A 2D-DIGE-based proteomic analysis reveals differences in the platelet releasate composition when comparing thrombin and collagen stimulations

Abstract

Upon stimulation, platelets release a high number of proteins (the releasate). There are clear indications that these proteins are involved in the pathogenesis of several diseases, such as atherosclerosis. In the present study we compared the platelet releasate following platelet activation with two major endogenous agonists: thrombin and collagen. Proteome analysis was based on 2D-DIGE and LC-MS/MS. Firstly, we showed the primary role of thrombin and collagen receptors in platelet secretion by these agonists; moreover, we demonstrated that GPVI is the primary responsible for collagen-induced platelet activation/aggregation. Proteomic analysis allowed the detection of 122 protein spots differentially regulated between both conditions. After excluding fibrinogen spots, down-regulated in the releasate of thrombin-activated platelets, 84 differences remained. From those, we successfully identified 42, corresponding to 37 open-reading frames. Many of the differences identified correspond to post-translational modifications, primarily, proteolysis induced by thrombin. Among others, we show vitamin K-dependent protein S, an anticoagulant plasma protein, is up-regulated in thrombin samples. Our results could have pathological implications given that platelets might be playing a differential role in various diseases and biological processes through the secretion of different subsets of granule proteins and microvesicles following a predominant activation of certain receptors.

Figure 1. Effect of PAR-1, GPVI and α2β1 inhibitors on thrombin- and collagen-induced platelet aggregation.

(A) Representative platelet aggregation profiles following platelet activation with 0.75 U/mL Thrombin (shown in blue) or 30 μg/mL collagen (shown in black). (B) Effect of PAR-1 inhibition on thrombin-induced platelet aggregation, and of GPVI and α2β1 inhibition on collagen-induced platelet aggregation. Washed human platelets were pre-incubated with the inhibitors for 5 min, then 0.75 U/mL thrombin or 30 μg/mL collagen were added to trigger platelet aggregation. Results are presented as mean ± SE (n = 4–6). *p<0.05 (Mann-Whitney test). Coll: collagen; Thr: thrombin; Fab-OM2: Fab fragment of the anti-GPVI monoclonal antibody OM2; BTT: BTT 3033; SCH: SCH 79797.

Figure 2. High-resolution 2D-DIGE proteome analysis of platelet releasate from collagen and thrombin stimulated platelets.

Proteins were labeled with the corresponding Cy-dyes (see Methods section) and separated using isoelectric focusing (pH range 4–7, 24 cm) and 11% SDS-PAGE gels. (A) Representative image of the 2D-DIGE analysis where the fluorescence emission from Cy3 and Cy5 dyes is superimposed. Red-orange color spots are augmented in the releasate of thrombin-activated platelets whereas green color spots are augmented in the releasate of collagen-activated platelets. Main fibrinogen arrays are highlighted. (B) Representative image of the analysis in a gray scale with the differentially regulated spots (excluding fibrinogen) highlighted. Further information about protein identifications can be found in Table 1 and Supplementary Table 1.

Figure 3. Selection of protein features differentially regulated between collagen- and thrombin-induced releasate.

Enlargement of representative spots (i) with image analysis statistics (ii) for the following proteins: (A) MMRN1 (spot No. 1244); (B) PROS (spot No. 1839); (C) Coagulation Factor V(spot No. 1559); Thrombospondin-1 (spot No. 1226).

Figure 4. Vitamin K-dependent protein S (PROS) is elevated in the releasate of thrombin-stimulated platelets.

Representative western blot images of individual and pooled samples are shown (i) together with densitometry data (ii). Graph show mean values ± SE of band intensities. *p<0.05. IB: immunoblot; Coll: collagen; Thr: thrombin.