An age-progressive platelet differentiation path from hematopoietic stem cells causes exacerbated thrombosis.

Platelet dysregulation is drastically increased with advanced age and contributes to making cardiovascular disorders the leading cause of death of elderly humans. Here, we reveal a direct differentiation pathway from hematopoietic stem cells into platelets that is progressively propagated upon aging. Remarkably, the aging-enriched platelet path is decoupled from all other hematopoietic lineages, including erythropoiesis, and operates as an additional layer in parallel with canonical platelet production. This results in two molecularly and functionally distinct populations of megakaryocyte progenitors. The age-induced megakaryocyte progenitors have a profoundly enhanced capacity to engraft, expand, restore, and reconstitute platelets in situ and upon transplantation and produce an additional platelet population in old mice. The two pools of co-existing platelets cause age-related thrombocytosis and dramatically increased thrombosis in vivo. Strikingly, aging-enriched platelets are functionally hyper-reactive compared with the canonical platelet populations. These findings reveal stem cell-based aging as a mechanism for platelet dysregulation and age-induced thrombosis.

Cryo-EM structures of human arachidonate 12S-lipoxygenase bound to endogenous and exogenous inhibitors.

Human 12-lipoxygenase (12-LOX) is a key enzyme involved in platelet activation, and the regulation of its activity has been targeted for the treatment of heparin-induced thrombocytopenia. Despite the clinical importance of 12-LOX, the exact mechanisms by which it affects platelet activation are not fully understood, and the lack of structural information has limited drug discovery efforts. In this study, we used single-particle cryo-electron microscopy to determine high-resolution structures (1.7-2.8 Å) of human 12-LOX. Our results showed that 12-LOX can exist in multiple oligomeric states, from monomer to hexamer, which may affect its catalytic activity and membrane association. We also identified different conformations within the 12-LOX dimer, which likely represent different time points in its catalytic cycle. Furthermore, we identified small molecules bound to 12-LOX. The active site of the 12-LOX tetramer was occupied by an endogenous 12-LOX inhibitor, a long-chain acyl coenzyme A. In addition, we found that the 12-LOX hexamer can simultaneously bind to arachidonic acid and ML355, a selective 12-LOX inhibitor that has passed a phase 1 clinical trial for the treatment of heparin-induced thrombocytopenia and received a fast-track designation by the Food and Drug Administration. Overall, our findings provide novel insights into the assembly of 12-LOX oligomers, their catalytic mechanism, and small molecule binding, paving the way for further drug development targeting the 12-LOX enzyme.

The oxylipin analog CS585 prevents platelet activation and thrombosis through activation of the prostacyclin receptor.

Cardiovascular disease remains the primary cause of morbidity and mortality globally. Platelet activation is critical for maintaining hemostasis and preventing the leakage of blood cells from the vessel. There has been a paucity in the development of new drugs to target platelet reactivity. Recently, the oxylipin 12(S)-hydroxy-eicosatrienoic acid (12-HETrE), which is produced in platelets, was shown to limit platelet reactivity by activating the prostacyclin receptor. Here, we demonstrated the synthesis of a novel analog of 12-HETrE, known as CS585. Human blood and mouse models of hemostasis and thrombosis were assessed for the ability of CS585 to attenuate platelet activation and thrombosis without increasing the risk of bleeding. Human platelet activation was assessed using aggregometry, flow cytometry, western blot analysis, total thrombus formation analysis system, microfluidic perfusion chamber, and thromboelastography. Hemostasis, thrombosis, and bleeding assays were performed in mice. CS585 was shown to potently target the prostacyclin receptor on the human platelet, resulting in a highly selective and effective mechanism for the prevention of platelet activation. Furthermore, CS585 was shown to inhibit platelet function in human whole blood ex vivo, prevent thrombosis in both small and large vessels in mouse models, and exhibit long-lasting prevention of clot formation. Finally, CS585 was not observed to perturb coagulation or increase the risk of bleeding in the mouse model. Hence, CS585 represents a new validated target for the treatment of thrombotic diseases without the risk of bleeding or off-target activation observed with other prostaglandin receptor agonists.

The histone methyltransferase MLL1/KMT2A in monocytes drives coronavirus-associated coagulopathy and inflammation.

