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Research ArticleHematologyVascular biology
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10.1172/JCI163808
1Division of Cardiovascular Medicine and
2Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
3Cardiovascular Research Center,
4Department of Medicine, and
5Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
6Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA.
7Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
8Division of Nephrology and Departments of Internal Medicine and Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas, USA.
9Division of Hematology and Hematologic Malignancies, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Alec Schmaier, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 909, Boston, Massachusetts 02215, USA. Phone: 617.735.5274; Email: aschmaie@bidmc.harvard.edu. Or to: Sol Schulman, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 939, Boston, Massachusetts 02215, USA. Phone: 617.735.4049; sschulm1@bidmc.harvard.edu.
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1Division of Cardiovascular Medicine and
2Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
3Cardiovascular Research Center,
4Department of Medicine, and
5Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
6Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA.
7Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
8Division of Nephrology and Departments of Internal Medicine and Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas, USA.
9Division of Hematology and Hematologic Malignancies, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Alec Schmaier, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 909, Boston, Massachusetts 02215, USA. Phone: 617.735.5274; Email: aschmaie@bidmc.harvard.edu. Or to: Sol Schulman, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 939, Boston, Massachusetts 02215, USA. Phone: 617.735.4049; sschulm1@bidmc.harvard.edu.
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1Division of Cardiovascular Medicine and
2Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
3Cardiovascular Research Center,
4Department of Medicine, and
5Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
6Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA.
7Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
8Division of Nephrology and Departments of Internal Medicine and Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas, USA.
9Division of Hematology and Hematologic Malignancies, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Alec Schmaier, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 909, Boston, Massachusetts 02215, USA. Phone: 617.735.5274; Email: aschmaie@bidmc.harvard.edu. Or to: Sol Schulman, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 939, Boston, Massachusetts 02215, USA. Phone: 617.735.4049; sschulm1@bidmc.harvard.edu.
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1Division of Cardiovascular Medicine and
2Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
3Cardiovascular Research Center,
4Department of Medicine, and
5Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
6Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA.
7Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
8Division of Nephrology and Departments of Internal Medicine and Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas, USA.
9Division of Hematology and Hematologic Malignancies, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Alec Schmaier, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 909, Boston, Massachusetts 02215, USA. Phone: 617.735.5274; Email: aschmaie@bidmc.harvard.edu. Or to: Sol Schulman, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 939, Boston, Massachusetts 02215, USA. Phone: 617.735.4049; sschulm1@bidmc.harvard.edu.
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1Division of Cardiovascular Medicine and
2Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
3Cardiovascular Research Center,
4Department of Medicine, and
5Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
6Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA.
7Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
8Division of Nephrology and Departments of Internal Medicine and Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas, USA.
9Division of Hematology and Hematologic Malignancies, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Alec Schmaier, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 909, Boston, Massachusetts 02215, USA. Phone: 617.735.5274; Email: aschmaie@bidmc.harvard.edu. Or to: Sol Schulman, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 939, Boston, Massachusetts 02215, USA. Phone: 617.735.4049; sschulm1@bidmc.harvard.edu.
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1Division of Cardiovascular Medicine and
2Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
3Cardiovascular Research Center,
4Department of Medicine, and
5Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
6Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA.
7Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
8Division of Nephrology and Departments of Internal Medicine and Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas, USA.
9Division of Hematology and Hematologic Malignancies, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Alec Schmaier, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 909, Boston, Massachusetts 02215, USA. Phone: 617.735.5274; Email: aschmaie@bidmc.harvard.edu. Or to: Sol Schulman, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 939, Boston, Massachusetts 02215, USA. Phone: 617.735.4049; sschulm1@bidmc.harvard.edu.
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1Division of Cardiovascular Medicine and
2Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
3Cardiovascular Research Center,
4Department of Medicine, and
5Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
6Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA.
7Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
8Division of Nephrology and Departments of Internal Medicine and Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas, USA.
9Division of Hematology and Hematologic Malignancies, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Alec Schmaier, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 909, Boston, Massachusetts 02215, USA. Phone: 617.735.5274; Email: aschmaie@bidmc.harvard.edu. Or to: Sol Schulman, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 939, Boston, Massachusetts 02215, USA. Phone: 617.735.4049; sschulm1@bidmc.harvard.edu.
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1Division of Cardiovascular Medicine and
2Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
3Cardiovascular Research Center,
4Department of Medicine, and
5Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
6Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA.
7Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
8Division of Nephrology and Departments of Internal Medicine and Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas, USA.
9Division of Hematology and Hematologic Malignancies, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Alec Schmaier, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 909, Boston, Massachusetts 02215, USA. Phone: 617.735.5274; Email: aschmaie@bidmc.harvard.edu. Or to: Sol Schulman, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 939, Boston, Massachusetts 02215, USA. Phone: 617.735.4049; sschulm1@bidmc.harvard.edu.
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1Division of Cardiovascular Medicine and
2Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
3Cardiovascular Research Center,
4Department of Medicine, and
5Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
6Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA.
7Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
8Division of Nephrology and Departments of Internal Medicine and Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas, USA.
9Division of Hematology and Hematologic Malignancies, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Alec Schmaier, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 909, Boston, Massachusetts 02215, USA. Phone: 617.735.5274; Email: aschmaie@bidmc.harvard.edu. Or to: Sol Schulman, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 939, Boston, Massachusetts 02215, USA. Phone: 617.735.4049; sschulm1@bidmc.harvard.edu.
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1Division of Cardiovascular Medicine and
2Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
3Cardiovascular Research Center,
4Department of Medicine, and
5Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
6Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA.
7Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
8Division of Nephrology and Departments of Internal Medicine and Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas, USA.
9Division of Hematology and Hematologic Malignancies, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Alec Schmaier, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 909, Boston, Massachusetts 02215, USA. Phone: 617.735.5274; Email: aschmaie@bidmc.harvard.edu. Or to: Sol Schulman, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 939, Boston, Massachusetts 02215, USA. Phone: 617.735.4049; sschulm1@bidmc.harvard.edu.
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1Division of Cardiovascular Medicine and
2Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
3Cardiovascular Research Center,
4Department of Medicine, and
5Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
6Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA.
7Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
8Division of Nephrology and Departments of Internal Medicine and Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas, USA.
9Division of Hematology and Hematologic Malignancies, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Alec Schmaier, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 909, Boston, Massachusetts 02215, USA. Phone: 617.735.5274; Email: aschmaie@bidmc.harvard.edu. Or to: Sol Schulman, Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, CLS 939, Boston, Massachusetts 02215, USA. Phone: 617.735.4049; sschulm1@bidmc.harvard.edu.
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Published March 23, 2023 - More info
Published in Volume 133, Issue 11 on June 1, 2023
Related article:
Abstract
Endothelial cells form a constitutively anticoagulant surface under homeostasis. While loss of this anticoagulant property is a hallmark of many cardiovascular diseases, the molecular mechanisms underlying the procoagulant transition remain incompletely understood. In this issue of the JCI, Schmaier et al. identify the phospholipid scramblases TMEM16E and TMEM16F, which support endothelial procoagulant activity through phosphatidylserine (PS) externalization. Genetic deletion of TMEM16E or TMEM16F or treatment with TMEM16 inhibitors prevented PS externalization and reduced fibrin formation in the vessel wall independently of platelets in a murine laser-injury model of thrombosis. These findings reveal a role for endothelial TMEM16E in thrombosis and identify TMEM16E as a potential therapeutic target for preventing thrombus formation.
Authors
× AbstractEndothelial cells (ECs) normally form an anticoagulant surface under physiological conditions, but switch to support coagulation following pathogenic stimuli. This switch promotes thrombotic cardiovascular disease. To generate thrombin at physiologic rates, coagulation proteins assemble on a membrane containing anionic phospholipid, most notably phosphatidylserine (PS). PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid “scramblases,” such as TMEM16F. TMEM16F-dependent PS externalization is well characterized in platelets. In contrast, how ECs externalize phospholipids to support coagulation is not understood. We employed a focused genetic screen to evaluate the contribution of transmembrane phospholipid transport on EC procoagulant activity. We identified 2 TMEM16 family members, TMEM16F and its closest paralog, TMEM16E, which were both required to support coagulation on ECs via PS externalization. Applying an intravital laser-injury model of thrombosis, we observed, unexpectedly, that PS externalization was concentrated at the vessel wall, not on platelets. TMEM16E-null mice demonstrated reduced vessel-wall–dependent fibrin formation. The TMEM16 inhibitor benzbromarone prevented PS externalization and EC procoagulant activity and protected mice from thrombosis without increasing bleeding following tail transection. These findings indicate the activated endothelial surface is a source of procoagulant phospholipid contributing to thrombus formation. TMEM16 phospholipid scramblases may be a therapeutic target for thrombotic cardiovascular disease.
