platelet predictions of Syk activity in a hypothetical population of mouse and human platelets is supported by the experimental outcomes. A, The results of 200 simulations of the Virtual Platelet are shown. Each line represents a prediction of how Syk phosphorylation changes over time in response to protein copy numbers randomly selected across their normal range (Human: GPVI, 5000 +/−12%; Syk, 2763 +/−22%, c‐Cbl, 2581 +/−25%. Mouse: GPVI, 5586 +/−12%; Syk, 23 286 +/−22%, c‐Cbl, 3241 +/− 25%). Simulations representing Syk phosphorylation in a hypothetical mouse platelet (n = 100) shown in red or in a human platelet (n = 100) in blue. B, A summary of model predictions for the time to reach maximal peak Syk activity with each simulation denoted graphically by a circle. C, Representative time course of Syk Y525/Y526 phosphorylation in human and mouse washed platelets following GPVI ligation with 10 µg/mL CRP. Actin acts as a loading control. D, Quantification of mouse and human Syk Y525/Y526 phosphorylation time courses. Mean ± S.D, n = 5. E, Time to maximal Syk Y525/Y526 phosphorylation. Line indicates the mean ± S.D. P = .5796 Statistical analysis was performed with…

Figure 3. Mouse platelets are refractory to large reductions in the number of Syk molecules. Modelling suggests that the sensitivity of Syk phosphorylation to variation in protein copy numbers varies between human and mouse platelets. Time to maximal Syk phosphorylation in a hypothetical population of human and mouse platelets is shown, where the copy number of each protein was varied by 50% above and below their initial value. A positive sensitivity score represents an increase in the time to reach peak activity and a negative score represents a decrease

Figure 4. A, Genotyping strategy confirming the presence of both a Syk wild‐type (WT) allele and kinase dead (KD) version under the control of PF4‐Cre in the heterozygous mice (HT) and homozygous mice (HO). B, Total Syk protein levels were measured by western blot in control (WT) and Syk KD HT (HT) mice using two different anti‐Syk antibodies. C, Quantification of Syk levels in platelets from WT and Syk KD HT mice. Mean ± S.D, n = 3. 4D10 P = .146, N19 P = .305 Statistical analysis was performed with unpaired t test. D, Platelet aggregation of washed control (WT) and heterozygous (HT) mice induced by 10 and 3 µg/mL CRP (addition of agonist indicated by arrowhead). E, Quantification of % aggregation at 5 minutes of control (WT) and Syk KD HT (HT) platelets. Mean ± S.D, n ≥ 3. Statistical analysis was performed with a two‐way ANOVA with Bonferroni posttest (ns; P > .05). F, Quantification of time to peak of control (WT) and Syk KD HT (HT) platelets. Mean ± S.D, n ≥ 3. Statistical analysis was performed with a two‐way ANOVA with Bonferroni post‐test (ns; P > .05, *P < .05). G, Representative time course of Syk Y525/Y526 phosphorylation in WT and Syk KD HT mouse washed platelets following GPVI…

Figure 5. Spatial distribution of Syk in mouse and human platelets. Representative plot of clusters in a human platelet (A) and a mouse platelet (B) spread on CRP. Clusters were identified using DBSCAN with a radius of 25 nm and a minimum number of points as 10. Each cluster is identified by a different color, the black points are background points which do not meet the criteria to be included in a cluster. Quantification of the number of detections per cluster (C), cluster area (D), number of clusters per spread platelet area (E), and the detection density within clusters (F) in human and mouse platelets. Mean ± S.D, n = 3 *P ≤ .05 **P ≤ .01 Statistical analysis was performed with unpaired t test