Coronavirus-associated coagulopathy (CAC) is a morbid and lethal sequela of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. CAC results from a perturbed balance between coagulation and fibrinolysis and occurs in conjunction with exaggerated activation of monocytes/macrophages (MO/Mφs), and the mechanisms that collectively govern this phenotype seen in CAC remain unclear. Here, using experimental models that use the murine betacoronavirus MHVA59, a well-established model of SARS-CoV-2 infection, we identify that the histone methyltransferase mixed lineage leukemia 1 (MLL1/KMT2A) is an important regulator of MO/Mφ expression of procoagulant and profibrinolytic factors such as tissue factor (F3; TF), urokinase (PLAU), and urokinase receptor (PLAUR) (herein, "coagulopathy-related factors") in noninfected and infected cells. We show that MLL1 concurrently promotes the expression of the proinflammatory cytokines while suppressing the expression of interferon alfa (IFN-α), a well-known inducer of TF and PLAUR. Using in vitro models, we identify MLL1-dependent NF-κB/RelA-mediated transcription of these coagulation-related factors and identify a context-dependent, MLL1-independent role for RelA in the expression of these factors in vivo. As functional correlates for these findings, we demonstrate that the inflammatory, procoagulant, and profibrinolytic phenotypes seen in vivo after coronavirus infection were MLL1-dependent despite blunted Ifna induction in MO/Mφs. Finally, in an analysis of SARS-CoV-2 positive human samples, we identify differential upregulation of MLL1 and coagulopathy-related factor expression and activity in CD14+ MO/Mφs relative to noninfected and healthy controls. We also observed elevated plasma PLAU and TF activity in COVID-positive samples. Collectively, these findings highlight an important role for MO/Mφ MLL1 in promoting CAC and inflammation.

Novel Strategy to Combat the Procoagulant Phenotype in Heparin-Induced Thrombocytopenia Using 12-LOX Inhibition.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a major concern for all individuals that undergo cardiac bypass surgeries or require prolonged heparin exposure. HIT is a life- and limb-threatening adverse drug reaction with an immune response following the formation of ultra-large immune complexes that drive platelet activation through the receptor FcγRIIA. Thrombotic events remain high following the standard of care treatment with anticoagulants, while increasing risk of bleeding complications. This study sought to investigate a novel approach to treatment of HIT. Recent reports demonstrate increased procoagulant activity in HIT; however, these reports required analysis ex vivo, and relevance in vivo remains unclear. METHODS: Using human and mouse model systems, we investigated the cooperativity of PARs (protease-activated receptors) and FcγRIIA in HIT. We challenged humanized FcγRIIA transgenic mice with or without endogenous mouse Par4 (denoted as IIA-Par4+/+ or IIA-Par4-/-, respectively) with a well-established model IgG immune complex (anti [α]-CD9). Furthermore, we assessed the procoagulant phenotype and efficacy to treat HIT utilizing inhibitor of 12-LOX (12[S]-lipoxygenase), VLX-1005, previously reported to decrease platelet activation downstream of FcγRIIA and PAR4, using the triple allele HIT mouse model. RESULTS: IIA-Par4+/+ mice given αCD9 were severely thrombocytopenic, with extensive platelet-fibrin deposition in the lung. In contrast, IIA-Par4-/- mice had negligible thrombocytopenia or pulmonary platelet-fibrin thrombi. We observed that pharmacological inhibition of 12-LOX resulted in a significant reduction in both platelet procoagulant phenotype ex vivo, and thrombocytopenia and thrombosis in our humanized mouse model of HIT in vivo. CONCLUSIONS: These data demonstrate for the first time the need for dual platelet receptor (PAR and FcγRIIA) stimulation for fibrin formation in HIT in vivo. These results extend our understanding of HIT pathophysiology and provide a scientific rationale for targeting the procoagulant phenotype as a possible therapeutic strategy in HIT.

Loss of 12-Lipoxygenase Improves the Post-Transfusion Function of Stored Platelets.

BACKGROUND: Platelets for transfusion are stored for 5 to 7 days. Previous studies have shown that HETE levels in the storage bag negatively correlate with platelet performance in vivo, suggesting that the dysregulation of bioactive lipid mediators may contribute to the storage lesion. In the current study, we sought to understand how genetic deletion and pharmacological inhibition of 12-LOX (12-lipoxygenase) affects platelets during storage and after transfusion. METHODS: Platelets from 12-LOX+/+ (wild-type [WT]) and 12-LOX-/- mice were stored for 24 and 48 hours and profiled using liquid chromatography-tandem mass spectrometry-multiple reaction monitoring or transfused into thrombocytopenic hIL4R (human interleukin 4 receptor)-transgenic mice. Platelet function was assessed by flow cytometry and in vivo thrombosis and hemostasis models. To test the role of the COX-1 (cyclooxygenase-1) pathway, donor mice were treated with acetylsalicylic acid. Human platelets were treated with the 12-LOX inhibitor, VLX-1005, or vehicle, stored, and transfused to NOD/SCID (nonobese diabetic/severe combined immunodeficiency) mice. RESULTS: Polyunsaturated fatty acids increased significantly in stored platelets from 12-LOX-/- mice, whereas oxylipin concentrations were significantly higher in WT platelets. After transfusion to thrombocytopenic mice, we observed significantly more baseline αIIbß3 integrin activation in 12-LOX-/- platelets than in WT platelets. Stored platelets from 12-LOX-/- mice occluded vessels significantly faster than stored WT platelets. In hemostasis models, significantly more stored 12-LOX-/- than WT platelets accumulated at the site of venous injury leading to reduced blood loss. Inhibition of COX-1 abrogated both increased integrin activation and thromboxane generation in stored 12-LOX-/- platelets, highlighting the critical role of this pathway for improved post-transfusion function. Consistent with our mouse studies, human platelets stored with VLX-1005, showed increased integrin activation compared with vehicle-treated platelets after transfusion. CONCLUSIONS: Deleting 12-LOX improves the post-transfusion function of stored murine platelets by increasing thromboxane generation through COX-1-dependent arachidonic acid metabolism. Future studies should determine the feasibility and safety of 12-LOX-inhibited platelets transfused to humans.