Graphical Abstract
Introduction
Thrombotic disorders such as myocardial infarction, stroke, and venous thromboembolism are leading causes of mortality worldwide (1). Blood coagulation, and therefore thrombosis, depends at key stages on a membrane surface containing anionic phospholipid, most commonly phosphatidylserine (PS) (2). PS promotes the recruitment and activation of coagulation factor X by the tissue factor–factor VIIa (TF-VIIa) complex (3, 4) and assembly and activity of the factor Xa-Va-prothrombinase coagulation enzyme complex (5, 6), the initiating and ultimate steps in thrombin formation, respectively. Coagulation factor interaction with PS accelerates enzyme kinetics by at least 3 orders of magnitude to physiologic rates (6, 7). PS constitutes approximately 10%–15% of plasma membrane phospholipid (8), but under basal conditions is sequestered on the inner membrane leaflet and is therefore inaccessible to the extracellular environment. A sustained rise in intracellular calcium (Ca2+) triggers PS externalization via Ca2+-activated phospholipid scramblases (PLSs), transmembrane channels that allow PS to move down its concentration gradient to the outer leaflet of the plasma membrane (9–11). By preventing enzyme complex assembly (12, 13), PS-binding proteins such as annexin V and lactadherin inhibit coagulation reactions in vitro (14, 15) and decrease thrombosis in vivo (15–17). These findings suggest that PS exposure is an integral step in thrombosis and that targeting PS may be a viable antithrombotic strategy.
Following agonist stimulation in vitro, activated platelets readily externalize PS and support thrombin generation, providing an accessible means of studying cell-based PS exposure in coagulation (7, 10). It has therefore been suggested that activated platelets are the major source of procoagulant PS in vivo and thus the major site of coagulation enzyme-complex assembly for hemostasis and thrombosis (18). The identification of TMEM16F as a PLS required for PS externalization in platelets (19, 20) and the finding that mutations in TMEM16F underlie the mild-to-moderate bleeding disorder Scott syndrome (19, 21) led to further investigation of TMEM16F in platelet function. Platelets require TMEM16F to externalize PS in response to stimuli that raise intracellular Ca2+ (22). Loss of phospholipid scrambling in platelets protects from thrombosis or impairs hemostasis in some studies but not in others (22–25). These findings would suggest that sources of PS beyond the platelet and additional proteins beyond TMEM16F may regulate procoagulant phospholipid externalization.
Endothelial cells (ECs) form a constitutive anticoagulant surface under basal conditions to maintain blood flow. Loss of this anticoagulant property is considered a hallmark of cardiovascular disease, leading to thrombosis. However, the relative contribution of activated endothelium to blood clotting in vivo is not well understood. Several pathologic stimuli, including hypoxia, cytokines, and lipopolysaccharide, induce PS externalization on ECs (26–28), but the physiologic significance of EC phospholipid scrambling and the proteins that regulate it remain poorly characterized, although TMEM16F is likely involved (29). Inflammatory stimuli also promote TF expression and its procoagulant activity in ECs (30, 31). The majority of cell surface–expressed TF binds factor VII/VIIa, but exists in a deactivated state (32, 33). PS externalization is at least partly responsible for enhancing the TF-dependent catalytic activity of factor VIIa (3, 4, 34, 35). Therefore, identification of regulators of PS externalization will aid our understanding of TF activation and inflammatory thrombosis.
To identify regulators of membrane procoagulant activity in ECs, we performed a targeted screen of genes encoding proteins predicted to regulate transmembrane phospholipid transport for their contribution to factor VIIa–catalyzed activation of factor X. Using this approach, we identified 2 TMEM16 family members, TMEM16F and its closest paralog, TMEM16E (36), that support EC procoagulant activity via PS externalization. The TMEM16 family comprises Ca2+-activated transmembrane proteins that function as ion channels, PLSs, or, in some cases, both (36). TMEM16E is highly expressed in skeletal muscle, where it regulates muscle regeneration and repair (37, 38), but is not expressed in platelets (22, 39, 40) and has no previously known role in hemostasis or thrombosis. Our findings from intravital thrombosis assays suggest that the vessel wall is a major source of PS during thrombus formation. We demonstrate that both genetic deletion of TMEM16E and pharmacologic blockade of TMEM16 proteins inhibit fibrin formation in a vessel-wall–dependent manner during thrombosis in vivo. These results suggest that endothelial-derived PS contributes to thrombus formation and that small-molecule inhibition of TMEM16 may be a novel antithrombotic strategy.
