Platelets contain an invaginated, tubular membranous structure called the surface-connected open canalicular system (SCCS or OCS), which is contiguous with the plasma membrane and serves as a site for granule fusion and as a reservoir of membrane for platelet spreading. According to ultrastructural studies, platelets from some species lack OCS. In an attempt to correlate biochemical and functional attributes with the presence of an OCS, platelets from human, mouse and dog (OCS(+)), and from cow, camel and horse (OCS(-)) were analysed for differential protein expression and aggregation in response to thrombin. Among the 18 different cytoskeletal and regulatory proteins examined, five (Rac1, RhoA, Ras, calmodulin and Src) were expressed at higher levels in OCS(+) platelets (p < 0.05). Given the role of Arf6 in the formation of tubular invaginations in nucleated cells, the levels of Arf6-GTP were analysed in OCS(+) and OCS(-) platelets. There was no significant correlation between the presence of OCS and total Arf6 or Arf6-GTP levels. Comparison of platelet aggregation between different species suggests that OCS(-) platelets have delayed responses. This comparison of platelets from six different species, which differ in their OCS, shows the differential expression of known signaling components and foreshadows future studies focusing on OCS formation and function.