Investigating the catalytic efficiency of C22-Fatty acids with LOX human isozymes and the platelet response of the C22-oxylipin products.

Polyunsaturated fatty acids (PUFAs) have been extensively studied for their health benefits because they can be oxidized by lipoxygenases to form bioactive oxylipins. In this study, we investigated the impact of double bond placement on the kinetic properties and product profiles of human platelet 12-lipoxygenase (h12-LOX), human reticulocyte 15-lipoxygenase-1 (h15-LOX-1), and human endothelial 15-lipoxygenase-2 (h15-LOX-2) by using 22-carbon (C22) fatty acid substrates with differing double bond content. With respect to kcat/KM values, the loss of Δ4 and Δ19 led to an 18-fold loss of kinetic activity for h12-LOX, no change in kinetic capability for h15-LOX-1, but a 24-fold loss for h15-LOX-2 for both C22-FAs. With respect to the product profiles, h12-LOX produced mainly 14-oxylipins. For h15-LOX-1, the 14-oxylipin production increased with the loss of either Δ4 and Δ19, however, the 17-oxylipin became the major species upon loss of both Δ4 and Δ19. h15-LOX-2 produced mostly the 17-oxylipin products throughout the fatty acid series. This study also investigated the effects of various 17-oxylipins on platelet activation. The results revealed that both 17(S)-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-DHA (17-HDHA) and 17-hydroxy-4Z,7Z,10Z,13Z,15E-DPAn6 (17-HDPAn6) demonstrated anti-aggregation properties with thrombin or collagen stimulation. 17-hydroxy-7Z,10Z,13Z,15E,19Z-DPAn3 (17-HDPAn3) exhibited agonistic properties, and 17-hydroxy-7Z,10Z,13Z,15E-DTA (17-HDTA) showed biphasic effects, inhibiting collagen-induced aggregation at lower concentrationsbut promoting aggregation at higher concentrations. Both 17-hydroxy-13Z,15E,19Z-DTrA (17-HDTrA), and 17-hydroxy-13Z,15E-DDiA (17-HDDiA) induced platelet aggregation. In summary, the number and placement of the double bonds affect platelet activation, with the general trend being that more double bonds generally inhibit aggregation, while less double bonds promote aggregation. These findings provide insights into the potential role of specific fatty acids and their metabolizing LOX isozymes with respect to cardiovascular health.

Biochemical and hydrogen-deuterium exchange studies of the single nucleotide polymorphism Y649C in human platelet 12-lipoxygenase linked to a bleeding disorder.

Human platelet 12-lipoxygenase (h12-LOX) is responsible for the formation of oxylipin products that play an important role in platelet aggregation. Single nucleotide polymorphisms (SNPs) of h12-LOX have been implicated in several diseases. In this study, we investigate the structural, dynamical, and functional impact of a h12-LOX SNP that generates a tyrosine-to-cysteine mutation at a buried site (Y649C h12-LOX) and was previously ascribed with reduced levels of 12(S)-hydroxyeicosatetraenoic acid (12S-HETE) production in isolated platelets. Herein, in vitro Michaelis-Menten kinetics show reduced catalytic rates for Y649C compared to WT h12-LOX at physiological or lower temperatures. Both proteins exhibited similar melting temperatures, metal content, and oligomerization state. Liposome binding for both proteins was also dependent upon the presence of calcium, temperature, and liposome composition; however, the Y649C variant was found to have lowered binding capacity to liposomes compared to WT at physiological temperatures. Further, hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments revealed a regional defined enhancement in the peptide mobility caused by the mutation. This increased instability for the mutation stemmed from a change in an interaction with an arched helix that lines the substrate binding site, located ≥15 Å from the mutation site. Finally, differential scanning calorimetry demonstrated a reduced protein (un)folding enthalpy, consistent with the HDX results. Taken together, these results demonstrate remarkable similarity between the mutant and WT h12-LOX, and yet, subtle changes in activity, membrane affinity and protein stability may be responsible for the significant physiological changes that the Y649C SNP manifests in platelet biology.

