TRAP Induces More Intense Tyrosine Phosphorylation than Thrombin with Differential Ultrastructural Features

Abstract

We have analyzed modifications on platelet ultrastructural morphology, cytoskeletal assembly, and tyrosine phosphorylation developing in platelets activated by both thrombin and the thrombin receptoractivating peptide (TRAP). Washed platelets exposed to various concentrations of thrombin or TRAP, for different periods, were: fixed and examined by electron microscopy, or lysed and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under similar activating conditions, thrombin and TRAP induced different sequences of activation causing distinctive morphological and biochemical changes. Platelets exposed to thrombin showed centralized organelles encircled by constricted microtubule coils and granules secreting their contents through narrow channels of the open canalicular system. In contrast, activation by TRAP induced swelling of the open canalicular system with organelles remaining randomly dispersed and microtubules peripherally distributed. Compared to thrombin activation, TRAP induced higher rates of actin polymerization; increased association of actin-binding protein, myosin, and ␣-actinin; and higher association of tyrosine-phosphorylated proteins with the insoluble cytoskeletal fraction. Secretion of intragranule substances, measured as expression of P-selectin and lysosomal integral membrane protein at the surface level, were similar for both agonists at equivalent concentrations. Platelet response depends on the nature and degree of the activating stimulus. Thrombin is one of the most powerful platelet-activating agents and is able to promote a full range of platelet responses, including the increase in the concentration of cytoplasmic Ca ϩϩ , leading to the activation of signaling cascades with shape change, internal contraction, and secretion. Platelet response to thrombin activation is guaranteed by the presence of at least two types of receptors, with high and moderate affinity for thrombin. The former is located in the N-terminal domain of the GPIb␣, 1-3 and the later with moderate affinity belong to the G protein-coupled seven transmembrane domain superfamily, known as protease-activated receptors (PARs). 4,5 Cleavage of PAR-1 by ␣-thrombin yields an agonistic new amino terminus with the initial sequence SFLLRN. This peptide sequence, termed thrombin receptor-activating peptide (TRAP), can also activate this receptor. TRAP is able to induce aggregation and secretion by similar mechanisms than thrombin, 4 it is also involved in inositol phosphatase metabolism, 6 in inhibition of adenylcyclase, 7 and in protein phosphorylation. 8 Although it is generally accepted that TRAP acting on PAR-1 has the ability to induce many of the cellular responses observed after thrombin activation, comparative studies show that TRAP seems to be less efficient to induce the whole sequence of responses caused by thrombin. 4,9 -13 Considering that thrombin has an additional receptor on GPIb␣, differential responses to both agonists should be expected.The present study has comparatively investigated morph...

Recent advances in blood‐related proteomics

“…Both inside-out and outside-in signaling are regulated by positive and/or negative phosphorylation [125]. The activation state of platelets has been studied by in vitro activation of platelets with the thrombin receptor activating peptide [126]. In another study, Garcia et al [127] detected 62 proteins differentially expressed in activated and inactivated platelets, either appearing, disappearing, or being down-or up-regulated between the two states.…”

Differential proteome analysis of TRAP-activated platelets: involvement of DOK-2 and phosphorylation of RGS proteins

“…This peptide sequence, termed thrombin receptor-activating peptide (TRAP), is also able to activate the noncleaved receptor and replace thrombin in in vitro experiments allowing for controlled cell stimulation. 2,7,8 Signaling by PAR-1 has been extensively studied. 7,9,10 However, a full picture of all of the signaling molecules in the PAR-1 pathway is not available.…”

“…2,7,8 Signaling by PAR-1 has been extensively studied. 7,9,10 However, a full picture of all of the signaling molecules in the PAR-1 pathway is not available. This information is important to fully understand the events underlying platelet activation by PAR-1 and for identification of potential drug targets and future therapeutic modulation of platelet activation processes.…”

Interactions between the megakaryocyte/platelet-specific β1 tubulin and the secretory leukocyte protease inhibitor SLPI suggest a role for regulated proteolysis in platelet functions

“…We treated wild-type platelets with thrombin and stained them with antibodies against ␤ tubulin and either VWF to detect residual ␣-granules or SLPI. Platelet activation normally results in central accumulation of platelet granules 37,38 ; indeed, we detected VWF-containing ␣-granules in the platelet center, surrounded by the condensed MT coil ( Figure 5). SLPI was found in the same Figure 5.…”