Study On The Role And Mechanism Of Platelet Glycoproten GPIbα In Regulating Platelet Function And Tumor Metastasis

Part Ⅰ The role and mechanism of platelet glycoproten Iba in platelet function and tumor metastasisPlatelets generated from megakaryocytes（MKs）play an important role in regulating thrombosis and hemostasis in the circulation.GPIba is a main subunit of GPIb-Ⅸ complex and plays an important role in platelet function.The nitrogen terminus of GPIbα contains the VWF and thrombin binding sites.However,the binding of VWF to GPIb-Ⅸ is strictly controlled and usually occurs only at the site of vascular injury or under pathological high shear stress.Because shear stress can cause changes in VWF and platelets.In vitro,ristocetin and botroccetin are commonly used to induce VWF binding to GPIb-Ⅸ and to simulate the effects of subcutaneous matrix and shear stress on VWF and/or GPIb-Ⅸ.The platelet pattern in hemostasis and thrombosis involves a priming step that relies on platelet membrane receptors binding to ligands on the surface of damaged or inflammatory blood vessels.Once bound to the surface,platelets provide a unique microenvironment that supports the aggregation of more platelets and the formation of fibrin-rich networks produced by clotting factors.The role of platelets in tumorigenesis and inflammation goes beyond the hemostasis/thrombosis model.Our recent study showed that pKa-dependent phosphorylation of GPIba at Ser166 negatively regulates the binding function of GPIb-Ⅸto VWF.The 14-3-3 ζ is an important signaling molecule in cells,combined with GPIb-Ⅸis regulated by phosphorylation serine residues.GPIb alpha carbon end and the combination of the 14-3-3ζ need RYSGHpS⁶⁰⁹L sequence,and Ser⁶⁰⁹ phosphorylation on GPIb alpha carbon high affinity with the 14-3-3ζ plays a key role.In conclusion,GPIbα plays an important role in the regulation of platelet function.Platelets have an inherent ability to adhere and have long been considered necessary for hemostasis and thrombosis.In addition to the platelet paradigm used for blood clotting and thrombosis,it is increasingly clear that platelet adhesion potential influences pathological events and not just its role in state maintenance.In fact,the new hypothesis suggests that the platelet pattern in hemostasis and thrombosis,namely platelet accumulation and fibrin matrix formation,may provide the mechanism for circulating tumor cell metastasis.Experimental evidence of platelet influence on tumor metastasis has been provided in experimental models that reduce circulating platelet count and reduce metastasis.Following the early observation of atypical and protumorigenic aggregates of tumor cells and platelets,knowledge of how tumor cells use platelets to survive,block,and eventually leak from blood vessels to distant organs has increased greatly in the past few decades.Then,it was found that a variety of molecules involved in this process,such as adhesion receptor p-select element,GPIb-Ⅸ or integrin αⅡbβⅢ,P2Y12 platelet activation receptors or protease activated receptor 1（PAR-1）,or the source of platelet growth factors.Activation of phospholipase C,phospholipase A2,and phosphatidylinositol 3-kinase（PI3K）and other kinases,when VWF binds to GPIb-Ⅸ-Ⅴ,can be initiated in cooperation or independently with the Fc receptor FcyRIIA or FCRy chain.Subsequently,the activated protein kinase or second messenger is involved in downstream activation of integrin αⅡBβⅢ.In in vitro,the addition of glioblastoma,neuroepithelioma,or breast cancer cells was found to alleviate aggregation by preincubation of platelets with GPIb antibodies.A 15-fold reduction in the number of metastases was detected in GPIb-Ⅸ-Ⅴ deficient mice and GPIb receptor fused with the IL-4 receptor.These results clearly demonstrate the role of GPIb-Ⅸ-Ⅴ in lung metastasis of melanoma,and the GPIb-Ⅸ-Ⅴ mediated signaling pathway appears to be indispensable.GPIba did not significantly promote cell metastasis in P-selectin knockout mice.Therefore,the role of GPIb-Ⅸ-Ⅴ in early metastasis of melanoma remains unclear and may require further study in GPIba knockout mice with spontaneous tumor development.It has been demonstrated that GPIba plays an important role in tumor metastasis,but the mechanism remains unclear.Objective:Study on the role and mechanism of platelet glycoproten Iba in platelet function and tumor metastasisMethods:CRISPR-Cas9 technique was used to delete the total protein（GPIbα-/-）or 10 amino acid from C-terminal（GPIbα10aa-/-）of GPIba gene in mice.Blood was collected from the orbital venous plexus of wild-type（WT）,GPIbα-/-and GPIbα10aa-/-mice using capillary glass tubes for