GPR56/ADGRG1 is a platelet collagen-responsive GPCR and hemostatic sensor of shear force.

Circulating platelets roll along exposed collagen at vessel injury sites and respond with filipodia protrusion, shape change, and surface area expansion to facilitate platelet adhesion and plug formation. Various glycoproteins were considered to be both collagen responders and mediators of platelet adhesion, yet the signaling kinetics emanating from these receptors do not fully account for the rapid platelet cytoskeletal changes that occur in blood flow. We found the free N-terminal fragment of the adhesion G protein-coupled receptor (GPCR) GPR56 in human plasma and report that GPR56 is the platelet receptor that transduces signals from collagen and blood flow-induced shear force to activate G protein 13 signaling for platelet shape change. Gpr56-/- mice have prolonged bleeding, defective platelet plug formation, and delayed thrombotic occlusion. Human and mouse blood perfusion studies demonstrated GPR56 and shear-force dependence of platelet adhesion to immobilized collagen. Our work places GPR56 as an initial collagen responder and shear-force transducer that is essential for platelet shape change during hemostasis.

Inhibitory Investigations of Acyl-CoA Derivatives against Human Lipoxygenase Isozymes.

Lipid metabolism is a complex process crucial for energy production resulting in high levels of acyl-coenzyme A (acyl-CoA) molecules in the cell. Acyl-CoAs have also been implicated in inflammation, which could be possibly linked to lipoxygenase (LOX) biochemistry by the observation that an acyl-CoA was bound to human platelet 12-lipoxygenase via cryo-EM. Given that LOX isozymes play a pivotal role in inflammation, a more thorough investigation of the inhibitory effects of acyl-CoAs on lipoxygenase isozymes was judged to be warranted. Subsequently, it was determined that C18 acyl-CoA derivatives were the most potent against h12-LOX, human reticulocyte 15-LOX-1 (h15-LOX-1), and human endothelial 15-LOX-2 (h15-LOX-2), while C16 acyl-CoAs were more potent against human 5-LOX. Specifically, oleoyl-CoA (18:1) was most potent against h12-LOX (IC50 = 32 µM) and h15-LOX-2 (IC50 = 0.62 µM), stearoyl-CoA against h15-LOX-1 (IC50 = 4.2 µM), and palmitoleoyl-CoA against h5-LOX (IC50 = 2.0 µM). The inhibition of h15-LOX-2 by oleoyl-CoA was further determined to be allosteric inhibition with a Ki of 82 +/- 70 nM, an α of 3.2 +/- 1, a ß of 0.30 +/- 0.07, and a ß/α = 0.09. Interestingly, linoleoyl-CoA (18:2) was a weak inhibitor against h5-LOX, h12-LOX, and h15-LOX-1 but a rapid substrate for h15-LOX-1, with comparable kinetic rates to free linoleic acid (kcat = 7.5 +/- 0.4 s-1, kcat/KM = 0.62 +/- 0.1 µM-1s-1). Additionally, it was determined that methylated fatty acids were not substrates but rather weak inhibitors. These findings imply a greater role for acyl-CoAs in the regulation of LOX activity in the cell, either through inhibition of novel oxylipin species or as a novel source of oxylipin-CoAs.

In Vitro Biosynthetic Pathway Investigations of Neuroprotectin D1 (NPD1) and Protectin DX (PDX) by Human 12-Lipoxygenase, 15-Lipoxygenase-1, and 15-Lipoxygenase-2.

