Abstract
Abstract 379
Polo-like kinase (Plk) family members are serine/threonine kinases involved in cell cycle regulation. Their expression and function in platelets are not known. We identified the presence of Plk3, a member of this family, in human and mouse platelets. We found that Plk3 is localized to the filopodia of activated platelets. Furthermore, it co-immunoprecipitates with integrin aIIbb3 in an aggregation-dependent manner. To understand the physiological function of Plk3 in thrombosis, we obtained Plk3−/− mice in C57Bl/6 background, examined the tail bleeding time of Plk3−/− mice, and compared it to the Plk3+/+ (WT) mice of the same genetic background. We found that the average tail bleeding time for WT mice was about 130 s, consistent with the values reported in the literature. Interestingly, the Plk3−/− mice had a significantly (P<0.05) delayed average tail bleeding time (325 s), suggesting that Plk3 deficiency results in a bleeding phenotype. These results suggest that there may be defects in the thrombotic process in these mice. To evaluate the in vivo thrombotic phenotype, we performed a 10% FeCl3-induced carotid artery injury and observed any differences in time of occlusion or unstable occlusions in Plk3−/− mice compared to WT mice. Consistent with our finding of extended tail bleeding time in Plk3−/− mice, our results showed that the WT mouse vessel occluded within 7–9 min, whereas Plk3−/− mouse took nearly twice that time (∼14 min) to initiate vessel occlusion (P<0.001). We also performed a collagen/epinephrine-induced pulmonary thromboembolism assay to investigate the role of platelet Plk3 in thrombosis. Our result suggests a marked protection from thromboembolism in Plk3−/− mice, since significantly more (P<0.0004) survived compared to WT mice. By assessing the ability of Evans blue dye to pass through the pulmonary circulation, we determined that this better survival rate in Plk3−/− mice is due to the failure of occlusion of pulmonary vessels in these mice. This was further supported by the histological examination of the lungs of these mice, which showed decreased size of the emboli and the reduced extent of arterial occlusion compared to WT. Ex vivo platelet functional studies suggested that thrombin-induced generation of TxA2, a potent activator of platelet function, was significantly attenuated (P<0.03) in Plk3−/− mice compared to WT. When tested for activation of cPLA2, a key enzyme in TxA2 generation, we found that the phosphorylation of cPLA2 is significantly attenuated (P<0.05) in Plk3 null platelets. Furthermore, thrombin-induced secretion of both a- and d- granules was significantly reduced (P<0.007) in Plk3−/− mouse platelets compared to WT, consistent with the observed anti-thrombotic phenotype in vivo. Surprisingly, however, platelet aggregation by low dose of thrombin or PAR4 peptide was significantly augmented (P<0.02) in Plk3 null platelets compared to WT. This was further supported by the significantly increased (P<0.05) fibrinogen receptor exposure on platelets. To determine the molecular mechanism of the observed hyperaggregation, we analyzed signaling events such as ERK1/2 and Akt, an upstream regulator of integrin aIIbb3 activation. Interestingly, we found that agonist-induced activation of ERK2 and Akt (both T308 and S473 phosphorylation) is significantly enhanced in the absence of Plk3. Furthermore, we found that levels of PTEN, a negative regulator of PI3-K/Akt pathway is reduced in the absence of Plk3. The severity of the anti-thrombotic phenotype in Plk3−/− mice may have been dampened due to the opposing role of Plk3. We next asked if the integrin outside-in signaling is also enhanced in these mice. As expected, platelet adhesion to immobilized fibrinogen was significantly increased (P<0.05) in the absence of Plk3. Surprisingly, when analyzed for fibrin clot retraction, we found that Plk3 null platelets failed to retract fibrin clot. These results suggest that Plk3, a mitotic kinase, plays a significant role in regulation of platelet function such as TxA2 generation, granular secretion, and clot retraction, thus affecting the process of thrombosis.
Disclosures:
No relevant conflicts of interest to declare.
Factor XI contributes to thrombin generation in the absence of factor XII
