Rong Yan

The glycoprotein Ibα–von Willebrand factor interaction induces platelet apoptosis

Summary.  Background: The interaction of glycoprotein (GP) Ibα with von Willebrand factor (VWF) initiates platelet adhesion, and simultaneously triggers intracellular signaling cascades leading to platelet aggregation and thrombus formation. Some of the signaling events are similar to those occurring during apoptosis, however, it is still unclear whether platelet apoptosis is induced by the GPIbα–VWF interaction. Objectives: To investigate whether the GPIbα–VWF interaction induces platelet apoptosis and the role of 14‐3‐3ζ in apoptotic signaling. Methods: Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing wild‐type (1b9) or mutant GPIb–IX interacting with VWF by flow cytometry or western blotting. Results: Ristocetin‐induced GPIbα–VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF; however, the shear‐induced apoptosis was eliminated by the anti‐GPIbα antibody AK2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb–IX with GPIbα truncated at residue 551 or a serine‐to‐alanine mutation at the 14‐3‐3ζ‐binding site in GPIbα. Conclusions: This study demonstrates that the GPIbα–VWF interaction induces apoptotic events in platelets, and that the association of 14‐3‐3ζ with the cytoplasmic domain of GPIbα is essential for apoptotic signaling. This finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases.

Identification of a Novel 14-3-3ζ Binding Site Within the Cytoplasmic Domain of Platelet Glycoprotein Ibα That Plays a Key Role in Regulating the von Willebrand Factor Binding Function of Glycoprotein Ib-IX

Rationale: The interaction between platelet glycoprotein (GP) Ib-IX and von Willebrand factor (VWF) is initiated by conformational changes in immobilized VWF and is also regulated by the intraplatelet proteins 14-3-3&zgr; and filamin A. Both 14-3-3&zgr; and filamin A associate with the cytoplasmic domain of GPIb&agr;, whereas little is known about their relationship in regulating the VWF binding function of GPIb-IX. Objective: To explore the mechanism underlying the roles of 14-3-3&zgr; and filamin A in regulating the VWF binding function of GPIb-IX. Methods and Results: A truncation mutant of GPIb&agr; (&Dgr;565) deleting the C-terminal 14-3-3&zgr; binding sites retained 14-3-3&zgr; binding function, in contrast, deletion of the C-terminal residues 551 to 610 of GPIb&agr; totally abolished 14-3-3&zgr; binding, indicating that the residues 551 to 564 of GPIb&agr; are important in the interaction between 14-3-3&zgr; and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIb&agr; suggesting phosphorylation of a population of GPIb&agr; molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), R557GpSLP561 corresponding to residues 557 to 561 of GPIb&agr; eliminated the association of 14-3-3&zgr; with &Dgr;565. MP-P also promoted GPIb-IX association with the membrane skeleton, and inhibited ristocetin-induced platelet agglutination, VWF binding to platelets and platelet adhesion to immobilized VWF. Furthermore, a GPIb-IX mutant replacing Ser559 of GPIb&agr; with alanine showed an enhanced association with the membrane skeleton, reduced ristocetin-induced VWF binding, and diminished ability to mediate cell adhesion to VWF under flow conditions. Conclusions: These data suggest a phosphorylation-dependent binding of 14-3-3&zgr; to central filamin A binding site of GPIb&agr;, and the dimeric 14-3-3&zgr; binding to both the C-terminal site and central RGpSLP site inhibits GPIb-IX association with the membrane skeleton and promotes the VWF binding function of GPIb-IX.

Calmodulin antagonists induce platelet apoptosis

Calmodulin (CaM) antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. In platelets, CaM has been found to bind directly to the cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet functions. However, it is still unknown whether CaM antagonists induce platelet apoptosis. Here we show that CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), tamoxifen (TMX), and trifluoperazine (TFP) induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, and phosphatidylserine exposure. CaM antagonists did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP-, botrocetin-, and alpha-thrombin-induced platelet aggregation, platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonists. Furthermore, cytosolic Ca(2+) levels were obviously elevated by both W7 and TMX, and membrane-permeable Ca(2+) chelator BAPTA-AM significantly reduced apoptotic events in platelets induced by W7. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis. The elevation of the cytosolic Ca(2+) levels may be involved in the regulation of CaM antagonists-induced platelet apoptosis.

Role of cAMP-dependent protein kinase in the regulation of platelet procoagulant activity

The membrane microparticle (MP) formation and phosphatidylserine (PS) exposure evoked by platelet activation provide catalytic surfaces for thrombin generation. Several reports have indicated the effects of cAMP-elevating agents on agonist-induced MP formation and PS exposure; however, the mechanism still remains unclear. Here we show that inhibition of basal cyclic AMP-dependent protein kinase (PKA) activity incurred platelet MP formation and PS exposure. Pretreatment of platelets with cAMP-elevating agent, forskolin, abolished thrombin plus collagen-induced MP formation and PS exposure, and obviously decreased calcium ionophore-evoked MP shedding. Moreover, the inhibitory effects of forskolin on agonists-induced MP formation and PS exposure were reversed by the PKA inhibitor H89. PKA inhibitor-induced MP formation was dose-dependently inhibited by calpain inhibitor MDL28170, and forskolin abrogated thrombin plus collagen-induced calpain activation. In conclusion, PKA plays key roles in the regulation of platelet MP formation and PS exposure. PKA-mediated MP shedding is dependent on calpain activation.

