Kesheng Dai

Podoplanin requires sialylated O-glycans for stable expression on lymphatic endothelial cells and for interaction with platelets

O-glycosylation of podoplanin (PDPN) on lymphatic endothelial cells is critical for the separation of blood and lymphatic systems by interacting with platelet C-type lectin-like receptor 2 during development. However, how O-glycosylation controls endothelial PDPN function and expression remains unclear. In this study, we report that core 1 O-glycan-deficient or desialylated PDPN was highly susceptible to proteolytic degradation by various proteases, including metalloproteinases (MMP)-2/9. We found that the lymph contained activated MMP-2/9 and incubation of the lymph reduced surface levels of PDPN on core 1 O-glycan-deficient endothelial cells, but not on wild-type ECs. The lymph from mice with sepsis induced by cecal ligation and puncture, which contained bacteria-derived sialidase, reduced PDPN levels on wild-type ECs. The MMP inhibitor, GM6001, rescued these reductions. Additionally, GM6001 treatment rescued the reduction of PDPN level on lymphatic endothelial cells in mice lacking endothelial core 1 O-glycan or cecal ligation and puncture-treated mice. Furthermore, core 1 O-glycan-deficient or desialylated PDPN impaired platelet interaction under physiological flow. These data indicate that sialylated O-glycans of PDPN are essential for platelet adhesion and prevent PDPN from proteolytic degradation primarily mediated by MMPs in the lymph.

Calpain Activator Dibucaine Induces Platelet Apoptosis

Calcium-dependent calpains are a family of cysteine proteases that have been demonstrated to play key roles in both platelet glycoprotein Ibα shedding and platelet activation and altered calpain activity is associated with thrombotic thrombocytopenic purpura. Calpain activators induce apoptosis in several types of nucleated cells. However, it is not clear whether calpain activators induce platelet apoptosis. Here we show that the calpain activator dibucaine induced several platelet apoptotic events including depolarization of the mitochondrial inner transmembrane potential, up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation and phosphatidylserine exposure. Platelet apoptosis elicited by dibucaine was not affected by the broad spectrum metalloproteinase inhibitor GM6001. Furthermore, dibucaine did not induce platelet activation as detected by P-selectin expression and PAC-1 binding. However, platelet aggregation induced by ristocetin or α-thrombin, platelet adhesion and spreading on von Willebrand factor were significantly inhibited in platelets treated with dibucaine. Taken together, these data indicate that dibucaine induces platelet apoptosis and platelet dysfunction.

Aspirin Induces platelet apoptosis

Aspirin is widely used in the treatment of a number of clinical conditions. Although aspirin is being thought to be a relatively “safe” medicine, it also has some side effects, particularly the risk of bleeding which may be severe and lead to death. The mechanisms, however, are not totally understood. It has been reported recently that aspirin induces apoptosis in many cell types. Thus, the aim of the current study is to explore whether aspirin induces platelet apoptosis. The data show that mitochondrial transmembrane potential (ΔΨm) depolarizations and phosphatidylserine (PS) exposures were dose-dependently induced by aspirin in platelets. To further confirm that aspirin incurs platelet apoptosis, caspase-3 activity was measured in platelets, and the result indicated that aspirin induced caspase-3 activation. Furthermore, the mean volume of platelets incubated with aspirin was obviously reduced. Caspase inhibitor z-VAD-fmk inhibited aspirin induced apoptotic platelet shrinkage and ΔΨm depolarization, but had no effect on PS exposure. In addition, platelets incubated with cyclooxygenase inhibitor indomethacin did not incur ΔΨm depolarazation and PS exposure. Taken together, the data indicate that aspirin induces platelet apoptosis via caspase-3 activation.

Effects of microgravity and hypergravity on platelet functions

Many serious thrombotic and haemorrhagic diseases or fatalities have been documented in human being exposed to microgravity or hypergravity environments, such as crewmen in space, roller coaster riders, and aircrew subjected to high-G training. Some possible related organs have been examined to explore the mechanisms underlying these gravity change-related diseases. However, the role of platelets which are the primary players in both thrombosis and haemostasis is unknown. Here we show that platelet aggregation induced by ristocetin or collagen and platelet adhesion to von Willebrand factor (VWF) were significantly decreased after platelets were exposed to simulated microgravity. Conversely, these platelet functions were increased after platelets were exposed to hypergravity. The tail bleeding time in vivo was significantly shortened in mice exposed to high-G force, whereas, was prolonged in hindlimb unloaded mice. Furthermore, three of 23 mice died after 15 minutes of -8 Gx stress. Platelet thrombi disseminated in the heart ventricle and blood vessels in the brain, lung, and heart from the dead mice. Finally, glycoprotein (GP) Ibalpha surface expression and its association with the cytoskeleton were significantly decreased in platelets exposed to simulated microgravity, and obviously increased in hypergravity-exposed platelets. These data indicate that the platelet functions are inhibited in microgravity environments, and activated under high-G conditions, suggesting a novel mechanism for gravity change-related haemorrhagic and thrombotic diseases. This mechanism has important implications for preventing and treating gravity change-related diseases, and also suggests that special attentions should be paid to human actions under different gravity conditions.