In this paper, human platelet 12-lipoxygenase [h12-LOX (ALOX12)], human reticulocyte 15-lipoxygenase-1 [h15-LOX-1 (ALOX15)], and human epithelial 15-lipoxygenase-2 [h15-LOX-2 (ALOX15B)] were observed to react with docosahexaenoic acid (DHA) and produce 17S-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid (17S-HpDHA). The kcat/KM values with DHA for h12-LOX, h15-LOX-1, and h15-LOX-2 were 12, 0.35, and 0.43 s-1 µM-1, respectively, which demonstrate h12-LOX as the most efficient of the three. These values are comparable to their counterpart kcat/KM values with arachidonic acid (AA), 14, 0.98, and 0.24 s-1 µM-1, respectively. Comparison of their product profiles with DHA demonstrates that the three LOX isozymes produce 11S-HpDHA, 14S-HpDHA, and 17S-HpDHA, to varying degrees, with 17S-HpDHA being the majority product only for the 15-LOX isozymes. The effective kcat/KM values (kcat/KM × percent product formation) for 17S-HpDHA of the three isozymes indicate that the in vitro value of h12-LOX was 2.8-fold greater than that of h15-LOX-1 and 1.3-fold greater than that of h15-LOX-2. 17S-HpDHA was an effective substrate for h12-LOX and h15-LOX-1, with four products being observed under reducing conditions: protectin DX (PDX), 16S,17S-epoxy-4Z,7Z,10Z,12E,14E,19Z-docosahexaenoic acid (16S,17S-epoxyDHA), the key intermediate in neuroprotection D1 biosynthesis [NPD1, also known as protectin D1 (PD1)], 11,17S-diHDHA, and 16,17S-diHDHA. However, h15-LOX-2 did not react with 17-HpDHA. With respect to their effective kcat/KM values, h12-LOX was markedly less effective than h15-LOX-1 in reacting with 17S-HpDHA, with a 55-fold lower effective kcat/KM in producing 16S,17S-epoxyDHA and a 27-fold lower effective kcat/KM in generating PDX. This is the first direct demonstration of h15-LOX-1 catalyzing this reaction and reveals an in vitro pathway for PDX and NPD1 intermediate biosynthesis. In addition, epoxide formation from 17S-HpDHA and h15-LOX-1 was negatively affected via allosteric regulation by 17S-HpDHA (Kd = 5.9 µM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (Kd = 2.5 µM), and 17S-hydroxy-13Z,15E,19Z-docosatrienoic acid (17S-HDTA) (Kd = 1.4 µM), suggesting a possible regulatory pathway in reducing epoxide formation. Finally, 17S-HpDHA and PDX inhibited platelet aggregation, with EC50 values of approximately 1 and 3 µM, respectively. The in vitro results presented here may help advise in vivo PDX and NPD1 intermediate (i.e., 16S,17S-epoxyDHA) biosynthetic investigations and support the benefits of DHA rich diets.

Platelet Signaling and Disease: Targeted Therapy for Thrombosis and Other Related Diseases.

Platelets are essential for clotting in the blood and maintenance of normal hemostasis. Under pathologic conditions such as atherosclerosis, vascular injury often results in hyperactive platelet activation, resulting in occlusive thrombus formation, myocardial infarction, and stroke. Recent work in the field has elucidated a number of platelet functions unique from that of maintaining hemostasis, including regulation of tumor growth and metastasis, inflammation, infection, and immune response. Traditional therapeutic targets for inhibiting platelet activation have primarily been limited to cyclooxygenase-1, integrin αIIbß3, and the P2Y12 receptor. Recently identified signaling pathways regulating platelet function have made it possible to develop novel approaches for pharmacological intervention in the blood to limit platelet reactivity. In this review, we cover the newly discovered roles for platelets as well as their role in hemostasis and thrombosis. These new roles for platelets lend importance to the development of new therapies targeted to the platelet. Additionally, we highlight the promising receptor and enzymatic targets that may further decrease platelet activation and help to address the myriad of pathologic conditions now known to involve platelets without significant effects on hemostasis.

Biosynthesis of the Maresin Intermediate, 13S,14S-Epoxy-DHA, by Human 15-Lipoxygenase and 12-Lipoxygenase and Its Regulation through Negative Allosteric Modulators.

Human reticulocyte 15-lipoxygenase-1 (h15-LOX-1 or ALOX15) and platelet 12-lipoxygenase (h12-LOX or ALOX12) catalysis of docosahexaenoic acid (DHA) and the maresin precursor, 14S-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (14S-HpDHA), were investigated to determine their product profiles and relative rates in the biosynthesis of the key maresin intermediate, 13S,14S-epoxy-4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid (13S,14S-epoxy-DHA). Both enzymes converted DHA to 14S-HpDHA, with h12-LOX having a 39-fold greater kcat/KM value (14.0 ± 0.8 s-1 µM-1) than that of h15-LOX-1 (0.36 ± 0.08 s-1 µM-1) and a 1.8-fold greater 14S-HpDHA product selectivity, 81 and 46%, respectively. However, h12-LOX was markedly less effective at producing 13S,14S-epoxy-DHA from 14S-HpDHA than h15-LOX-1, with a 4.6-fold smaller kcat/KM value, 0.0024 ± 0.0002 and 0.11 ± 0.006 s-1 µM-1, respectively. This is the first evidence of h15-LOX-1 to catalyze this reaction and reveals a novel in vitro pathway for maresin biosynthesis. In addition, epoxidation of 14S-HpDHA is negatively regulated through allosteric oxylipin binding to h15-LOX-1 and h12-LOX. For h15-LOX-1, 14S-HpDHA (Kd = 6.0 µM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (Kd = 3.5 µM), and 14S-hydroxy-7Z,10Z,12E,16Z,19Z-docosapentaenoic acid (14S-HDPAω-3) (Kd = 4.0 µM) were shown to decrease 13S,14S-epoxy-DHA production. h12-LOX was also shown to be allosterically regulated by 14S-HpDHA (Kd = 3.5 µM) and 14S-HDPAω-3 (Kd = 4.0 µM); however, 12S-HETE showed no effect, indicating for the first time an allosteric response by h12-LOX. Finally, 14S-HpDHA inhibited platelet aggregation at a submicrololar concentration, which may have implications in the benefits of diets rich in DHA. These in vitro biosynthetic pathways may help guide in vivo maresin biosynthetic investigations and possibly direct therapeutic interventions.

