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.

Internalization of IgG-Coated Targets Results in Activation and Secretion of Soluble CD40 Ligand and RANTES by Human Platelets

“…The fact that cytochalasin D inhibited platelet activation (measured by CD62P) and release of sCD40L and RANTES suggests that both responses are dependent upon conformational alteration of the actin cytoskeleton. However, cytocha- lasin D has been observed to affect platelet activation (measured by CD62P, CD62, and annexin V) in response to collagen as an agonist but not thrombin, suggesting that cytochalasin blocks actin-dependent signal transduction but not vesicle release (10). Therefore, we believe that our results are due to cytochalasin D blocking internalization of IgG-coated targets and not by blocking vesicle fusion.…”

Critical roles for the actin cytoskeleton and cdc42 in regulating platelet integrinα2β1

“…For example it has recently been shown that 2 1 may signal to IIb 3 through Rap1b [10] and reciprocally that IIb 3 forms an essential step in regulating the affinity state of 2 1 [11]. A signal that has been shown to be important for downstream signaling from 2 1 is actin polymerization, since in the presence of cytochalasins 2 1 -mediated protein tyrosine phosphorylation, secretion and aggregation are each ablated [12]. Consequently it has been long established that actin polymerization is required for collagen-induced activation of platelets [12,13].…”

Effects of Lys-form of plasminogen on platelet actin cytoskeleton

“…Інгібу-вання процесів полімеризації актину та ре-організації актинового цитоскелета порушує адгезивні властивості інтегринів, що призво-дить до втрати тромбоцитами агрегативної здатності, як це показано в експериментах з використанням цитохалазинів [6]. Інформа-ція про те, що деякі кринглвмісні фрагменти плазміногену (ангіостатини) пригнічують міграцію моноцитів-макрофагів завдяки дезорганізації актинового цитоскелета цих клітин [23], стала додатковим стимулом для проведення цього дослідження.…”

“…В експериментах Diaz-Ricart та співавт. [6] показано, що в результаті об-робки тромбоцитів агентами-інгібіторами полімеризації актину (цитохалазін B, ла-трункулін А), ці клітини втрачають здатність розпізнавати адгезивні ліганди та агрегувати навіть за наявності потужних індукторів. У свою чергу взаємодія GP IIbIIIa з адге-зивними молекулами стабілізує цитоскелет тромбоцитів і сприяє організації філаментів у стрес-фібрили, характерні для фокальних контактів [15].…”