4?��?6.0 and 19.2?��?9.2, respectively. In the DKO platelets, the average surface area was estimated as 9.8?��?3.8 and 14.3?��?6.1 at 1.5 and 15?min, respectively. To further investigate the relative extent of platelet spreading at each time point, we compared the percentage of platelets of each genotype that had spread either greater or equal to the average surface area of WT and calpain-1 null platelets. After 1.5?min of spreading, ?34% of WT platelets showed a surface area that was greater than their average surface area, whereas only ?12% of WT platelets spread more than the average surface area of calpain-1 null platelets. In calpain-1 null mice, ?36% of platelets spread greater than Unoprostone
their average surface area, whereas ?74% of calpain-1 null platelets showed a surface area that is greater than the average WT platelet surface area. Similarly, in the double knockout mice, ?75% of platelets displayed a surface area that is greater than the average surface area of WT platelets, but only ?30% of platelets showed a surface area that is greater than the average surface area of calpain-1 null platelets. A one-way anova analysis of variance (P?<?0.0001) and Student��s t-test indicated significant differences between these groups. A similar analysis was performed on platelets after 15?min of spreading on the fibrinogen surface, and spreading for 1.5 and <a href="http://www.selleckchem.com/screening/protease-inhibitor-library.html
">Small molecule library 15?min on the collagen surface (Table?1). The treatment of WT mouse platelets with MDL enhanced the platelet spreading phenotype on glass- (Fig.?S2A), collagen- (Fig.?S2C) and fibrinogen-coated surfaces (Fig.?5). Notably, the mouse platelet spreading phenotype was more pronounced on the ECM-coated surfaces as compared with the glass. These results are consistent see more
with the enhanced platelet spreading phenotype in the calpain-1 null mice. Surprisingly, human platelets treated with MDL under similar conditions showed inhibition of spreading on glass- (Fig.?S2B, top), fibrinogen- (Fig.?S2B, lower) and collagen-coated surface (Fig.?S2D). In addition, pre-incubation of MDL-treated WT platelets (Fig.?S2C) and human platelets (Fig.?S2D,E) with thrombin also produced opposite phenotypes on both collagen and fibrinogen. Thus, pharmacological inhibition of both calpains with MDL exerts opposite effects on spreading in mouse and human platelets under these conditions. The precise molecular basis of the differential effects of MDL on platelets is not known at this stage. It is to be noted that pharmacological inhibition of calpains by MDL is predicated on the assumption that this inhibitor shows strict specificity for platelet calpains in two species. A potential caveat of utilizing only the pharmacological approach for calpain inhibition is illustrated by calpeptin, another peptidyl calpain inhibitor similar to MDL, which is known to inhibit SHP-2, a major tyrosine phosphatase, in addition to calpains [28,29].