Nowadays, a number of researchers or scientists are dedicated to discover a novel drug or new therapeutic strategy that not only prevents thrombosis-relevant events, such as stroke and heart attack, but also has minimal bleeding effect. Moreover, several potential targets, such as glycoprotein VI, were reported [1
]. In addition, cell therapy is also a promising therapeutic option. Due to its immunomodulatory and regenerative activities, mesenchymal stem cells were also investigated and were used to treat ischemic stroke and retinal injury in preclinical settings [2
]. Thus, it is important to find a new drug or therapeutic approach to prevent thrombosis-relevant events.
The phospholipase D (PLD) enzyme can catalyze the hydrolysis of phosphatidylcholine to choline and phosphatidic acid under a variety of stimuli, such as growth factors and neurotransmitters. Phosphatidic acid is a second messenger that can function in vesicular trafficking, cytoskeleton reorganization, and different signaling pathways [5
]. There are two classical PLD isoforms in mammals, PLD1 and PLD2, which were proposed to be involved in many physiological and pathological processes in cancer, immunity, and thrombus formation [5
]. In mice, the deficiency of PLD1, not PLD2, can impair thrombus formation in pulmonary thromboembolism, aortic occlusion and ferric chloride (FeCl3
)-induced carotid artery injury models [6
]. Thielmann et al. [7
] also reported that these two isoforms have partially redundant functions in thrombus formation and that Pld1−/−/Pld2−/−
mice display reduced granule release and enhanced integrin activation. Moreover, Stegner et al. reported that pharmacological inhibition of both PLD1 and PLD2 by 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) can diminish granule release as well [8
]. Further, these reports showed that platelets from PLD1 or PLD2 knockout mice or FIPI-treated platelets did not show impairment of platelet aggregation. However, our previous study revealed that 5 μM PLD1 inhibitor (VU0155069; VU1) or PLD2 inhibitor (VU0364739; VU2) led to a complete inhibition of platelet aggregation induced by collagen in humans, but not in mice, suggesting that PLD plays differential regulatory roles between mouse and human platelets [9
Notably, studies revealed different results obtained from Pld1−/−
mice in different animal models. For example, Pld1−/−
mice revealed considerable protection against lethal pulmonary embolization, FeCl3
-induced injury of carotid artery, and mechanical injury of the abdominal aorta [6
], but demonstrated non-significant protection against FeCl3
-induced injury of small mesenteric arterioles [7
]. However, compared to Pld1−/−/Pld2−/−
or FIPI (3 mg/kg)-treated mice, Pld1−/−
mice demonstrated similar or even better improvement in infarct size induced by transient middle cerebral artery (MCA) occlusion (MCAO), [6
]. Moreover, our previous study revealed that pharmacological inhibition of PLD1, but not PLD2, affords a protective effect against thrombosis in mesenteric microvessels of mice [9
]. These results revealed that PLD1 plays a more crucial role in thrombus formation in mice.
Owing to these diversities, we used the selective pharmacological PLD1 inhibitor VU1 and PLD2 inhibitor VU2 to further determine or confirm the role of PLD1 and PLD2 in thrombosis-relevant events, including pulmonary thrombosis and transient MCAO-induced brain injury.
The significance of the current study was that it further defined the roles of PLD1 and PLD2 in thrombosis-relevant events in mice. Here, we confirmed that PLD1 might play more important roles than PLD2 and that both PLD1 and PLD2 act synergistically, or partially redundantly, in regulating thrombosis-relevant events, including acute pulmonary thrombosis and ischemic stroke, in mice.
PLD is involved in various cell biological processes, such as regulated exocytosis, endocytosis, cell migration, proliferation, apoptosis, and autophagy [12
]. PLD also participates in pathological processes, such as cancer and Alzheimer’s disease [13
]. However, the role of PLD in thrombosis remains unclear. Deficiency of PLD1, but not PLD2, was reported to impair thrombus formation in the models of pulmonary thromboembolism, aortic occlusion, and FeCl3
-induced carotid artery injury [6
]. Moreover, PLD1 is a regulator of platelet-mediated inflammation [17
]. This report also showed that adhesion of PLD1-deficient but not PLD2-deficient platelets on activated endothelial cells obviously decreased under high shear rates, suggesting that PLD1 plays a major role in platelet-mediated inflammation under high shear rates [17
]. In addition, another study reported that PLD1 could regulate LPS-induced sepsis, which might be due to the reduced thrombin generation on PLD1-deficient platelets and the subsequent reduced fibrin formation and platelet consumption, eventually reducing the risk of disseminated intravascular coagulation [18
]. By contrast, Thielmann et al. reported that Pld1−/−
mice did not show a significant protection against thrombus formation in small mesenteric arterioles induced by FeCl3
, as compared to wild-type mice, but Pld1−/−/Pld2−/−
mice demonstrated a prolonged time of full occlusion. Moreover, Pld1−/−/Pld2−/−
mice displayed reduced granule release and enhanced integrin activation. Thus, the authors suggest that these two isoforms show partially redundant functions in thrombus formation and granule release [7
]. These two studies using PLD knockout mice revealed a discrepancy in the role of PLD1 among several different thrombotic models of mice. This needs to be clarified in further research.
