The relative role of PLCbeta and PI3Kgamma in platelet activation

Abstract

Stimulation of platelet G protein-coupled receptors results in the cleavage of phosphatidylinositol 4,5-trisphosphate (PIP(2)) into inositol 1,4,5-trisphosphate and 1,2-diacylglycerol by phospholipase C (PLCbeta). It also results in the phosphorylation of PIP2 by the gamma isoform of phosphatidylinositol 3-kinase (PI3Kgamma) to synthesize phosphatidylinositol 3,4,5-trisphosphate. To understand the role of PIP2 in platelet signaling, we evaluated knock-out mice lacking 2 isoforms of PLCbeta (PLCbeta2 and PLCbeta3) or lacking the G(betagamma)-activated isoform of PI3K (PI3Kgamma). Both knock-out mice were unable to form stable thrombi in a carotid injury model. To provide a functional explanation, knock-out platelets were studied ex vivo. PLCbeta2/beta3-/- platelets failed to assemble filamentous actin, had defects in both secretion and mobilization of intracellular calcium, and were unable to form stable aggregates following low doses of agonists. Platelets lacking PI3Kgamma disaggregated following low-dose adenosine diphosphate (ADP) and had a mildly impaired ability to mobilize intracellular calcium. Yet, they exhibited essentially normal actin assembly and secretion. Remarkably, both PLCbeta2/beta3-/- and PI3Kgamma-/- platelets spread more slowly upon fibrinogen. These results suggest substantial redundancy in platelet signaling pathways. Nonetheless, the diminished ability of knock-out platelets to normally spread after adhesion and to form stable thrombi in vivo suggests that both PLCbeta2/beta3 and PI3Kgamma play vital roles in platelet cytoskeletal dynamics.

Figure 1.

Immunoblots of PLCβ isoforms in wild-type and PLCβ2/β3-null platelets. Total cell lysates containing human or murine platelets were fractionated by gel electrophoresis and immunoblotted with PLCβ isoform-specific polyclonal antibodies. (A) Murine platelets have relatively less PLCβ2 and relatively more PLCβ1. (B) Murine platelets lacking PLCβ2 and PLCβ3 did not have a compensatory increase in PLCβ1 or PLCβ4.

Figure 2.

Effect of PI3Kγ- or PLCβ2/β3-null mutations on in vivo thrombosis. Carotid injury was induced by application of FeCl₃-soaked filter paper for 2 minutes, and vessel occlusion was monitored by a Doppler ultrasound. The Doppler tracings show flow (milliliters per minute) on the y axis and time on the x axis. Control mice typically developed stable occlusions within their injured arteries. In contrast, PLCβ2/β3 and PI3Kγ knock-out mice both failed to form stable arterial occlusions in response to chemical injury.

Figure 3.

Platelet cytoplasmic calcium concentration in PLCβ2/β3-null platelets. Washed murine platelets were incubated with a calcium fluorophore (Fura-2 AM) and then stimulated with either 1 U/mL thrombin or 10 mM ADP. The concentration of cytoplasmic calcium (nM) was quantitated as a function of time. (A) Representative fluorimetry tracings of experiments using thrombin- or ADP-stimulated wild-type and PLCβ2/β2-null platelets. (B) A graph displaying the mean ± SEM of the cytosolic calcium concentration for 6 experiments. (C) A graph demonstrating the influence of the extracellular calcium concentration on the ability of platelets to raise their cytoplasmic calcium concentration in response to 1 U/mL thrombin. The change in cytosolic calcium was normalized to the response seen in wild-type cells. The graphs show the mean ± SEM for 3 experiments.

Figure 4.

Platelet cytoplasmic calcium concentration in PI3Kγ-null platelets. Washed murine platelets were incubated with a calcium fluorophore (Fura-2 AM) and then stimulated with either 1 U/mL thrombin or 10 mM ADP. The concentration of cytoplasmic calcium (nM) was quantitated as a function of time. (A) Representative fluorimetry tracings of experiments using thrombin- or ADP-stimulated wild-type and PI3Kγ-null platelets. (B) A graph displaying the mean ± SEM of the cytosolic calcium concentration for 3 experiments. (C) A graph demonstrating the influence of the extracellular calcium concentration on the ability of platelets to raise their cytoplasmic calcium concentration in response to 1 U/mL thrombin. The change in cytosolic calcium was normalized to the response seen in wild-type cells. The graph shows the mean ± SEM for 3 experiments.

Figure 5.

Platelet ATP secretion in response to agonist stimulation. Secretion of washed platelets was measured at 37°C by the turbidometric method in a Lumi-dual Aggregometer following the addition of various agonists. Secretion induced by low doses of ADP or U46619 (thromboxane A2 analog) was impaired in PLCβ2/β3 knock-out platelets (A). Shown are means ± standard error of 3 experiments. Similar results were seen in 7 experiments analyzing secretion in platelet-rich plasma. (B) A graph showing an analysis of the agonist-induced secretion response of PI3Kγ-null platelets compared with control cells. Paired student t testing revealed no significant defect in PI3Kγ knock-out platelets with any agonist. Shown are the means ± SEM normalized in 2 experiments. A similar lack of secretion defect was found in 3 experiments analyzing secretion in platelet-rich plasma (not shown).

Figure 6.

Aggregation tracings of platelets lacking PLCβ2/β3. Murine platelets lacking PLCβ2/β3 were analyzed after agonist stimulation in a Lumi-dual Aggregometer. Platelets lacking both PLCβ2 and PLCβ3 have a defect in aggregation in response to ADP and low doses of thrombin and exhibit impairment in the second wave of aggregation. Results are representative of 9 experiments performed to date.

Figure 7.

Aggregation tracings of platelets lacking PI3Kγ. Murine platelets lacking the γ isoform of PI3K were analyzed after agonist stimulation. Platelets lacking PI3Kγ had a mild defect in agonist-mediated aggregation associated with an impaired second wave of aggregation. Results are representative of 7 experiments performed to date.

Figure 8.

Actin assembly in response to thrombin. Following stimulation of washed murine platelets with 1 U/mL thrombin, platelets were fixed, permeabilized, and stained with fluorescent phalloidin. Flow cytometry was used to quantitate phalloidin binding in 100 000 cells, and analysis was performed using CELLQuest software. Shown is the mean ± SEM for 3 experiments. Platelet actin assembly in response to thrombin requires PLCβ2/β3 (A) but does not require PI3Kγ (B).

Figure 9.

Platelet spreading on immobilized fibrinogen. Washed platelets were layered at a density of 5 × 10⁷/mL onto fibrinogen-coated slides. After several time points, cells were fixed and incubated with the membrane stain, vybrant DiO. Platelets lacking either PLCβ2β3 or PI3Kγ had impaired spreading compared with wild-type platelets. The spreading defect was most apparent at earlier time points. This demonstrates that second messengers generated by both PLCβ2β3 and PI3Kγ are required for platelet spreading. Results are representative for 3 experiments performed to date.