Phosphatidylserine surface expression and integrin αIIbβ3 activity on thrombin/convulxin stimulated platelets/particles of different sizes (original) (raw)
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Phosphorylation Sites in the Integrin beta 3 Cytoplasmic Domain in Intact Platelets
Journal of Biological Chemistry, 1999
Protein seryl/threonyl phosphatase inhibitors such as calyculin A block inside-out and outside-in platelet signaling. Our studies demonstrate that the addition of calyculin A blocks platelet adhesion and spreading on fibrinogen, responses that depend on integrin ␣ IIb  3 signaling. We hypothesized that this reflects a change in ␣ IIb  3 structure caused by a specific state of phosphorylation. We show that addition of calyculin A leads to increased phosphorylation of the  3 subunit, and phosphoamino acid analysis reveals that only threonine residues become phosphorylated; sequence analysis by Edman degradation established that threonine 753 became stoichiometrically phosphorylated during inhibition of platelet phosphatases by calyculin A. This region of  3 is linked to outside-in signaling such as platelet spreading responses. The effect of calyculin A on platelet adhesion and spreading and on the phosphorylation of T-753 in  3 is reversed by the calcium ionophore A23187, demonstrating that these effects of calyculin A are not generally toxic ones. We propose that phosphorylation of  3 on threonine 753, a region of  3 linked to outside-in signaling, may be a mechanism by which integrin ␣ IIb  3 function is regulated.
2020
We would like to acknowledge our supervisors Professor, Alberto Redaelli, for giving us the possibility to work on this project and Professor Marvin J. Slepian for welcoming us as part of the team. We also would like to thank Silvia Bozzi, PhD, for the support and technical advices she gave us during the work. We would also like to acknowledge the Sarver Heart Centre team, Daniel, Sparky, Allegra, Kaitlyn, Adriana, Yana and Sam for their help and their friendship. A special acknowledgment goes to our families for the opportunity they gave us to follow our dreams and for supporting us during this experience. A kindly thought goes to all our friends, for the lovely time we've spent together and all their support. Finally, we would like to thank each other for supporting, affection, patience and understanding.
Activation induced morphological changes and integrin αIIbβ3 activity of living platelets
Methods, 2013
Platelets are essential in hemostasis. Upon activation they undergo a shape-change accompanied with receptor presentation. Atomic force microscopy (AFM) imaging and single molecule force spectroscopy (SMFS) were used as powerful tools for exploring morphological changes as well as receptor activities of platelets. Imaging time series was accomplished with and without fixation steps at the single platelet level. Hereby the response of mechanical stimulation of the platelet by the AFM cantilever tip was directly observed. We demonstrate that living and fixed platelets develop filopodia after a short activation time followed by their disappearance including cellular bleb formation. Thereafter a second filopodia formation (filopodia extrusion) was observed; those filopodia subsequently disappeared again, and finally platelets detached from the support due to cell death. We determined the influence of mechanical stress on the chronology of morphological changes of platelets and demonstrated shear force induced filopodia formation. Through recordings over several hours, topographical AFM images over the full platelet lifetime -from early activation up to apoptosis -are presented. SMFS measurements on living platelets allowed determining the activation state of the most prominent membrane receptor integrin aIIbb3 at all different phases of activation. aIIbb3 was fully activated, independent of the morphological state.
Arteriosclerosis, Thrombosis, and Vascular Biology, 2005
Objective— Previous work has shown that platelets stimulated with the combination of thrombin and convulxin, a glycoprotein VI agonist, develop 2 populations with different levels of α-granule factor V bound to the platelet surface. To evaluate whether this phenomenon is restricted to α-granule components or is a feature that can be generalized to other coagulation factors, we studied the binding of factors V, VIII, IX, and X on the surface of platelets stimulated by convulxin and thrombin. Methods and Results— The relative amount of factors V, VIII, IX, and X on the surface of platelets stimulated with thrombin or convulxin plus thrombin was measured using flow cytometry. Simultaneous measurements of factor Xa and thrombin generation were obtained and correlated with the binding of coagulation factors on the platelet surface. The binding of factors V, VIII, IX, and X all increased on a subpopulation of platelets when stimulated with the combined agonists. The development of this pl...
Archives of biochemistry and …, 2002
In a physiological milieu platelets continue to be exposed to agonists long after clot formation. We studied the regulation of postaggregation events consequent on protease-activated receptor (PAR) 1 ligation with either thrombin or the thrombin receptor-activating peptide (TRAP). Stimulation with TRAP (20 M) but not with thrombin (1 U/ml) for 15 min evoked platelet disaggregation by about 30% and downregulation of high-affinity fibrinogen binding sites on integrin ␣ IIb  3 to nearly prestimulation levels. Concurrently, only TRAP disorganized the actin-based cytoskeleton, with decrease in the cytoskeletal content of focal contact-associated proteins like integrin ␣ IIb  3 , Src, and focal adhesion kinase (FAK). While protein tyrosine kinases were activated during the initial period of platelet aggregation with either agonist, stimulation of protein tyrosine phosphatases determined the successive phase of reduced phosphotyrosine content. SHP-1, an abundant protein tyrosine phosphatase in the platelets, was tyrosine phosphorylated on challenge of PAR-1 and coprecipitated with two unidentified tyrosine phosphorylated proteins of 140 and 60 kDa; in addition, SHP-1 tyrosine phosphorylation (which is associated with enhanced phosphatase activity) was sustained until 15 min. Activity of calpain was upregulated following incubation with thrombin and not with TRAP. Collectively, these data suggest that signaling pathways elicited by PAR-1 agonists thrombin and TRAP are markedly different, which could have important implications on late platelet responses.
Platelets: Physiology and Biochemistry
Seminars in Thrombosis and Hemostasis, 2005
Platelets are specialized blood cells that play central roles in physiologic and pathologic processes of hemostasis, inflammation, tumor metastasis, wound healing, and host defense. Activation of platelets is crucial for platelet function that includes a complex interplay of adhesion and signaling molecules. This article gives an overview of the activation processes involved in primary and secondary hemostasis, for example, platelet adhesion, platelet secretion, platelet aggregation, microvesicle formation, and clot retraction/stabilization. In addition, activated platelets are predominantly involved in cross talk to other blood and vascular cells. Stimulated ''sticky'' platelets enable recruitment of leukocytes at sites of vascular injury under high shear conditions. Platelet-derived microparticles as well as soluble adhesion molecules, sP-selectin and sCD40L, shed from the surface of activated platelets, are capable of activating, in turn, leukocytes and endothelial cells. This article focuses further on the new view of receptor-mediated thrombin generation of human platelets, necessary for the formation of a stable platelet-fibrin clot during secondary hemostasis. Finally, special emphasis is placed on important stimulatory and inhibitory signaling pathways that modulate platelet function.