Role of Human 15-Lipoxygenase-2 in the Biosynthesis of the Lipoxin Intermediate, 5S,15S-diHpETE, Implicated with the Altered Positional Specificity of Human 15-Lipoxygenase-1.

The oxylipins, 5S,12S-dihydroxy-6E,8Z,10E,14Z-eicosatetraenoic acid (5S,12S-diHETE) and 5S,15S-dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid (5S,15S-diHETE), have been identified in cell exudates and have chemotactic activity toward eosinophils and neutrophils. Their biosynthesis has been proposed to occur by sequential oxidations of arachidonic acid (AA) by lipoxygenase enzymes, specifically through oxidation of AA by h5-LOX followed by h12-LOX, h15-LOX-1, or h15-LOX-2. In this work, h15-LOX-1 demonstrates altered positional specificity when reacting with 5S-HETE, producing 90% 5S,12S-diHETE, instead of 5S,15S-diHETE, with kinetics 5-fold greater than that of h12-LOX. This is consistent with previous work in which h15-LOX-1 reacts with 7S-HDHA, producing the noncanonical, DHA-derived, specialized pro-resolving mediator, 7S,14S-diHDHA. It is also determined that oxygenation of 5S-HETE by h15-LOX-2 produces 5S,15S-diHETE and its biosynthetic kcat/KM flux is 2-fold greater than that of h15-LOX-1, suggesting that h15-LOX-2 may have a greater role in lipoxin biosynthesis than previously thought. In addition, it is shown that oxygenation of 12S-HETE and 15S-HETE by h5-LOX is kinetically slow, suggesting that the first step in the in vitro biosynthesis of both 5S,12S-diHETE and 5S,15S-diHETE is the production of 5S-HETE.

15-Lipoxygenase-1 biosynthesis of 7S,14S-diHDHA implicates 15-lipoxygenase-2 in biosynthesis of resolvin D5.

The two oxylipins 7S,14S-dihydroxydocosahexaenoic acid (diHDHA) and 7S,17S-diHDHA [resolvin D5 (RvD5)] have been found in macrophages and infectious inflammatory exudates and are believed to function as specialized pro-resolving mediators (SPMs). Their biosynthesis is thought to proceed through sequential oxidations of DHA by lipoxygenase (LOX) enzymes, specifically, by human 5-LOX (h5-LOX) first to 7(S)-hydroxy-4Z,8E,10Z,13Z,16Z,19Z-DHA (7S-HDHA), followed by human platelet 12-LOX (h12-LOX) to form 7(S),14(S)-dihydroxy-4Z,8E,10Z,12E,16Z,19Z-DHA (7S,14S-diHDHA) or human reticulocyte 15-LOX-1 (h15-LOX-1) to form RvD5. In this work, we determined that oxidation of 7(S)-hydroperoxy-4Z,8E,10Z,13Z,16Z,19Z-DHA to 7S,14S-diHDHA is performed with similar kinetics by either h12-LOX or h15-LOX-1. The oxidation at C14 of DHA by h12-LOX was expected, but the noncanonical reaction of h15-LOX-1 to make over 80% 7S,14S-diHDHA was larger than expected. Results of computer modeling suggested that the alcohol on C7 of 7S-HDHA hydrogen bonds with the backbone carbonyl of Ile399, forcing the hydrogen abstraction from C12 to oxygenate on C14 but not C17. This result raised questions regarding the synthesis of RvD5. Strikingly, we found that h15-LOX-2 oxygenates 7S-HDHA almost exclusively at C17, forming RvD5 with faster kinetics than does h15-LOX-1. The presence of h15-LOX-2 in neutrophils and macrophages suggests that it may have a greater role in biosynthesizing SPMs than previously thought. We also determined that the reactions of h5-LOX with 14(S)-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-DHA and 17(S)-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-DHA are kinetically slow compared with DHA, suggesting that these reactions may be minor biosynthetic routes in vivo. Additionally, we show that 7S,14S-diHDHA and RvD5 have anti-aggregation properties with platelets at low micromolar potencies, which could directly regulate clot resolution.