Mechanism of platelet functional changes and effects of anti-platelet agents on in vivo hemostasis under different gravity conditions

Serious thrombotic and hemorrhagic problems or even fatalities evoked by either microgravity or hypergravity occur commonly in the world. We recently reported that platelet functions are inhibited in microgravity environments and activated under high-G conditions, which reveals the pathogenesis for gravity change-related hemorrhagic and thrombotic diseases. However, the mechanisms of platelet functional variations under different gravity conditions remain unclear. In this study we show that the amount of filamin A coimmunoprecipitated with GPIbalpha was enhanced in platelets exposed to modeled microgravity and, in contrast, was reduced in 8 G-exposed platelets. Hypergravity induced actin filament formation and redistribution, whereas actin filaments were reduced in platelets treated with modeled microgravity. Furthermore, intracellular Ca2+ levels were elevated by hypergravity. Pretreatment of platelets with the cell-permeable Ca2+ chelator BAPTA-AM had no effect on cytoskeleton reorganization induced by hypergravity but significantly reduced platelet aggregation induced by ristocetin/hypergravity. Two anti-platelet agents, aspirin and tirofiban, effectively reversed the shortened tail bleeding time and reduced the death rate of mice exposed to hypergravity. Furthermore, the increased P-selectin surface expression was obviously reduced in platelets from mice treated with aspirin/hypergravity compared with those from mice treated with hypergravity alone. These data suggest that the actin cytoskeleton reorganization and intracellular Ca2+ level play key roles in the regulation of platelet functions in different gravitational environments. The results with anti-platelet agents not only further confirm the activation of platelets in vivo but also suggest a therapeutic potential for hypergravity-induced thrombotic diseases.

Role of 14-3-3ζ in Platelet Glycoprotein Ibα-von Willebrand Factor Interaction-Induced Signaling

The interaction of platelet glycoprotein (GP) Ib-IX with von Willebrand factor (VWF) exposed at the injured vessel wall or atherosclerotic plaque rupture initiates platelet transient adhesion to the injured vessel wall, which triggers intracellular signaling cascades leading to platelet activation and thrombus formation. 14-3-3ζ has been verified to regulate the VWF binding function of GPIb-IX by interacting with the cytoplasmic domains of GPIb-IX. However, the data regarding the role of 14-3-3ζ in GPIb-IX-VWF interaction-induced signaling still remain controversial. In the present study, the data indicate that the S609A mutation replacing Ser609 of GPIbα with alanine (S609A) significantly prevented the association of 14-3-3ζ with GPIbα before and after the VWF binding to GPIbα. GPIb-IX-VWF interaction-induced activations of Src family kinases and protein kinase C were clearly reduced in S609A mutation. Furthermore, S609A mutation significantly inhibited GPIb-IX-VWF interaction-induced elevation of cytoplasmic Ca2+ levels in flow cytometry analysis. Taken together, these data indicate that the association of 14-3-3ζ with the cytoplasmic domain of GPIbα plays an important role in GPIb-IX-VWF interaction-induced signaling.

Integrin-αIIbβ3-mediated outside-in signalling activates a negative feedback pathway to suppress platelet activation.

Integrin-αIIbβ3-mediated outside-in signalling is widely accepted as an amplifier of platelet activation; accumulating evidence suggests that outside-in signalling can, under certain conditions, also function as an inhibitor of platelet activation. The role of integrin-αIIbβ3-mediated outside-in signalling in platelet activation is disputable. We employed flow cytometry, aggregometry, immunoprecipitation, and immunoblotting to investigate the role of integrin-αIIbβ3-mediated outside-in signalling in platelet activation. Integrin αIIbβ3 inhibition enhances agonist-induced platelet ATP secretion. Human platelets lacking expression of αIIbβ3 exhibited more platelet ATP secretion than their wild-type counterparts. Moreover, integrin-αIIbβ3-mediated outside-in signals activate SHIP-1, which in turn mediates p-Akt dephosphorylation, leading to inactivation of PI3K/Akt signalling. Furthermore, 3AC (SHIP-1 inhibitor) inhibits platelet disaggregation, and promotes platelet ATP secretion. Upon ADP stimulation, Talin is recruited to αIIbβ3, and it is dissociated from αIIbβ3 when platelets disaggregate. In addition, treatment with RUC2, an inhibitor of αIIbβ3, which blocks αIIbβ3-mediated outside-in signalling, can markedly prevent the dissociation of talin from integrin. SHIP1 Inhibitor 3AC inhibits the dissociation of talin from integrin-β3. These results suggest that integrin-αIIbβ3-mediated outside-in signalling can serve as a brake to restrict unnecessary platelet activation by activated SHIP-1, which mediated the disassociation of talin from β3, leading to integrin inactivation and blocking of PI3K/Akt signalling to restrict platelet ATP secretion.