ResultsIdentification of transmembrane phospholipid transporters predicted to regulate EC procoagulant activity. We used siRNA to evaluate genes implicated in regulation of membrane phospholipid asymmetry and measured coagulation initiation on TNF-α–stimulated ECs. Primary human umbilical vein ECs (HUVECs) were transfected with gene-specific pools of 4 distinct siRNAs and cultured for 72 hours prior to TNF-α stimulation. The cells were then assayed for their ability to support factor VIIa–catalyzed conversion of factor X to factor Xa in a chromogenic assay (Figure 1A). Since membrane PS composition is a critical determinant of factor X activation by the TF-VIIa complex (3, 34, 35), our approach was able to identify regulators of transmembrane phospholipid exchange that influenced coagulation. We focused on 13 validated genes known to affect the outer leaflet expression of PS in biological membranes, including 6 members of the TMEM16 family of Ca2+-activated PLSs, 3 members of the Xk-related family of caspase-activated PLSs (41), and 3 members of the P4-ATPase family of phospholipid flippases (42), including their cofactor, CDC50A. We identified TMEM16E, Xkr9, and TMEM16F as significant regulators of factor VIIa–catalyzed activation of factor X in ECs (P = 0.0004, 0.0016, and 0.0113, respectively, as compared with control siRNA; Figure 1A). TMEM16E is the closest paralog of TMEM16F, which is the canonical Ca2+-activated PLS (36). Both TMEM16E and TMEM16F have been shown to have PLS activity (37,43). Primary human ECs from multiple tissues expressed both TMEM16E and TMEM16F in a manner independent of TNF-α stimulation (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/JCI163808DS1). In contrast, we could not verify gene expression of XKR9 in multiple types of ECs using quantitative PCR (qPCR). Therefore, we focused our attention on TMEM16E and TMEM16F as PLSs that may promote coagulation on ECs.
Figure 1TMEM16E and TMEM16F regulate EC procoagulant activity. (A) Heatmap illustrating the relative positive or negative regulation of factor VIIa–catalyzed activation of factor X following silencing of the indicated genes in HUVECs. Indicated genes were silenced with a pool of 4 distinct siRNAs and tested in triplicate. Each box represents an independent experimental plate, with scale bar depicting percentage of factor Xa generated compared with cells transfected with untargeted control siRNA. Lighter color indicates lower percentage factor Xa generation compared with control. (B–G) HUVECs were transfected with individual siRNAs for 72 hours and assayed for their ability to support factor VIIa–catalyzed activation of factor X (B–D) or thrombin generation in plasma-treated ECs (E–G). Cells were stimulated with TNF-α (10 ng/mL) for 3.5 hours (B, C, E, and F), TNF-α for 3.5 hours plus Ca2+ ionophore A23187 (6 μM) for 20 minutes (D), or A23187 alone for 20 minutes (G). Representative experiments are depicted as mean absorbance for factor Xa generation (B) or the first derivative of arbitrary fluorescent units for thrombin generation (E) as a function of time. 16E, 16F, and TF denote siRNA targeting TMEM16E, TMEM16F, and TF, respectively, and #1 and #2 denote distinct siRNA sequences. When indicated, lactadherin (100 nM) was added to cells treated with control siRNA. n = 3–5 independent experiments. Error bars indicate mean ± SEM (B and E) or mean ± SD (C, D, F, and G). Asterisks denoting significance are in reference to control siRNA, unless otherwise specified with brackets to indicate pairwise comparison, ANOVA with Tukey’s post test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
