Abstract
The cytoskeleton participates in the coordinated regulation of intracellular signaling molecules, following agonist stimulation of cells. We have demonstrated that von Willebrand factor (vWF) induced the cytoskeletal association and activation of phosphatidylinositol 3-kinase (PtdIns 3-kinase) in human platelets. The activation of PtdIns 3-kinase coincided with the tyrosine phosphorylation of multiple platelet proteins, as assessed by anti-phosphotyrosine immunoblotting. One of these tyrosine-phosphorylated proteins, pp60c-src, became specifically enriched in the cytoskeletal fraction of vWF-stimulated platelets. The vWF-stimulated cytoskeletal association of PtdIns 3-kinase and pp60c-src required platelet stirring and aggregation, was specifically blocked by an anti-GPIb monoclonal antibody, and was not observed in platelets lacking the glycoprotein Ib/IX complex (Bernard-Soulier syndrome). Pretreatment of normal platelets with 5 mM EDTA (37 degrees C for 90 min) or RGDS (2 mM), which disrupts the binding of various adhesive proteins to platelet integrins and inhibits fibrinogen-mediated platelet aggregation, did not alter the vWF-stimulated activation and cytoskeletal association of PtdIns 3-kinase and pp60c-src. Pretreatment of platelets with acetylsalicylic acid (1 mM) completely abolished vWF-stimulated production of thromboxane A2, dense granule release, and the activation of protein kinase C, without altering the activation and cytoskeletal translocation of PtdIns 3-kinase and pp60c-src. Our results suggest that vWF binding to the platelet adhesion receptor glycoprotein Ib/IX can mediate activation and translocation of both tyrosine and lipid kinase(s) independent of other agonists.
Highlights
VON WILLEBRAND FACTOR STIMULATES THE CYTOSKELETAL ASSOCIATIONAND ACTIVATION OF PHOSPHATIDYLINOSITOL 3-KINASE AND ~ ~ 6 0 ' " " ~ HUI NMAN PLATELETS*
Stimulation of Platelet Aggregation bvyWF and Ristocetin in the Presence of EDTA or Anti-GPZb mAb A.lG"The attachment and immobilization of von Willebrandfactor (vWF) to the subendothelial matrix is essential for its interaction with the plateletadhesion receptor
Ristocetin-induced bindingof vWF to platelets stimulates the cytoskeletal associationof pp60'"". a, washed platelets were exposed to control buffer ; vWF (10pg/ml) and ristoceti(n1 mg/ml) without stirring ;thrombin (1uniffml) ;vWF and GPIbAX (Roth, 1991).In contrast thbeinding of soluble vWF to GPIbAX under conditions of low shear stress requires tphreesence of cationic molecules such as the macrolide antibiotic ristocetin or the snake venom protein, botrocetin (Ruggeri and Zimmerman, 1987)
Summary
Sion, but transduce signalsfrom the extracellular environment t o specific cytoplasmic signaling enzymes (Hynes,1992). Preparation of Washed Platelets-Plateletswereobtained from some studies, cytoskeletal-associated proteins were extractedfrom the healthy volunteers who had not taken anti-platelet medication in the 15,000 x g pellet using radioimmunoprecipitation assay (RIPA)buffer preceding 2 weeks and washed using a modified method of Baenziger (10 mM NaH,PO,, pH 7.0, 150 mM NaCI, 2 mM EDTA, 2 mM phenyland Majerus (1974). 24.3 mM NaH,PO,,4.3 mM %HPO,, pH 6.5, 113 mM NaCl, 5.5 mM each reaction mixture, and the enzyme assay incubated for 20 min at glucose, 0.5% bovine serum albumin, and[10] mM theophylline, washed room temperature. Quantitation of PtdIns 3-Kinase Activity-PtdIns 3-kinase activity thrombin (1unitlml) or vWF (10 pg/ml) and ristocetin (1mg/ml) were was quantitated on platelet extracts diluted l/lOOO-fold by measuring performed in thepresence of CaCI, without fibrinogen.Bovine plasma thenzymatiicncorporation of 32iPntPotdIns(4,5)P2, forming (100 pl, 1:4 dilution) was addedto washed platelets in the absenceof PtdIns(”2P-3,4,5)P3, a s described previously (Susaetal., 1992). Protein concentrations were determined using the Bio-Rad protein assay with bovine serum albumin as a standard. vWF was purifiefdrom cryoprecipitate and fibrinogen was purified from fresh frozen plasma as described previously (Montgomery and Zimmerman, 1978; Jakobsen and Koerulf, 1973)
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