Our previous study using the selective pharmacological inhibitor of PLD1 VU1 (2.7 mg/kg) or of PLD2 VU2 (2.5 mg/kg) also demonstrated that only PLD1 inhibition, but not PLD2 inhibition, could significantly delay thrombus formation, indicating that PLD1 might be more crucial than PLD2 in the thrombotic events [9
]. Therefore, in the present study, we further used two different thrombotic models of acute pulmonary thrombosis and ischemic stroke, to define the roles of PLD1 and PLD2. The results revealed that only PLD1 inhibition, but not PLD2 inhibition, could partially improve pulmonary thrombosis-induced death, which is consistent with the results reported by Elvers et al. [6
]. Moreover, simultaneous PLD1 and PLD2 inhibition led to a considerable improvement in the survival rate of mice. Likewise, only PLD1 inhibition, but not PLD2 inhibition, could partially improve ischemic stroke, and inhibition of both PLD1 and PLD2 could afford considerable protection against ischemic stroke. Post-stroke behavior of the mice was also evaluated using mNSS, rotarod test, and the open-field test. These tests also revealed that the inhibition of either PLD1 or both PLD1 and PLD2 affords protective effects against neurological deficit after stroke. This finding was also in agreement with that of a previous study, which showed that the pharmacological inhibition of both PLD1 and PLD2 by FIPI could effectively prevent ischemic stroke [8
]. Although we did not exclude the possibility that pharmacological inhibitors might have off-target effects, our previous [9
] and present study revealed rather consistent findings that inhibition of only PLD1, but not PLD2, could afford a protective effect against thrombosis-relevant events and that enhanced protective effects appeared when PLD1 and PLD2 inhibitors were used simultaneously. These results were also consistent with those of previous studies in which PLD-knockout mice and nonselective PLD inhibitor FIPI were used [6
]. However, among all of these studies (including ours) that investigated the role of PLD, only one study showed that the absence of PLD1 was not resistant to thrombus formation in small mesenteric arterioles induced by 20% FeCl3
]. In general, PLD1, therefore, was involved in thrombus formation.
We previously demonstrated that PLD1 and PLD2 are essential for maintaining platelet activation in humans [9
]. However, other studies reported that PLD1 is required for full integrin activation of platelets [6
] and that it plays a major role in regulating platelet-mediated inflammation [17
], but absence of PLD2 has no effect on platelet activation [7
] in mice. Thus, PLD has different regulatory effects on platelet activation in humans and mice. This discrepancy might be attributed to the difference between the two species. Meanwhile, genetic deletion or pharmacological inhibition of PLD1, PLD2, or both of PLD1 and PLD2, did not result in a prolonged bleeding time, suggesting that PLD inhibition might be a safe strategy to prevent cardiovascular diseases [9
]. However, in the current study, there is a limitation that we cannot exclude the possibility of off-target effect of inhibitors. It is hard to find the volunteers with PLD1 or PLD2 deficiency to validate the difference between mice and human. Moreover, previous studies suggested that it is crucial to understand the differences between humans and mice [20
]. This might also contribute to causality of the failure of clinical trials, which is inconsistent with the results of the preclinical study. Therefore, the PLD enzyme might need extensive investigation to understand the difference of PLD enzyme between mice and humans in the future. Moreover, targeting PLD might provide a therapeutic strategy to develop a novel antiplatelet drug. Nowadays, every scientist is dedicated to finding a drug or an approach to prevent a stroke or heart attack, but also hopes that this drug does not affect hemostasis [1
]. This issue is critical because the side effect of bleeding remains a limitation of clinical antiplatelet drugs, which has limited their use.
Excepting platelet-mediated inflammation and the thrombotic events in mouse models in which PLD1 was reported to play a major role, we previously demonstrated that both PLD1 and PLD2 are crucial regulators in human platelet activation [9
]. Similarly, both PLD1 and PLD2 reportedly regulate macrophage phagocytosis [22
], Alzheimer’s disease [16
], and tumor growth [23
]. Collectively, PLD isoforms might play different roles in different species and diseases that involve corresponding cells or tissues.
On the other hand, cell therapy is also a promising therapeutic option in ischemic stroke. For example, dental pulp stem cells (DPSCs) were reported to provide a tempting prospect for stroke treatment [25
]. However, the safety issues of DPSCs, such as an in vitro expansion and in vivo delivery are a concern. It is important to use quality-controlled, and potentially advantageous supplements to establish a preparatory study for regenerative medicine applications [26
], such as stroke treatment.