Inhibition of cytoskeletal assembly by cytochalasin B prevents signaling through tyrosine phosphorylation and secretion triggered by collagen but not by thrombin

Abstract

Activation of platelets leads to cytoskeletal assembly that is responsible for platelet motility and internal contraction. We have evaluated the involvement of the cytoskeleton in platelet activation by two strong agonists, collagen and thrombin. Activation was assessed by measuring changes in cytoskeletal assembly, externalization of activation-dependent markers and expression of procoagulant activity, and tyrosine phosphorylation of proteins, in both the absence and the presence of cytochalasin B. Activation of platelets with collagen and thrombin induced morphological changes and increased the expression of CD62P, CD63, glycoprotein IV, and binding of annexin V to platelets. Moreover, both activating agents induced actin polymerization, increased the association of other contractile proteins, and promoted tyrosine phosphorylation of multiple proteins, some of which were associated with the cytoskeleton. The presence of cytochalasin B blocked the previous events when collagen was used as the activating agent, although binding of annexin V still occurred. In contrast, platelet response to thrombin was not completely prevented by the presence of cytochalasin B. Thus, activation by collagen requires a functional cytoskeleton to trigger signaling through tyrosine phosphorylation and secretion. This is not the case for thrombin, which is capable of activating signaling mechanisms in the presence of strong inhibitors of cytoskeletal assembly. Moreover, the expression of a procoagulant surface in platelets still occurs even when platelet motility has been inhibited.

Figure 1.

Effect of Cyt-B on platelet activation by collagen. Electron microscopy images (original magnifications, ×23,000) and electrophoretic profiles (8% SDS-PAGE) corresponding to resting platelets (A) and platelets activated with collagen (20 μg/ml) in the absence (B) and in the presence (C) of Cyt-B (50 μmol/L), respectively. Profiles are representative of eight different experiments.

Figure 2.

Effect of Cyt-B on platelet activation by thrombin. Electron microscopy images (original magnifications, ×23,000) and electrophoretic profiles (8% SDS-PAGE) corresponding to resting platelets (A) and platelets activated with thrombin (0.1 U/ml) in the absence (B) and in the presence (C) of Cyt-B (50 μmol/L), respectively. Profiles are representative of eight different experiments.

Figure 3.

Changes in the expression of antigens after activation of platelets with collagen (A) and thrombin (B): effect of Cyt-B. Bar graphs showing the changes observed in the activation markers measured using flow cytometry. Results correspond to platelets before activation (open bars), after activation in the absence of Cyt-B (filled bars), and after activation in the presence of Cyt-B (50 μmol/L) (slashed bars). Activation was performed with collagen (20 μg/ml) (A) and with thrombin (0.1 U/ml) (B). Results are expressed as percent of positive platelets (mean ± SEM, n = 5). Asterisk indicates statistically significant differences (P < 0.05) after activation, and plus indicates statistically significant differences (P < 0.05) when Cyt-B was present.

Figure 4.

Tyrosine-phosphorylated proteins present in platelet lysates (I) and associated with the polymerized cytoskeletal fraction (II) after collagen activation: effect of Cyt-B. Protein profiles corresponding to resting platelets (B), platelets activated with 20 μg/ml of collagen (Col) in the absence and in the presence of Cyt-B (50 μmol/L). After activation, platelets were lysated to analyze whole platelet lysates (I) or processed to extract the polymerized cytoskeletal fraction (II). Samples were resolved by 8% SDS-PAGE and proteins transferred to nitrocellulose membranes. Phosphotyrosine proteins were detected by a specific antibody and the enhanced chemiluminescence technique. Images are representative of eight different experiments.

Figure 5.

Tyrosine-phosphorylated proteins present in platelet lysates (I) and associated with the polymerized cytoskeletal fraction (II) after thrombin activation: effect of Cyt-B. Protein profiles corresponding to resting platelets (B), platelets activated with 0.1 U/ml of thrombin (T) in the absence and in the presence of Cyt-B (50 μmol/L). After activation, platelets were lysated to analyze whole platelet lysates (I) or processed to extract the polymerized cytoskeletal fraction (II). Samples were resolved by 8% SDS-PAGE and proteins transferred to nitrocellulose membranes. Phosphotyrosine proteins were detected by a specific antibody and the enhanced chemiluminescence technique. Images are representative of eight different experiments.