Barbara Kandler

Activated platelets increase fibrinolysis of mesenchymal progenitor cells.

Bone regeneration is initiated by the formation of a blood clot. Activated platelets within this fibrin‐rich matrix release signaling molecules that can attract mesenchymal progenitor cells. To gain insight into the cellular mechanism by which activated platelets can support the immigration of mesenchymal progenitors, we have tested the hypothesis that platelet‐released signaling molecules increase the capacity of bone marrow stromal cells (BMSC) to activate plasminogen. We report herein that platelet‐released supernatants (PRS) elevate total urokinase‐type plasminogen activator (uPA) and total plasminogen activator inhibitor‐1 (PAI‐1) levels in BMSC, as assessed by immunoassay. Quantitative polymerase chain reaction showed an upregulation of uPA, uPA receptor, and PAI‐1. Zymography and kinetic analysis based on casein hydrolysis revealed enhanced activity of cell‐associated uPA upon exposure of BMSC to PRS. Inhibiting c‐Jun N‐terminal kinase (JNK) and phosphatidylinositol 3‐kinase (PI3K) signaling reduced uPA production and decreased plasminogen activation. Corresponding Western blot analysis showed increased phosphorylation of JNK and AKT in BMSC treated with PRS. These results suggest that activated platelets can enhance the plasminogen activation capacity of mesenchymal progenitors through the stimulation of uPA production, requiring JNK and PI3K/AKT signaling. By this mechanism platelets may contribute to the organization of the blood clot during bone regeneration.

Activated platelets retain their potential to induce osteoclast-like cell formation in murine bone marrow cultures.

Supernatants immediately obtained after platelet activation can induce osteoclast-like cell formation in murine bone marrow cultures. Here we report that activated platelets retain their potential to induce osteoclast-like cell formation over a 3-day period with repeated washing, when co-cultured with murine bone marrow cells. Supernatants obtained from washed platelets 3 days following their activation with thrombin, caused the differentiation of haematopoietic progenitors into osteoclast-like cells. The platelet-derived soluble factor(s) responsible for the induction of osteoclastogenesis can be retained in an ultrafilter with a nominal molecular weight limit of 10 kDa, and loose their activity when incubated at 99°C. Indomethacin, which inhibits cyclooxygenase activity, and osteoprotegerin, a decoy receptor for receptor activator of nuclear factor-κB ligand (RANKL), suppressed the formation of osteoclast-like cells in this model. The in vitro findings presented here suggest that activated platelets can induce osteoclast-like cell formation via a prostaglandin and RANKL-dependent mechanism over a time period corresponding to the existence of a blood clot.