p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo

Abstract

Platelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-alpha and GPV. We recently demonstrated that tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37 degrees C or 22 degrees C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

Figure 1

TACE^ΔZn/ΔZn platelets are protected from storage-induced shedding of GPIb-α and increased posttransfusion clearance. (A) Surface expression of GPIb-α was determined by flow cytometry on freshly isolated or 15-hour stored platelet-rich plasma (PRP) from TACE^+/+ and TACE^ΔZn/ΔZn mice. Results are mean ± SEM; n = 6. n.s. indicates not significant. (B) Platelets from stored PRP from TACE^+/+ and TACE^ΔZn/ΔZn mice were washed in Tyrode-N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffer and labeled with CFSE. A total of 10⁸ platelets/10 g of body weight was infused intravenously into TACE^+/+ mice. Blood was drawn at the indicated time points, and platelets were immediately analyzed by flow cytometry. Results are mean percentage CFSE-labeled platelets ± SEM; n = 4. ***P < .001. *P < .05.

Figure 2

p38 MAPK inhibition prevents the shedding of GPIb-α from stored mouse and human platelets. GPIb-α surface expression was assessed by flow cytometry. (A) Wild-type mouse PRP was stored 1, 3, 6, 9, 12, and 15 hours at 37°C in the presence of DMSO (vehicle) or the p38 MAPK inhibitor SB203580 (40μM); n = 8. ***P < .001. (Inset) Western blot for GPIb-α in lysates from freshly isolated platelets and platelets stored for 16 hours in the presence or absence of SB203580. The results are representative of 3 independent experiments. (B) PRP was analyzed immediately (fresh) or after storage at 22°C or 37°C in the presence of DMSO (vehicle) or SB203580 (40μM); n = 10. (C) Washed platelets were treated for 60 minutes at 37°C with DMSO (untreated) or 100μM CCCP in the presence of DMSO (vehicle), SB203580 (40μM), or SB202190 (40μM); n = 9. (D) PRP isolated from human blood was analyzed immediately (fresh) or after 72 hours of storage at 37°C in the presence of DMSO (vehicle) or SB203580 (40μM); n = 8. Results are mean ± SEM. n.s. indicates not significant.

Figure 3

Phosphorylation and activity of p38 MAPK are increased during in vitro storage of platelets. Mouse platelets were isolated from fresh PRP or PRP stored in the presence of SB203580 (40μM) or DMSO (vehicle), washed in Tyrode-N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffer, and lysed immediately. (A) Immunoprecipitated phospho-p38 MAPK was detected by Western blot analysis using a specific antibody for p38 MAPK and quantified (mean ± SEM); n = 4. (B) The activity of phosphorylated p38 MAPK was assessed in lysates from 2 different preparations of freshly isolated or stored platelets by its ability to phosphorylate the substrate fusion protein ATF-2. (C) Phosphorylation of p38 and ATF-2 in platelets that were treated with 100μM CCCP for the indicated times. Total p38 MAPK was assessed in platelet lysates by Western blot analysis using a specific antibody for p38 MAPK.

Figure 4

p38 MAPK inhibition does not affect integrin-mediated aggregation and adhesion to collagen under flow of freshly isolated and stored murine platelets. (A) PRP stored in the presence of DMSO (vehicle) or SB203580 (40μM) was examined in standard aggregometry after addition of 5μM ADP or 1mM PAR4-AP. (B) Freshly isolated murine PRP was activated with the indicated agonists in the presence or absence of SB203580. Results are representative of 3 separate experiments. (C) Platelets in freshly isolated murine blood were labeled by the addition of an Alexa488-labeled antibody to GPIX. Whole blood, in the presence or absence of SB203580, was perfused over collagen at a shear rate of 1200 seconds. Adhesion and thrombus formation of fluorescently labeled platelets were monitored over time. (Left) Representative images. (Right) Fluorescence intensity (platelet adhesion) measured over time. Results are mean ± SEM; n = 6 (3 different blood preparations).

Figure 5

Inhibition of p38 MAPK during storage improves the posttransfusion recovery and hemostatic function of platelets. (A) Platelets from fresh PRP or PRP stored in the presence of DMSO (stored) or SB203580 (40μM; stored/SB203580) were washed and labeled with 2 μg/mL CFSE. A total of 10⁸ platelets/10 g of body weight was infused intravenously into a wild-type mouse. At the indicated time points, blood was drawn and analyzed by flow cytometry. Results are mean percentage of labeled platelets ± SEM; n = 6. (B) FeCl₃-induced thrombosis was studied in mesenteric arterioles of mice transfused with 0.5 × 10⁸ SB203580/stored platelets labeled with calcein-red/orange and 1.5 × 10⁸ vehicle/stored platelets labeled with calcein-green. Platelet adhesion (tethering) and thrombus growth were monitored until complete vessel occlusion occurred (occlusion). Representative images are shown. (C) IL4Rα/GPIb-α-tg mice were transfused with the indicated platelet preparations (10⁸ platelets/10 g of body weight). The tail bleeding time was measured 1 hour after platelet transfusion. n.s. indicates not significant.