Hyaluronan synthases, or HAS’s, produce the body’s hyaluronan (HA). The role of HA production in blood’s function has yet to be investigated. We strove to investigate how HA synthesis alters the production and function of the cells responsible for blood clotting: platelets.

Our lab produced double-knockout mice (dKO mice), so named for knocking out HAS1 and HAS3. Then we examined the bone marrow of dKO under the microscope to search for any notable changes. To our surprise, we found an increase in megakaryocytes in our dKO mice compared to our wild type mice. As megakaryocytes produce platelets, we measured circulating platelets. Platelet numbers were similar between our wild type mice and dKO mice. Whatever changes in hyaluronidase activity had led to altered megakaryocyte numbers,

But were these dKO platelets changes on a signaling level? We performed aggregometry experiments, allowing us to measure the platelet’s abilities to clot. Our dKO platelets clotted, or “aggregated,” less in the presence of thrombin. They also displayed fewer signs of activation through PAR-4 receptor stimulation when we measured integrin and p-selectin via flow cytometry. Both integrin and p-selectin are surface receptors that allow platelets to bind to either other platelets or the outside of other cells under flow. When we ran these platelets over integrin’s binding partner fibrinogen at varying flow rates, we shockingly found that dKO bound less to fibrinogen at slower venous flow conditions when compared to our wild type mice. These data suggest that HAS1 and HAS3 play a role in integrin activation, but only when the receptor PAR4 binds to and is activated by thrombin.

We decided to examine the signaling pathways downstream of PAR4 activation. We measured a molecule named AKT, a well-known signaling molecule in platelet activation and clotting. When AKT is phosphorylated, the platelet activates/clots.  Using flow cytometry, we found that AKT was significantly less phosphorylated (and therefore less activated) in our dKO during thrombin stimulation. This seems to explain the reduced integrin activation in our platelets; reduced activation of AKT results in reduced overall cell activation.

Our study finds that HAS receptors act beyond the production of hyaluronan. In platelets, hyaluronan synthases—and by extension, hyaluronan production—serves a key role in thrombin signaling upstream of integrin activation.