Coordinate interactions of Csk, Src, and Syk kinases with [alpha]IIb[beta]3 initiate integrin signaling to the cytoskeleton

Abstract

Integrins regulate cell adhesion and motility through tyrosine kinases, but initiation of this process is poorly understood. We find here that Src associates constitutively with integrin alphaIIbbeta3 in platelets. Platelet adhesion to fibrinogen caused a rapid increase in alphaIIbbeta3-associated Src activity, and active Src localized to filopodia and cell edges. Csk, which negatively regulates Src by phosphorylating Tyr-529, was also constitutively associated with alphaIIbbeta3. However, fibrinogen binding caused Csk to dissociate from alphaIIbbeta3, concomitant with dephosphorylation of Src Tyr-529 and phosphorylation of Src activation loop Tyr-418. In contrast to the behavior of Src and Csk, Syk was associated with alphaIIbbeta3 only after fibrinogen binding. Platelets multiply deficient in Src, Hck, Fgr, and Lyn, or normal platelets treated with Src kinase inhibitors failed to spread on fibrinogen. Inhibition of Src kinases blocked Syk activation and inhibited phosphorylation of Syk substrates (Vav1, Vav3, SLP-76) implicated in cytoskeletal regulation. Syk-deficient platelets exhibited Src activation upon adhesion to fibrinogen, but no spreading or phosphorylation of Vav1, Vav3, and SLP-76. These studies establish that platelet spreading on fibrinogen requires sequential activation of Src and Syk in proximity to alphaIIbbeta3, thus providing a paradigm for initiation of integrin signaling to the actin cytoskeleton.

Figure 1.

Association of tyrosine kinases with αIIbβ3. (A) Washed human platelets were plated on fibrinogen (Fib) for 45 min or maintained in suspension in a BSA-coated dish (BSA). Cells were then lysed in NP-40 detergent, the detergent-soluble fraction was immunoprecipitated with an antibody to β3 or normal rabbit serum (NRS) as a control, and immunoprecipitates were probed on Western blots with antibodies to Src and other tyrosine kinases, as indicated. (B) Platelets in suspension (BSA) were lysed in NP-40, and the presence of Src in β3 and GP Ibα immunoprecipitates was compared. Results are representative of five separate experiments.

Figure 2.

Effect of platelet adhesion to fibrinogen on Src activation. As described in the legend to Fig. 1, lysates from fibrinogen-adherent and -nonadherent platelets were immunoprecipitated with an antibody to β3 (A) or FAK (B), and immunoprecipitates were probed on Western blots as indicated. (C) An analysis of the pool of Src that did not coimmunoprecipitate with αIIbβ3 after two sequential immunoprecipitations with the anti-β3 antibody. In all panels, platelets were pretreated for 10 min with buffer, cytochalasin D (CD), or diluent (DMSO) before plating. (D) The raw data from the single experiment in A as Src “activity” associated with β3, expressed as the normalized ratio of Src pTyr-418/pTyr-529. (E) The means ± SEM of this ratio for five experiments. (F) The effects of platelet adhesion on Src activity in β3 immunoprecipitates, measured by an in vitro kinase assay as described in Materials and methods. Results represent means ± SEM of four experiments.

Figure 3.

Effect of soluble fibrinogen binding to platelets on αIIbβ3-associated Csk and Src. Platelets were incubated as indicated in the presence or absence of 250 μg/ml fibrinogen, 0.5 mM MnCl₂, and 2 mM RGDS for 20 min. Then the presence of Csk and Src in β3 immunoprecipitates was analyzed as described in the legends to Figs. 1 and 2. Results are representative of two experiments.

Figure 4.

Distribution of activated Src and total Src in fibrinogen-adherent platelets. Cells were plated on fibrinogen-coated coverslips for 45 min and prepared for confocal microscopy as described in Materials and methods. Images represent four platelets in various stages of spreading. In the merged images, activated Src (Src pTyr-418) is red and total Src is green. Arrowheads point to some of the filopodia that stained heavily for activated Src. The results are from a single experiment representative of three so performed. Bar, 10 μm.

Figure 5.

Effect of PP2 on Src activity and Src association with αIIbβ3. Platelets were preincubated for 30 min with PP2, plated on fibrinogen or suspended over BSA for 45 min, and Src in αIIbβ3 immunoprecipitates was analyzed as described in the legend to Fig. 1. Results are representative of two experiments.

Figure 6.

Role of Src family kinases in platelet spreading on fibrinogen. (A) Human platelets were preincubated for 30 min with buffer, 5 μM PP2, or 5 μM PP3. After adhesion to fibrinogen for 45 min, cells were fixed, permeabilized, stained for F-actin (red) and phosphotyrosine (green), and analyzed by confocal microscopy. Arrowheads orient the reader to filopodia and arrows to the peripheral rims of some of the spreading platelets. Results are representative of three experiments. (B) Platelets from wild-type or chimeric mice were plated on fibrinogen for 45 min, stained as above, and analyzed by confocal microscopy. Note that murine platelets are smaller than human platelets. Bars, 10 μm.

Figure 7.

Effect of inhibition of Src kinases on Syk and its substrates. Platelets were processed as described in the legends to Figs. 1 and 5. (A) The effect of 5 μM PP2 on tyrosine phosphorylation of Syk. (B) The effect on tyrosine phosphorylation of Vav1, Vav3 and SLP-76. (C) The effect on adhesion-dependent association of Syk with αIIbβ3. Results are representative of two experiments.

Figure 8.

Platelet spreading on fibrinogen is defective in syk ^−/− murine platelets. Platelets were obtained from syk^−/− and wild-type mice and plated on fibrinogen for 45 min with no agonist; with a combination of PAR-4 receptor–activating peptide (1 mM), ADP (50 μM), and epinephrine (50 μM); or with collagen (10 μg/ml). Cells were then fixed, permeabilized and stained for F-actin (red) and phosphotyrosine (green), and analyzed by confocal microscopy. Arrowheads point to filopodia and arrows to the peripheral rims of some of the spreading platelets. Results are representative of three experiments. Bar, 10 μm.

Figure 9.

Effect of Syk deficiency on adhesion-dependent tyrosine phosphorylation in platelets. After plating on BSA or fibrinogen for 45 min, wild-type and syk^−/− platelets were subjected to immunoprecipitation and Western blotting as indicated. Results are representative of three experiments.

Figure 10.

Effect of Syk deficiency on adhesion-dependent activation of Src. Wild-type and syk^−/− platelets were plated on fibrinogen or suspended over BSA for 45 min, and β3 immunoprecipitates were analyzed on Western blots as indicated. Results are representative of three experiments.