An Antithrombotic Strategy by Targeting Phospholipase D in Human Platelets

Abstract

Phospholipase D (PLD) is involved in many biological processes. PLD1 plays a crucial role in regulating the platelet activity of mice; however, the role of PLD in the platelet activation of humans remains unclear. Therefore, we investigated whether PLD is involved in the platelet activation of humans. Our data revealed that inhibition of PLD1 or PLD2 using pharmacological inhibitors effectively inhibits platelet aggregation in humans. However, previous studies have showed that PLD1 or PLD2 deletion did not affect mouse platelet aggregation in vitro, whereas only PLD1 deletion inhibited thrombus formation in vivo. Intriguingly, our data also showed that the pharmacological inhibition of PLD1 or PLD2 does not affect mouse platelet aggregation in vitro, whereas the inhibition of only PLD1 delayed thrombus formation in vivo. These findings indicate that PLD may play differential roles in humans and mice. In humans, PLD inhibition attenuates platelet activation, adhesion, spreading, and clot retraction. For the first time, we demonstrated that PLD1 and PLD2 are essential for platelet activation in humans, and PLD plays different roles in platelet function in humans and mice. Our findings also indicate that targeting PLD may provide a safe and alternative therapeutic approach for preventing thromboembolic disorders.

Keywords: clot retraction; phospholipase D; platelet activation; thrombus formation.

Figure 1

Effects of phospholipase D (PLD)1 and PLD2 on platelet aggregation and thrombus formation. Washed (A) human platelets (3.6 × 10⁸ cells/mL) and (B) mouse platelets (1 × 10⁸ cells/mL) were preincubated with DMSO (solvent control), the PLD1 inhibitor VU1 (2 or 5 μM), or the PLD2 inhibitor VU2 (2 or 5 μM) and then stimulated using collagen (1 μg/mL) to trigger platelet aggregation. (C) Mice received an intravenous bolus of DMSO, VU1 (2.7 mg/kg), or VU2 (2.5 mg/kg) for 30 min before the administration of sodium fluorescein; subsequently, mesenteric venules were irradiated to induce microthrombus formation. Arrows indicate thrombus. (D) Bleeding was induced by severing the tail at 3 mm from the tail tip, and the bleeding tail stump was immersed in saline. The bleeding time was continually recorded until no sign of bleeding was observed for at least 10 s. Each point in the scatter plot graph represents a mouse. The profiles (A,B) are representative examples of three similar experiments. Data (C,D) are presented as the mean ± standard error of the mean (SEM) (C, n = 6; D, n = 8). *** p < 0.001, compared with the DMSO group.

Figure 2

Pharmacological inhibition of PLD mediated the reduction of human platelet aggregation and granule release. (A,B) Washed human platelets (3.6 × 10⁸ cells/mL) were preincubated with DMSO (solvent control), VU1 (10 and 25 μM), or VU2 (10 and 25 μM) and then stimulated using thrombin (0.01 U/mL) and U46619 (1 μM) to trigger (A) platelet aggregation and the (B) phosphorylation of PLD1 and 2. Effects of VU1 (25 μM) and VU2 (25 μM) on (C) thrombin-induced adenosine triphosphate (ATP) release and (D) P-selectin secretion were characterized by the detection of chemiluminescent emission from the luciferin–luciferase reaction and the fluorescence of P-selectin–fluorescein isothiocyanate (FITC)–antibody through flow cytometry, respectively. The profiles (A–D) are representative examples of three similar experiments.

Figure 3

Effects of VU1 on the phosphorylation of Akt and mitogen-activated protein kinases (MAPKs). Washed platelets (1.2 × 10⁹ cells/mL) were preincubated with DMSO or VU1 (10 and 25 μM), and thrombin (0.01 U/mL) was subsequently added to trigger the phosphorylation of Akt, extracellular signal-regulated protein kinase (ERK), anti-c-Jun N-terminal kinase (JNK), and p38 MAPK. Cells were then collected, and subcellular extracts were analyzed through Western blotting. Data (A–D) are presented as means ± SEM (n = 3). * p < 0.05 and ** p < 0.01, compared with the resting group; # p < 0.05 and ## p < 0.01, compared with the thrombin (positive) group.

Figure 4

Effects of VU2 on the phosphorylation of Akt and MAPKs. Washed platelets (1.2 × 10⁹ cells/mL) were preincubated with DMSO or VU2 (10 and 25 μM), and thrombin (0.01 U/mL) was subsequently added to trigger the phosphorylation of Akt, ERK, JNK, and p38 MAPK. Cells were then collected, and subcellular extracts were analyzed through Western blotting. Data (A–D) are presented as means ± SEM (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001, compared with the resting group; # p < 0.05, ## p < 0.01, and ### p < 0.001, compared with the thrombin (positive) group.

Figure 5

Regulatory effects of PLD on platelet adhesion, spreading, and clot retraction. (A,B) Washed platelets (3 × 10⁷ cells/mL) were preincubated with DMSO, VU1 (10 and 25 μM), or VU2 (10 and 25 μM) and allowed to adhere and spread on immobilized fibrinogen for 1.5 h. After fixation, cells were stained with FITC-phalloidin, and images were obtained using a confocal microscope. Scale bar: 10 μm. The inset indicates the high magnification image of the region marked by dashed box. (C) Platelet suspensions (3.6 × 10⁸ cells/mL) were pretreated with DMSO, VU1 (10 and 25 μM), or VU2 (10 and 25 μM) for 3 min, and subsequently, clot retraction was initiated using thrombin (0.01 U/mL) in the presence of fibrinogen and CaCl₂. Clot retraction was allowed to proceed at 37 °C in an aggregometer tube and photographed at the indicated times. Data (A,B) are presented as means ± SEM (n = 3). * p < 0.05, compared with the DMSO (solvent control) group. Profiles (C) are representative examples of three similar experiments.

Figure 6

Hypothetical scheme of PLD (PLD1 and PLD2) involvement in human platelet activation. PLD may be involved in the activation of Akt and MAPK, leading to granule release and subsequent inside-out signaling-mediated integrin αIIbβ3 activation. It may also be involved in outside-in signaling of integrin αIIbβ3 and support platelet adhesion, spreading, and plug formation, thus stabilizing thrombus formation.