Formation and Resolution of Pial Microvascular Thrombosis in a Mouse Model of Thrombotic Thrombocytopenic Purpura.

OBJECTIVE: Microvascular thrombosis is the hallmark pathology of thrombotic thrombocytopenic purpura (TTP), a rare life-threatening disease. Neurological dysfunction is present in over 90% of patients with TTP, and TTP can cause long-lasting neurological damage or death. However, the pathophysiology of microvascular thrombosis in the brain is not well studied to date. Here, we investigate the formation and resolution of thrombosis in pial microvessels. Approach and Results: Using a cranial intravital microscopy in well-established mouse models of congenital TTP induced by infusion of recombinant VWF (von Willebrand factor), we found that soluble VWF, at high concentration, adheres to the endothelium of the vessel wall, self-associates, and initiates platelet adhesion resulting in the formation of pial microvascular thrombosis in ADAMTS13-/- (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) mice. Importantly, VWF-mediated pial microvascular thrombosis occurred without vascular injury to the brain, and thrombi consisted of resting platelets adhered onto ultra-large VWF without fibrin in the brain in rVWF (recombinant VWF) challenged ADAMTS13-/- mice. Prophylactic treatment with recombinant ADAMTS13 (BAX930) effectively prevented the onset of the VWF-mediated microvascular thrombosis and therapeutic treatment with BAX930 acutely resolved the preexisting or growing thrombi in the brain of ADAMTS13-/- mice after rVWF challenge. The absence of platelet activation and fibrin formation within VWF-mediated thrombi and efficacy of BAX930 was confirmed with an endothelial-driven VWF-mediated microvascular thrombosis model in mice. CONCLUSIONS: Our results provide important insight into the initiation and development of microvascular thrombi in mouse models that mimics TTP and indicate that rADAMTS13 could be an effective interventional therapy for microvascular thrombosis, the hallmark pathology in TTP.

KLF11 (Krüppel-Like Factor 11) Inhibits Arterial Thrombosis via Suppression of Tissue Factor in the Vascular Wall.

Objective- Mutations in Krüppel like factor-11 ( KLF11), a gene also known as maturity-onset diabetes mellitus of the young type 7, contribute to the development of diabetes mellitus. KLF11 has anti-inflammatory effects in endothelial cells and beneficial effects on stroke. However, the function of KLF11 in the cardiovascular system is not fully unraveled. In this study, we investigated the role of KLF11 in vascular smooth muscle cell biology and arterial thrombosis. Approach and Results- Using a ferric chloride-induced thrombosis model, we found that the occlusion time was significantly reduced in conventional Klf11 knockout mice, whereas bone marrow transplantation could not rescue this phenotype, suggesting that vascular KLF11 is critical for inhibition of arterial thrombosis. We further demonstrated that vascular smooth muscle cell-specific Klf11 knockout mice also exhibited significantly reduced occlusion time. The expression of tissue factor (encoded by the F3 gene), a main initiator of the coagulation cascade, was increased in the artery of Klf11 knockout mice, as determined by real-time quantitative polymerase chain reaction and immunofluorescence. Furthermore, vascular smooth muscle cells isolated from Klf11 knockout mouse aortas showed increased tissue factor expression, which was rescued by KLF11 overexpression. In human aortic smooth muscle cells, small interfering RNA-mediated knockdown of KLF11 increased tissue factor expression. Consistent results were observed on adenovirus-mediated overexpression of KLF11. Mechanistically, KLF11 downregulates F3 at the transcriptional level as determined by reporter and chromatin immunoprecipitation assays. Conclusions- Our data demonstrate that KLF11 is a novel transcriptional suppressor of F3 in vascular smooth muscle cells, constituting a potential molecular target for inhibition of arterial thrombosis.

Anti-inflammatory ω-3 endocannabinoid epoxides.

