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.

Shear-induced platelet adhesion through the interaction of glycoprotein (GP) Ibα with von Willebrand factor (VWF) exposed at the injured vessel wall or atherosclerotic plaque rupture is a prerequisite for the physiological hemostatic process or pathological thrombus formation in stenosed arteries. Here we show that shear-induced interaction of platelets with immobilized VWF results in GPIbα ectodomain shedding. Washed platelets were exposed to VWF-coated glass capillary or cone-and-plate viscometer at different shear rates, and GPIbα ectodomain was shed from platelets, while a small mass of GPIbα COOH-terminal peptide, ～17 kDa, was increased correspondingly. The extent of GPIbα shedding was enhanced with the concentration of immobilized VWF and the time duration of constant shear stress, whereas it was obviously reduced with the decreased number of adherent platelets. Pretreatment of platelets with membrane-permeable calpain inhibitors and metalloproteinase inhibitor abolished shearinduced GPIbα shedding. Furthermore, GPIbα shedding was obviously diminished by anti-integrin-αIIbβ3 monoclonal antibody SZ21, phosphatidylinositol 3-kinase inhibitor wortmannin, and cell-permeable calcium chelator 1, 2-bis(o-aminophenoxy)ethane-N, N, N′, N′-tetraacetic acid. These results indicate that shear-induced platelet-VWF interaction results in calpain and metalloproteinase-dependent GPIb ectodomain shedding. These findings not only have a physiological implication in understanding the presence of glycocalicin in normal circulation, but also suggest a novel mechanism for the negative regulation of platelet function and the limitation of platelet thrombus infinite formation under pathophysiological flow conditions.

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.

Introduction: The interaction of platelet glycoprotein (GP) Ibα with von Willebrand factor (VWF) exposed at the injured vessel wall initiates platelet adhesion and thrombus formation. Thus GPIbα 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 GPIbα shedding. Here we show that prolonged inhibition of protein kinase A (PKA) results in metalloproteinase-dependent GPIbα shedding. Methods and Results: GPIbα was shed from platelets prolongedly incubated with PKA inhibitors in a dosedependent manner. In platelets treated with PKA inhibitor H89, the level of GPIbα shedding was significantly higher than that in calcium ionophore or α-thrombin treated platelets, however, P-selectin surface expression was significantly lower. PKA inhibition mediated GPIbα 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 GPIbα shedding, indicating that itwasmetalloproteinasedependent. Time course experiments revealed that the maximum GPIbαshedding 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 inmetalloproteinase-dependent platelet GPIbα 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.