TMEM16E and TMEM16F are required for procoagulant activity in ECs. To corroborate these findings, we tested the requirement of TMEM16E and TMEM16F in supporting coagulation on ECs. We validated 2 distinct siRNAs for their ability to inhibit expression of TMEM16E or TMEM16F in ECs (Supplemental Figure 2). We silenced TMEM16E or TMEM16F in primary HUVECs with these individual siRNAs and stimulated cells with TNF-α to induce expression of TF. Cells were then tested for their ability to support factor VIIa–catalyzed activation of factor X in a kinetic factor Xa generation assay. Silencing of either TMEM16E or TMEM16F resulted in approximately 50% reduction of factor Xa generation compared with addition of nontargeting control siRNA (Figure 1, B and C). Silencing of TMEM16E or TMEM16F also inhibited factor VIIa–catalyzed activation of factor X that was augmented by addition of ionophore A23187 (Figure 1D), which promotes PS externalization by raising intracellular Ca2+ (9, 10). In both cases, the degree of inhibition following silencing of TMEM16E and TMEM16F was similar to that observed following treatment with lactadherin, which neutralizes externalized PS, suggesting that TMEM16E and TMEM16F may promote procoagulant activity through PS externalization (Figure 1, C and D). Dual silencing of TMEM16E and TMEM16F did not further suppress factor Xa generation beyond silencing of either gene product alone (Figure 1, C and D; see Discussion). We observed a similar dependence on both TMEM16E and TMEM16F for factor VIIa–catalyzed activation of factor X following stimulation with lipopolysaccharide (Supplemental Figure 3, A and B). To uncouple the effects of TNF-α on TF expression and PS externalization, we used an Ea.hy926 cell line stably expressing TF (28). In Ea.hy926-TF cells, TMEM16E and TMEM16F were required for Ca2+ ionophore–induced augmentation of factor VIIa–catalyzed activation of factor X (Supplemental Figure 3C).
ECs stimulated with TNF-α also required TMEM16E and TMEM16F to support thrombin generation in human plasma (Figure 1, E and F). In this assay, EC-mediated thrombin generation was completely abolished by lactadherin (Figure 1F). Treatment with Ca2+ ionophore A23187 alone promoted thrombin generation as well and was inhibited following silencing of TMEM16E or TMEM16F or addition of lactadherin (Figure 1G). Together, these results demonstrate that both TMEM16E and TMEM16F were necessary to support maximal procoagulant activity on the EC surface.
TMEM16E and TMEM16F regulate PS externalization on ECs. TMEM16E and TMEM16F function as Ca2+-activated PLSs, disrupting membrane phospholipid asymmetry by allowing PS and other anionic phospholipids to move down their concentration gradients from the inner to the outer membrane leaflet (11, 37, 44). Indeed, PS externalization in response to TNF-α or Ca2+ ionophore was markedly inhibited in HUVECs following silencing of TMEM16E or TMEM16F, as detected by annexin V binding and immunofluorescence microscopy (Figure 2, A and B) or flow cytometry (Figure 2C). PS externalization is a common end point of apoptosis and other cell death pathways, but we did not observe an increase in dead cells after exposure to TNF-α and ionophore at the concentrations used in this study (Supplemental Figure 4). Therefore, PS externalization in these experiments was not due to cell death. Silencing of TMEM16E or TMEM16F had no effect on TNF-α–induced expression of TF on the cell surface (Figure 2D). TMEM16 proteins can also function as ion channels and have been implicated in regulating intracellular Ca2+ flux in response to G protein–coupled receptor signaling (20, 44, 45). Therefore, TMEM16E or TMEM16F could affect PS externalization indirectly through regulation of Ca2+ transients. To test this possibility, we measured intracellular Ca2+ following stimulation with thrombin, which induces rapid Ca2+ elevation in ECs. Silencing of TMEM16E or TMEM16F did not significantly affect intracellular Ca2+ flux (Figure 2, E and F). Although endothelial TF activity is negatively regulated by the TF pathway inhibitor (TFPI), silencing of TMEM16E or TMEM16F did not significantly affect the expression of TFPI (Figure 2G; see supplemental material for full, uncut gels). These results indicate that TMEM16E and TMEM16F regulate EC procoagulant activity via PS externalization.