Clinical studies suggest that diets rich in ω-3 polyunsaturated fatty acids (PUFAs) provide beneficial anti-inflammatory effects, in part through their conversion to bioactive metabolites. Here we report on the endogenous production of a previously unknown class of ω-3 PUFA-derived lipid metabolites that originate from the crosstalk between endocannabinoid and cytochrome P450 (CYP) epoxygenase metabolic pathways. The ω-3 endocannabinoid epoxides are derived from docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to form epoxyeicosatetraenoic acid-ethanolamide (EEQ-EA) and epoxydocosapentaenoic acid-ethanolamide (EDP-EA), respectively. Both EEQ-EAs and EDP-EAs are endogenously present in rat brain and peripheral organs as determined via targeted lipidomics methods. These metabolites were directly produced by direct epoxygenation of the ω-3 endocannabinoids, docosahexanoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA) by activated BV-2 microglial cells, and by human CYP2J2. Neuroinflammation studies revealed that the terminal epoxides 17,18-EEQ-EA and 19,20-EDP-EA dose-dependently abated proinflammatory IL-6 cytokines while increasing anti-inflammatory IL-10 cytokines, in part through cannabinoid receptor-2 activation. Furthermore the ω-3 endocannabinoid epoxides 17,18-EEQ-EA and 19,20-EDP-EA exerted antiangiogenic effects in human microvascular endothelial cells (HMVEC) and vasodilatory actions on bovine coronary arteries and reciprocally regulated platelet aggregation in washed human platelets. Taken together, the ω-3 endocannabinoid epoxides' physiological effects are mediated through both endocannabinoid and epoxyeicosanoid signaling pathways. In summary, the ω-3 endocannabinoid epoxides are found at concentrations comparable to those of other endocannabinoids and are expected to play critical roles during inflammation in vivo; thus their identification may aid in the development of therapeutics for neuroinflammatory and cerebrovascular diseases.

Genetic Variant in Human PAR (Protease-Activated Receptor) 4 Enhances Thrombus Formation Resulting in Resistance to Antiplatelet Therapeutics.

OBJECTIVE: Platelet activation after stimulation of PAR (protease-activated receptor) 4 is heightened in platelets from blacks compared with those from whites. The difference in PAR4 signaling by race is partially explained by a single-nucleotide variant in PAR4 encoding for either an alanine or threonine at amino acid 120 in the second transmembrane domain. The current study sought to determine whether the difference in PAR4 signaling by this PAR4 variant is because of biased Gq signaling and whether the difference in PAR4 activity results in resistance to traditional antiplatelet intervention. APPROACH AND RESULTS: Membranes expressing human PAR4-120 variants were reconstituted with either Gq or G13 to determine the kinetics of G protein activation. The kinetics of Gq and G13 activation were both increased in membranes expressing PAR4-Thr120 compared with those expressing PAR4-Ala120. Further, inhibiting PAR4-mediated platelet activation by targeting COX (cyclooxygenase) and P2Y12 receptor was less effective in platelets from subjects expressing PAR4-Thr120 compared with PAR4-Ala120. Additionally, ex vivo thrombus formation in whole blood was evaluated at high shear to determine the relationship between PAR4 variant expression and response to antiplatelet drugs. Ex vivo thrombus formation was enhanced in blood from subjects expressing PAR4-Thr120 in the presence or absence of antiplatelet therapy. CONCLUSIONS: Together, these data support that the signaling difference by the PAR4-120 variant results in the enhancement of both Gq and G13 activation and an increase in thrombus formation resulting in a potential resistance to traditional antiplatelet therapies targeting COX-1 and the P2Y12 receptor.

In vivo modeling of docosahexaenoic acid and eicosapentaenoic acid-mediated inhibition of both platelet function and accumulation in arterial thrombi.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are associated with a variety of cellular alterations that mitigate cardiovascular disease. However, pinpointing the positive therapeutic effects is challenging due to inconsistent clinical trial results and overly simplistic in vitro studies. Here we aimed to develop realistic models of n-3 PUFA effects on platelet function so that preclinical results can better align with and predict clinical outcomes. Human platelets incubated with the n-3 PUFAs docosahexaenoic acid and eicosapentaenoic acid were stimulated with agonist combinations mirroring distinct regions of a growing thrombus. Platelet responses were then monitored in a number of ex-vivo functional assays. Furthermore, intravital microscopy was used to monitor arterial thrombosis and fibrin deposition in mice fed an n-3 PUFA-enriched diet. We found that n-3 PUFA treatment had minimal effects on many basic ex-vivo measures of platelet function using agonist combinations. However, n-3 PUFA treatment delayed platelet-derived thrombin generation in both humans and mice. This impaired thrombin production paralleled a reduced platelet accumulation within thrombi formed in either small arterioles or larger arteries of mice fed an n-3 PUFA-enriched diet, without impacting P-selectin exposure. Despite an apparent lack of robust effects in many ex-vivo assays of platelet function, increased exposure to n-3 PUFAs reduces platelet-mediated thrombin generation and attenuates elements of thrombus formation. These data support the cardioprotective value of-3 PUFAs and strongly suggest that they modify elements of platelet function in vivo.