Cbl-b is a novel physiologic regulator of glycoprotein VI-dependent platelet activation

Abstract

Cbl-b, a member of the Cbl family of E3 ubiquitin ligases, plays an important role in the activation of lymphocytes. However, its function in platelets remains unknown. We show that Cbl-b is expressed in human platelets along with c-Cbl, but in contrast to c-Cbl, it is not tyrosine-phosphorylated upon glycoprotein VI (GPVI) stimulation. Cbl-b, unlike c-Cbl, is not required for Syk ubiquitylation downstream of GPVI activation. Phospholipase Cgamma2 (PLCgamma2) and Bruton's tyrosine kinase (BTK) are constituently associated with Cbl-b. Cbl-b-deficient (Cbl-b(-/-)) platelets display an inhibition in the concentration-response curve for GPVI-specific agonist-induced aggregation, secretion, and Ca(2+) mobilization. A parallel inhibition is found for activation of PLCgamma2 and BTK. However, Syk activation is not affected by the absence of Cbl-b, indicating that Cbl-b acts downstream of Syk but upstream of BTK and PLCgamma2. When Cbl-b(-/-) mice were tested in the ferric chloride thrombosis model, occlusion time was increased and clot stability was reduced compared with wild type controls. These data indicate that Cbl-b plays a positive modulatory role in GPVI-dependent platelet signaling, which translates to an important regulatory role in hemostasis and thrombosis in vivo.

FIGURE 1.

Cbl-b in platelets. A, detection of Cbl-b in platelet lysates and PBMCs. Platelets and PBMCs, at the amounts indicated, were separated by SDS-PAGE, transferred to Immobilon, and probed for the presence of Cbl-b. The arrows indicate the expected position of Cbl-b. B, lack of tyrosine phosphorylation of Cbl-b in convulxin-activated platelets. c-Cbl or Cbl-b was immunoprecipitated (IP) from platelets activated with convulxin (100 ng/ml); immunoprecipitates were subjected to SDS-PAGE and transferred to Immobilon. The blot was probed with anti-phosphotyrosine (pY) antibody (4G10), anti-Cbl-b, or anti-c-Cbl, as indicated. Rabbit IgG was used as a negative control.

FIGURE 2.

Comparison of expression levels of signaling proteins in WT versus Cbl-b^−/− murine platelets. Proteins were prepared from an equal number of WT and Cbl-b^−/− platelets. Proteins were separated by SDS-PAGE, transferred to Immobilon, and probed for the indicated proteins. p85 is a subunit of PI 3-kinase.

FIGURE 3.

Lack of involvement of Cbl-b in Syk ubiquitylation. WT, c-Cbl^−/−, or Cbl-b^−/− platelets were activated with 100 ng/ml convulxin for the indicated times; perchloric acid extracts were subjected to SDS-PAGE and transferred to Immobilon. The blot was probed with an antibody to phospho-Syk (Tyr(P)⁵⁰⁹/Tyr(P)⁵¹⁰) (pY509/510) or total Syk. The arrows indicate the suspected location of ubiquitylated Syk.

FIGURE 4.

Association of Cbl-b with PLC γ2and BTK. A and B, human washed platelets were incubated with 200 ng/ml convulxin or vehicle as indicated. After preclearing, proteins were immunoprecipitated (IP) with either anti-Cbl-b (A) or anti-BTK (B). IgG was used as a negative control in both cases. Immunoprecipitates were analyzed by Western blotting using the indicated antibodies. C, platelet lysates were prepared as above and treated with agarose containing either GST or GST-Cbl-b. The agarose-bound material was analyzed by Western blotting. Blots were probed for either Cbl-b (GST-Cbl-b used for pull-down), PLCγ2, BTK, or Syk as labeled. Each experiment is representative of at least three similar experiments.

FIGURE 5.

Effect of Cbl-b deficiency on platelet responses. A, platelets were prepared from WT, Cbl-b^−/−, or Cbl-b-RFm mice for aggregation and stimulated with the indicated concentrations of convulxin or CRP. B, platelet ATP secretion was measured in WT, Cbl-b^−/−, or Cbl-b RFm platelets. A and B, representative traces from at least three experiments. C, representative traces from Ca²⁺ mobilization experiments. D–F, agonist-dependent Ca²⁺ mobilization from WT or Cbl-b^−/− platelets in response to various doses of CRP (n = 4) (D), convulxin (n = 6) (E), or AYPGKF (n = 6) (E). G, platelets were prepared from WT or Cbl-b RFm mice, and CRP-induced Ca²⁺ mobilization (n = 3) was measured. Bars (A–C), 1 min. Error bars (D–G), S.E.

FIGURE 6.

Site-specific phosphorylation downstream of GPVI activation in Cbl-b^−/− murine platelets. Platelets were prepared from WT or Cbl-b^−/− mice and were activated with various concentrations of convulxin for 1 min. Blots were probed for phospho-Syk (Tyr(P)⁵⁰⁹/Tyr(P)⁵¹⁰) (pY509/Y510) (A), phospho-BTK (Tyr(P)⁵⁵¹) (pY551) (B), or phospho-PLCγ2 (Tyr(P)⁷⁵³, Tyr(P)⁷⁵⁹) (pY753,759) (C). These data are representative of at least four experiments in each case. A–C show single representative immunoblots. For D, platelets were prepared from WT or Cbl-b^−/− mice and were activated with 200 ng/ml convulxin for the indicated times. Platelet samples were separated by SDS-PAGE and probed using antibodies for either phospho-AKT or phospho-ERK. The bar graphs are from three experiments. There was no statistical significance between WT or Cbl-b^−/− platelets for either ERK or AKT phosphorylation. Error bars, S.E. KO, knock-out.

FIGURE 7.

Cbl-b^−/− mice exhibit prolonged time to occlusion and less stable thrombi in the FeCl₃ injury model. The carotid arteries of WT or Cbl-b^−/− mice were subjected to FeCl₃ injury, as described under “Experimental Procedures.” A, time to 90% occlusion was calculated and graphed for WT (n = 7) and Cbl-b^−/− (n = 14). Statistical analysis was performed using unpaired Student's t test. B, clot stability assessed by determining the percentage of mice that form stable thrombi for at least 5 min.