Figure 2TMEM16E and TMEM16F are required for PS externalization on ECs. HUVECs were transfected with indicated siRNAs for 72 hours, stimulated with TNF-α (10 ng/mL) for 16 hours (A) or Ca2+ ionophore A23187 (6 μM) for 20 minutes (B), and stained with annexin V (green) to detect PS externalization and Zombie Red (red) to detect cell death. Total annexin V fluorescence was normalized to number of nuclei (blue) and dead cells. (C) PS externalization following treatment with ionophore A23187 was detected using annexin V by flow cytometry. Histograms were generated after gating on live (DAPI negative) cells only. (D) HUVECs were transfected with indicated siRNAs for 72 hours, stimulated with TNF-α (10 ng/mL) for 3.5 hours, and stained for TF (green). Mean fluorescent intensity (MFI) was normalized to background for each image. Representative images are shown. (E and F) Intracellular Ca2+ flux was measured with Calbryte 520 AM in siRNA-transfected HUVECs following stimulation with thrombin (1 U/mL). Silencing of the store-operated Ca2+ regulator STIM1 served as a positive control. Time course of Calbryte 520 fluorescence after thrombin stimulation normalized to background fluorescence (E) and AUC values (F) normalized to cells treated with control siRNA are shown. (G) TFPI protein was determined by SDS-PAGE and immunoblotting with anti-TFPI antibody in HUVECs transfected with indicated siRNAs. Numbers refer to fold-change normalized to GAPDH (± SD). Scale bars: 100 μm (A and B); 50 μm (D). n = 3–6 independent experiments. Error bars indicate mean ± SD (A–D and F) or mean ± SEM (E). Asterisks denoting significance are in reference to control siRNA, ANOVA with Tukey’s post test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
PS externalization during thrombotic injury occurs on the vessel wall. To directly observe the contribution of vascular PS to thrombus formation in vivo, we used intravital microscopy to monitor PS externalization and platelet and fibrin accumulation in arterioles following laser injury to the vessel wall. The kinetics, localization, and AUC for PS were assessed by injecting fluorescent-conjugated annexin V. Following laser injury, PS externalization was consistently detected along the vessel wall, not in the growing platelet aggregate, and increased gradually, usually plateauing by 180 seconds (Figure 3, A–E, Supplemental Figure 5, and Supplemental Video 1). PS externalization consistently extended proximal and distal to the site of laser ablation and spread to the opposite wall in approximately 20% to 33% of injuries (Supplemental Figure 5). Addition of the integrin α2bβ3 (glycoprotein IIb/IIIa) antagonist eptifibatide prevented platelet accumulation at the site of injury, but had no effect on total PS externalization (Figure 3, B–E, Supplemental Figure 5, and Supplemental Video 2). We employed annexin V at a dose of 0.025 μg/g of body weight, which was significantly lower than annexin V doses shown to inhibit thrombosis in other models (16, 17). Annexin V at this concentration resulted in a mild reduction in fibrin formation, with no effect on platelet accumulation (Supplemental Figure 6). Use of other PS probes such as lactadherin and pSIVA demonstrated an identical vessel wall pattern of PS exposure following laser injury (Figure 3, F and G).
Figure 3PS externalization visualized via intravital microscopy occurs on the vessel wall and is unaffected by platelet inhibition. Thrombus formation was monitored for 180 seconds in WT mice following laser injury of the cremasteric arteriole in the presence or absence of the platelet aggregation inhibitor eptifibatide (10 μg/g of body weight). (A) Representative images at indicated time points of the PS probe annexin V (red, Alexa Fluor 647), platelets (blue, anti-CD42b antibody, DyLight 405), and fibrin (green, anti-fibrin antibody, DyLight 488). Note annexin V positivity on the vessel wall and in the absence of platelet aggregation. Kinetics and magnitude of median integrated RFUs for platelet accumulation (B) and PS externalization (D) are shown following laser injury. AUC for fluorescence intensity was determined for platelets (C) and annexin V (E). Lines represent the median AUC for individual thrombi (vehicle n = 34, eptifibatide n = 35) analyzed by Mann-Whitney U test. ****P < 0.0001. A vessel-wall pattern for PS externalization is also observed using alternative PS probes pSIVA (F, red pseudocolor) and lactadherin-FITC (G, red pseudocolor), shown 180 seconds following laser injury. In both F and G, platelets are labeled blue, and representative images are shown from 10 individual thrombi. Arrowheads denote extent of vessel-wall injury and x indicates sites of laser ablation. Scale bars: 25 μm.
Mice lacking TMEM16E have reduced fibrin formation following vessel injury. Since we identified a role for TMEM16E in regulating EC procoagulant activity, we asked whether absence of TMEM16E in mice altered thrombosis. Encoded by Ano5, TMEM16E is highly expressed in skeletal muscle, but we have found it is also expressed in ECs (Supplemental Figure 1) (43, 46). TMEM16E–/– mice are overtly healthy but demonstrate defective muscle repair (38). Blood coagulation in TMEM16E–/– mice has not been studied. We used intrav
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