Pharmacological Inhibition of Phospholipase D Protects Mice From Occlusive Thrombus Formation and Ischemic Stroke—Brief Report

Abstract

Objective-We recently showed that mice lacking the lipid signaling enzyme phospholipase (PL) D1 or both PLD isoforms (PLD1 and PLD2) were protected from pathological thrombus formation and ischemic stroke, whereas hemostasis was not impaired in these animals. We sought to assess whether pharmacological inhibition of PLD activity affects hemostasis, thrombosis, and thrombo-inflammatory brain infarction in mice. Approach and Results-Treatment of platelets with the reversible, small molecule PLD inhibitor, 5-fluoro-2-indolyl deschlorohalopemide (FIPI), led to a specific blockade of PLD activity that was associated with reduced α-granule release and integrin activation. Mice that received FIPI at a dose of 3 mg/kg displayed reduced occlusive thrombus formation upon chemical injury of carotid arteries or mesenterial arterioles. Similarly, FIPI-treated mice had smaller infarct sizes and significantly better motor and neurological function 24 hours after transient middle cerebral artery occlusion. This protective effect was not associated with major intracerebral hemorrhage or prolonged tail bleeding times. Conclusions-These results provide the first evidence that pharmacological PLD inhibition might provide a safe therapeutic strategy to prevent arterial thrombosis and ischemic stroke. 11,23 Here, we show that pharmacological inhibition of PLD activity with FIPI specifically reduced PLDdependent α-granule release and integrin activation resulting in decreased thrombosis and infarct progression during acute stroke in mice without affecting hemostasis. Materials And MethodsMaterials and Methods are available in the online-only Supplement. Results FIPI Treatment Abolishes PLD Activity and Leads to Defective Integrin Activation and α-Granule ReleaseIt has been shown that platelets of Pld1−/− mice display defective integrin activation and α-granule release on platelet stimulation with intermediate concentration of thrombin.11 To assess whether the small, reversible PLD inhibitor FIPI specifically inhibits PLD and whether this leads to the same effects as observed in the PLD1/2 double-deficient mice, we analyzed PLD activity and platelet activation on FIPI treatment. Although platelet stimulation with thrombin triggered PLD activity in vehicle-treated platelets, FIPI dose-dependently inhibited PLD activity ( Figure 1A). In subsequent in vitro experiments we chose to use 100 nmol/L FIPI because this was the lowest concentration that abolished PLD activity, consistent with results for other cell types.14 Treatment of platelets with this concentration of FIPI did not alter glycoprotein expression on the platelet surface (data not shown). Integrin activation and P-selectin exposure, as a measurement for α-granule release, were analyzed flow cytometrically in vehicle-and FIPI-treated wild-type and Pld1−/− mice to test for potential off-target effects created by FIPI. The results revealed decreased integrin activation and α-granule release in FIPI-treated platelets upon stimulation with an intermediate concentration (3 mU/mL) of...

Functional Regulation of Phospholipase D Expression in Cancer and Inflammation

“…Although small molecule PLD inhibitors appear to have some value in cell culture systems, their usefulness for PLD inhibition in vivo in animal models is less well established. Recent studies have shown that pharmacologically and genetically induced PLD inhibition had no obvious side effects (8,11); thus, such a safe therapy could be particularly advantageous in clinical practice. Accordingly, highly selective PLD inhibitors with greater potency need to be developed and analyzed to enable optimized drug delivery and bioavailability.…”

“…The PLD signaling pathway provides a possible molecular link between inflammation and cancer; however, systematic investigation of PLD as a therapeutic target has only begun within the last few years with the development of small molecule PLD inhibitors and PLD knock-out mice (5)(6)(7)(8)(9)(10)(11)(12)(13). This review covers recent advances in the regulation of PLD expression and its role in cancer and inflammation.…”

Cellular and Physiological Roles for Phospholipase D1 in Cancer

“…To date, the inhibitors have not been associated with off-target or nonspecific effects, excepting one of the PLD1-selective compounds (66). FIPI has also been employed in vivo to study cancer growth and metastasis and platelet activation (22,35,67), which was achievable given its 5.5 h half-life and acceptable bioavailability and solubility (54). The subtypespecific PLD inhibitors currently available are less amenable for in vivo studies with shorter half-lives (45 min to 1 h) and efficient first-pass clearance (56), but with continued development could become invaluable tools for study in animal models and as potential therapeutics.…”

Thematic Review Series: Phospholipases: Central Role in Lipid Signaling and Disease

“…Roles for PLD2 in thrombotic disease ( 37,66 ), cancer ( 67, 68 ), Alzheimer's disease (AD) ( 69 ), and immune function ( 65 ), based on animal model studies, have recently been summarized ( 5 ). Other potential functions have been raised by tissue culture studies, some of which will be reviewed here.…”

“…Roles for PLD1 in thrombotic disease ( 28,37,38 ), cancer ( 39 ), and auto-immunity ( 40 ), as revealed through animal model studies, have recently been summarized ( 5 ). Many other possible roles are suggested by cellular studies that have not yet been addressed in vivo, some of which will be reviewed here.…”