title: A critical role for the regulation of Syk from agglutination to aggregation in human platelets.
abstract: Agglucetin, a tetrameric glycoprotein (GP) Ibα agonist from Formosan Agkistrodon acutus venom, has been characterized as an agglutination inducer in human washed platelets (WPs). In platelet-rich plasma (PRP), agglucetin dramatically elicits a biphasic response of agglutination and subsequent aggregation. For clarifying the intracellular signaling events from agglutination to aggregation in human platelets, we examined the essential signaling molecules involved through the detection of protein tyrosine phosphorylation (PTP). In WPs, an anti-GPIbα monoclonal antibody (mAb) AP1, but not a Src kinase inhibitor PP1, completely inhibited agglucetin-induced agglutination. However, PP1 but not AP1 had a potent suppression on platelet aggregation by a GPVI activator convulxin. The PTP analyses showed agglucetin alone can cause a weak pattern involving sequential phosphorylation of Lyn/Fyn, Syk, SLP-76 and phospholipase Cγ2 (PLCγ2). Furthermore, a Syk-selective kinase inhibitor, piceatannol, significantly suppressed the aggregating response in agglucetin-activated PRP. Analyzed by flow cytometry, the binding capacity of fluorophore-conjugated PAC-1, a mAb recognizing activated integrin αIIbβ3, was shown to increase in agglucetin-stimulated platelets. Again, piceatannol but not PP1 had a concentration-dependent suppression on agglucetin-induced αIIbβ3 exposure. Moreover, the formation of signalosome, including Syk, SLP-76, VAV, adhesion and degranulation promoting adapter protein (ADAP) and PLCγ2, are required for platelet aggregation in agglucetin/fibrinogen-activated platelets. In addition, GPIbα-ligation via agglucetin can substantially promote the interactions between αIIbβ3 and fibrinogen. Therefore, the signal pathway of Lyn/Fyn/Syk/SLP-76/ADAP/VAV/PLCγ2/PKC is sufficient to trigger platelet aggregation in agglucetin/fibrinogen-pretreated platelets. Importantly, Syk may function as a major regulator for the response from GPIbα-initiated agglutination to integrin αIIbβ3-dependent aggregation in human platelets.
authors: Chang Gung University of Science and Technology, Kwei-Shan, Tao-Yuan, Taiwan.
authors: Chun-Ho
authors: CH
authors: Shih
authors: Chang Gung University of Science and Technology, Kwei-Shan, Tao-Yuan, Taiwan.
authors: Tin-Bin
authors: TB
authors: Chiang
authors: Chang Gung University of Science and Technology, Kwei-Shan, Tao-Yuan, Taiwan. Electronic address: wjwang@gw.cgust.edu.tw.
authors: Wen-Jeng
authors: WJ
authors: Wang
mesh_terms: D000371:Agglutination / Q000502:physiology*; D001792:Blood Platelets / Q000502:physiology*; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D010974:Platelet Aggregation / Q000502:physiology*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D000072377:Syk Kinase
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D010980:Platelet Membrane Glycoproteins; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase
keywords: ADAP; Agglucetin; Convulxin; FITC; Fc receptor γ-chain; FcRγ; GP; Glycoprotein Ibα; Integrin αIIbβ3; LAT; PI3K; PKC; PLCγ2; PRP; PTP; SH2-domain-containing leucocyte protein of 76kDa; SLP-76; Syk; WPs; adhesion and degranulation promoting adapter protein; fluorescein isothiocyanate; glycoprotein; linker for activation of T-cells; mAb; monoclonal antibody; pAb; phosphoinositide 3-kinase; phospholipase Cγ2; platelet-rich plasma; polyclonal antibody; protein kinase C; protein tyrosine phosphorylation; vWf; von Willebrand factor; washed platelets