Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets

Abstract

Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the alpha-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Galphaq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72(syk) and stimulation of pp60(c-src) as well as in phosphorylation of myosin light chain (MLC) in Galphaq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Galphaq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase-mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase-dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change.

Figure 1

Scanning electron microscopy of inactivated and activated wild-type and Gα_q-deficient platelets. Wild-type platelets (A, B, E, F, I, and J) and Gα_q-deficient platelets (C, D, G, H, K, and L) were preincubated for 30 min in the absence (A–D) or presence of 10 μM Y-27632 (E–H) or were pretreated for 2 h with 50 μg/ml C3 exoenzyme (I–L). Thereafter, platelets were incubated for 5 s in the absence (A, C, E, G, I, and K) and presence of 5 μM U46619 (B, D, F, H, J, and L), fixed, and then analyzed as described in Materials and Methods. Platelets incubated for 2 h in C3-exoenzyme buffer alone showed normal shape change in response to U46619 (data not shown). Bar, 1 μm.

Figure 2

Aggregation response of wild-type and Gα_q-deficient platelets. Wild-type platelets (B and D) and platelets from Gα_q (−/−) mice (A, C, and E) were preincubated for 20 min with the indicated concentrations of 8-pCPT-cGMP (pCPT-cGMP) or Sp-5,6-DCl-cBIMPS (cBIMPS) (A and B) or for 30 min with the indicated concentrations of Y-27632 (C and D), and Gα_q-deficient platelets were preincubated for 2 h in the absence or presence of C3 exoenzyme (E). Thereafter, incubation was started by the addition of 5 μM U46619. Shown is the relative light transmission through the platelet suspension. 0% represents transmission through the platelet suspension before addition of U46619, and 100% represents transmission through the buffer alone. Upward movements of the curve show decreases in light transmission indicating platelet shape change. Addition of stimuli is signified by the arrows, 3-min traces are shown.

Figure 3

ADP ribosylation of Rho in C3-treated platelets. (A) Platelets from wild-type and Gα_q-deficient animals were preincubated for 2 h in the absence (−) or presence of the indicated concentrations of C3 exoenzyme. Thereafter, platelets were lysed and subjected to C3-exoenzyme catalyzed (³²P) ADP ribosylation as described under Materials and Methods. Shown is an autoradiogram with the position of the 21-kD standard protein on the left. (B) Densitometric evaluation of bands shown in A. Bars represent mean of two independent experiments as percent of control.

Figure 4

Determination of F-actin content. Wild-type and Gα_q-deficient platelets were preincubated for 30 min in the absence (c) or presence of 10 μM Y-27632 (Y) or were pretreated for 2 h with 50 μg/ml C3 exoenzyme (C3). Thereafter, platelets were incubated for 10 s in the absence (−) or presence of 5 μM U46619 (+), fixed, and then incubated with fluorescein isothiocyanate (FITC)-phalloidin. Platelets incubated for 2 h in C3 exoenzyme buffer alone showed full increase in F-actin content in response to U46619 (data not shown). F-actin content was determined as described in Materials and Methods. Shown are means ± SD of triplicates.

Figure 5

Receptor-dependent activation of G proteins in membranes of wild-type and Gα_q-deficient platelets. Membranes from wild-type (A) and Gα_q-deficient platelets (B) were photolabeled with [α-³²P]GTP azidoanilide in the absence (−) or presence of 5 μM U46619 or 5 U/ml thrombin (+). Membranes were solubilized and G protein α-subunits (Gα₁₂, Gα₁₃, Gα_q, and Gα_i) were immunoprecipitated as described under Materials and Methods. Anti-Gα₁₂, anti-Gα₁₃, anti-Gα_q/11 antisera, and an antiserum recognizing Gα_i1, Gα_i2, and Gα_i3 were used. Precipitated proteins were subjected to SDS-PAGE. Shown are autoradiograms of dried SDS gels with the position of the 43-kD standard protein shown on the left.

Figure 6

Effect of thrombin and U46619 on tyrosine phosphorylation and pp72^syk and pp60^c-src-activity in wild-type and Gα_q-deficient platelets. (A) Wild-type and Gα_q-deficient platelets were incubated for 30 s in the absence (−) or presence of 5 U/ml thrombin (Thr.) or 5 μM U46619 (TXA₂). (B) Wild-type and Gα_q-deficient platelets were incubated for the indicated times in the absence (−) or presence of 5 μM of the thromboxane A₂ mimetic U46619 (T). Cells were lysed and cellular proteins were separated by SDS-PAGE and blotted on nitrocellulose filters. Phosphotyrosine was detected by an antiphosphotyrosine antibody. Shown are autoluminograms with the position of standard proteins shown on the left. (C) Wild-type (left) and Gα_q-deficient platelets (right) were incubated with buffer (−) or 5 μM U46619 (+) for the indicated times. Platelets were lysed and incubated with agarose conjugates of anti-pp72^syk IgG (IP: Syk) or of anti-pp60^c-src IgG (IP: Src), and immunoprecipitates were analyzed by immunoblotting with antiphosphotyrosine antibodies (WB: P-Y), anti-pp72^syk antibodies (WB: Syk), anti-pp60^c-src antibodies (WB: Src), or were subjected to in vitro kinase assays using histone as a substrate (Histone-P).

Figure 7

U46619-induced MLC phosphorylation in wild-type and Gα_q-deficient platelets. (A) Wild-type and Gα_q-deficient platelets were incubated for the indicated time periods with buffer (c) or 5 μM U46619 (U). (B and C) Platelets were preincubated for 20 min without or with the synthetic cyclic nucleotides 8-pCPT-cGMP (cGMP) or Sp-5,6-DCl-cBIMPS (cAMP) at the indicated concentrations. Thereafter, platelets were incubated for 10 s with buffer (c) or 5 μM U46619 (U). (D and E) Platelets were preincubated for 30 min without or with the Rho-kinase inhibitor Y-27632 at the indicated concentrations (30 or 100 μM for wild-type and 10 or 30 μM for Gα_q-deficient platelets). Incubation was conducted for 10 s with buffer (c) or 5 μM U46619 (U). (F) Wild-type and Gα_q-deficient platelets were preincubated for 120 min without (−) or with 50 μg/ml C3 exoenzyme (+). Thereafter, platelets were incubated for 10 s with buffer (c) or 5 μM U46619 (U). Reactions were stopped by addition of perchloric acid, and phosphorylation of MLC was determined using urea/glycin gels as described in Materials and Methods. Shown are autoluminograms of anti-MLC immunoblots. Phosphorylation of MLC results in a faster mobility (lower position) of MLC on urea/glycin gels.