Prolonged inhibition of protein kinase A results in metalloproteinase-dependent platelet GPIbα shedding

INTRODUCTION The interaction of platelet glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) exposed at the injured vessel wall initiates platelet adhesion and thrombus formation. Thus GPIbalpha ectodomain shedding has important implications for thrombosis and hemostasis. A disintegrin and metalloproteinase 17 (ADAM17) was identified recently to play an essential role in agonist induced GPIbalpha shedding. Here we show that prolonged inhibition of protein kinase A (PKA) results in metalloproteinase-dependent GPIbalpha shedding. METHODS AND RESULTS GPIbalpha was shed from platelets prolongedly incubated with PKA inhibitors in a dose-dependent manner. In platelets treated with PKA inhibitor H89, the level of GPIbalpha shedding was significantly higher than that in calcium ionophore or alpha-thrombin treated platelets, however, P-selectin surface expression was significantly lower. PKA inhibition mediated GPIbalpha shedding was reversed by PKA activator forskolin and partially inhibited by membrane-permeable calpain inhibitors. Furthermore, the metalloproteinase inhibitor GM6001 or EDTA completely inhibited H89 induced GPIbalpha shedding, indicating that it was metalloproteinase-dependent. Time course experiments revealed that the maximum GPIbalpha shedding occurred at 30 minutes after treatment with PKA inhibitor. Platelets prolongedly treated with PKA inhibitor exhibited significant decrease in botrocetin-induced aggregation and shear-induced adhesion on VWF. CONCLUSIONS These data show that prolonged inhibition of PKA results in metalloproteinase-dependent platelet GPIbalpha ectodomain shedding. This finding has physiological implications for hemostasis and limiting thrombus infinite formation after platelet activation, and it also suggests a novel strategy to develop new drugs for thrombotic diseases.

The role of calpain in the regulation of ADAM17-dependent GPIbα ectodomain shedding

There are evidence that both a disintegrin and metalloproteinase 17 (ADAM17) and calpain are involved in platelet glycoprotein (GP)Ibalpha ectodomain cleavage. However, the relationship between the two enzymes in the shedding process remains unclear. Here we show that calcium ionophore A23187- and alpha-thrombin-induced GPIbalpha shedding is completely inhibited by the metalloproteinase inhibitor GM6001, whereas it is only partially inhibited by calpain inhibitors. Calpain activator dibucaine-induced GPIbalpha shedding was completely inhibited by both metalloproteinase and calpain inhibitors. On the other hand, calpain inhibitors did not inhibit GPIbalpha shedding induced by the reagents that specifically activate ADAM17. Furthermore, A23187-induced GPIbalpha shedding in Chinese hamster ovary cells expressing wild-type or mutant GPIb-IX was also partially inhibited by calpain inhibitors and almost completely inhibited by GM6001. Therefore, these data indicate that calpain plays an important role in the regulation of ADAM17-dependent GPIbalpha ectodomain shedding in both platelets and nucleated cells.

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.

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.

Hypergravity results in human platelet hyperactivity

Thrombotic diseases or fatalities have been reported to occasionally occur under conditions of hypergravity although the mechanism is still unclear. To investigate the effect of hypergravity on platelets that are the primary players in thrombus formation, platelet rich plasma (PRP) or washed platelets were exposed to hypergravity at 8 G for 15 minutes. No platelet aggregation was induced by 8 G alone, whereas ristocetin or collagen-induced platelet aggregation was significantly increased. The number of platelets adherent to immobilized fibrinogen and the area of platelets spreading on von Willbrand factor (VWF) matrix were increased simultaneously. Flow cytometry assay indicated that integrin αIIbß3 was partially activated in 8 Gexposed platelets, but there was no significant difference in P-selectin surface expression between platelets treated with 8 G and 1 G control. The results indicate that hypergravity leads to human platelet hyperactivity, but fails to incur essential platelet activation events, suggesting a novel mechanism for thrombotic diseases occurring under hypergravitional conditions.

The critical roles of cyclic AMP/cyclic AMP-dependent protein kinase in platelet physiology

Platelets are the primary players in both thrombosis and hemostasis. Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA) are important signaling molecules in the regulation of platelet function, such as adhesion, aggregation, and secretion. Elevation of intracellular cAMP, which induces the activation of PKA, results in the inhibition of platelet function. Thus, tight control of the intracellular cAMP/PKA signaling pathway has great implications for platelet-dependent hemostasis and effective cardiovascular therapy. In this review, we summarize the PKA substrates and their contributions to platelet function, especially the advancing understanding of the cAMP/PKA-dependent signaling pathway in platelet physiology. In addition, we suggest the possibility that cAMP/PKA is involved in the platelet procoagulant process and receptor ectodomain shedding.