hematological analysis.The platelet morphology of GPIbα-/-and GPIbα10aa-/-mice was observed by Reysch-Gimsa staining.Flow cytometry was used to detect GPIbα,GPIbβ,GPⅨ and GPⅡb/Ⅲa expression.Flow cytometry was used to detect GPIbα-/-and GPIbα10aa-/-mice platelet apoptosis（mitochondrial membrane potential depolarization,PS externalization）and activation（P-selectin and JON/A binding）.The platelet survival time in GPIbα-/-and GPIbα10aa-/-mice was determined by biotin-labeled platelets.The aggregation ability of GPIbα10aa-/-mouse platelets was detected by platelet aggregator.Western blot was used to detect the phosphorylation of Akt protein at Ser473 and Thr308 in WT and GPIbα10aa-/-mouse platelets stimulated by Collagen Ⅱ,PAR4-AP,U46619,ADP and botroccetin.Mouse melanoma B16F10 cells were injected into WT,GPIbα-/-,GPIbα10aa-/-and c-MPL-/-mice by tail vein injection,respectively,to observe the metastasis of melanoma B16F10 cells.Flow cytometry and confocal microscopy were used to detect the interaction of B16F10 cells with platelets in WT and GPIbα10aa-/-mice,respectively.The expression of WT and GPIbα10aa-/-platelet P-selectin induced by B16F10 cells was detected by flow cytometry.The release of TGF-β1 in serum was detected by enzyme-linked immunosorbent assay.Murine lung cancer cells（LLC）were subcutaneously injected into WT mice to observe the effect of MPac on lung metastasis.Results:1)GPIbα-/-mice showed severe thrombocytopenia and giant platelets,while there was no significant change in platelet morphology in GPIba10aa-/-mice.2)GPIbα-/mice platelet membrane surface GPIbα,GPIbβ and GPIbⅨ expression significantly reduced.GPIbα,GPIbβ and GPIbⅨ express were not obvious reduced in GPIbα10aa-/mice.3)Compared with WT control,the apoptosis and activation of GPIbα-/-and GPIbα10aa-/-mouse platelets stimulated by PAR4-AP,CRP,ADP and U46619 were significantly reduced.4)GPIba and GPIbα10aa gene knockout significantly prolonged the platelet lifetime in peripheral blood of mice.5)Thrombin,PAR4-AP,ADP,Collagen Ⅱ,U46619 and botrocetin induced platelet aggregation in GPIbα10aa-/-mice was decreased significantly.6)In GPIbα10aa-/-mice,the occlusion time of the mesenteric arteriole were significantly prolonged after ferric chloride injury.7)GPIbα and GPIbal0aa gene knockout decreased platelet interaction with tumor cells,and reduced the release of soluble p-selectin and TGF-β1,thereby inhibiting B16F10 cell metastasis.8)The peptide MPaC effectively inhibited the metastasis of B16F10 cells and LLC cells to the lung of mice.Conclusion:The platelet functions and tumor metastasis in GPIbα-/-and GPIbα10aa-/mice were significantly reduced,indicating the essential role of GPIbα in the regulation of platelet functions and metastasis.Part Ⅱ Actin polymerization regulates platelet membrane glycoprotein GPIba sheddingObjective:To investigate the role of actin polymerization in regulating the shedding of GPIbα from platelets.Methods:Isolated human or mouse platelets were incubated with jasplakinolide（actin polypeptide）or cytorelaxin B（cytoskeleton destroying agent）,and GPIba shedding was detected by flow cytometry and Western blot.Jasplakinolide was co-cultured with isolated human or mouse platelets,and the aggregation function of platelets after GPIba shedding was detected by stimulation with different concentrations of collagen,ADP,U46619,ristocetin/botrocetin and thrombin.The effects of jasplakinolide on platelet GPIbα shedding induced by thrombin or A23187 were analyzed by flow cytometry and Western blot.Flow cytometry and Western blot were used to investigate the role of calpsin activators dibuvacaine and calpsin inhibitor 1 in actin polymerization-induced GPIbα shedding.Both recombinant wild-type GPIb-Ⅸ（1B9）cells and mutant GPIB-Ⅸ（ΔABP）cells（containing filamin A binding site deficient mutant GPIB-Ⅸ）were used to test the role of the interaction between filamin A and the cytoplasmic domain of GPIba in regulating the shedding of GPIbα induced by actin polymerization.Results:1)Actin polymerization regulates ADAM17-mediated GPIbα shedding;2)Jasplakinolide increased thrombin and A23187-induced GPIbα shedding,while cytorelaxin B decreased thrombin induced GPIbα shedding.3)Actin polymerization activates calprotease to hydrolyze filamin A,resulting in ADAM 17-mediated GPIbα shedding;4)The interaction between filamin A and the cytoplasmic domain of GPIba plays A key role in the process of GPIbα shedding induced by actin polymerization.Conclusion:Our study shows that actin polymerization can regulate ADAM 17mediated GPIba shedding.The binding of filamin A to the cytoplasmic domain of GPIba is a necessary condition for the regulation.