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.

Calmodulin (CaM) antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some ofwhich have been extensively used as anti-cancer agents. In platelets, CaMhas 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. CaMantagonists did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP-, botrocetin-, and α-thrombin-induced platelet aggregation, platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pretreatedwith CaM antagonists. Furthermore, cytosolic Ca2+ levelswere obviously elevated by both W7and TMX, and membrane-permeable Ca2+ chelator BAPTA AMsignificantly reduced apoptotic events in platelets induced by W7. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis. The elevation of the cytosolic Ca2+ levels may be involved in the regulation of CaM antagonists-induced platelet apoptosis.

There are evidence that both a disintegrin and metalloproteinase 17 (ADAM17) and calpain are involved in platelet glycoprotein (GP)Iba ectodomain cleavage. However, the relationship between the two enzymes in the shedding process remains unclear. Here we show that calcium ionophore A23187-and a-thrombin-induced GPIba shedding is completely inhibited by the metalloproteinase inhibitor GM6001, whereas it is only partially inhibited by calpain inhibitors. Calpain activator dibucaine-induced GPIba shedding was completely inhibited by both metalloproteinase and calpain inhibitors. On the other hand, calpain inhibitors did not inhibit GPIba shedding induced by the reagents that specifically activate ADAM17. Furthermore, A23187-induced GPIba shedding in Chinese hamster ovary cells expressing wildtype 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 GPIba ectodomain shedding in both platelets and nucleated cells.

Background: The interaction of glycoprotein (GP) Iba 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 GPIba-VWF interaction. Objectives: To investigate whether the GPIba-VWF interaction induces platelet apoptosis and the role of 14-3-3f 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 GPIba-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-GPIba antibody AK2. Furthermore, apoptotic events occurred in 1b9 cells stimulated withVWFand ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb-IX with GPIba truncated at residue 551 or a serine-to-alanine mutation at the 14-3-3fbinding site in GPIba. Conclusions: This study demonstrates that the GPIba-VWF interaction induces apoptotic events in platelets, and that the association of 14-3-3f with the cytoplasmic domain of GPIba is essential for apoptotic signaling. This finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases.

The platelet receptor for von Willebrand factor (VWF), glycoprotein (GP) Ib–IX, mediates platelet adhesion and activation. The cytoplasmic domains of the GPIb α and β subunits contain binding sites for the phosphorylation-dependent signaling molecule, 14-3-3. Here we show that a novel membrane-permeable inhibitor of 14-3-3-GPIbα interaction, MPα C, potently inhibited VWF binding to platelets and VWF-mediated platelet adhesion under flow conditions. MPαC also inhibited VWF-dependent platelet agglutination induced by ristocetin. Furthermore, activation of the VWF binding function of GPIb-IX induced by GPIbβ dephosphorylation is diminished by mutagenic disruption of the 14-3-3 binding site in the C-terminal domain of GPIbα, mimicking MPα C-induced inhibition, indicating that the inhibitory effect of MPαC is likely to be caused by disruption of 14-3-3 binding to GPIbα. These data suggest a novel 14-3-3-dependent regulatory mechanism that controls the VWF binding function of GPIb-IX, and also suggest a new type of antiplatelet agent that may be potentially useful in preventing or treating thrombosis.