Endothelial nitric oxide synthase plays a minor role in inhibition of arterial thrombus formation

2005 ◽  
Vol 93 (06) ◽  
pp. 1161-1167 ◽  
Author(s):  
Burcin Özüyaman ◽  
Susanne Küsters ◽  
Elisabeth Kirchhoff ◽  
Rüdiger Scharf ◽  
Jürgen Schrader ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Surface Expression ◽  
No Synthase ◽  
Minor Role ◽  
Endothelial No Synthase ◽  
Tail Tip ◽  
A Minor

SummaryEndothelial NO synthase (eNOS) expressed in the vascular en-dothelium or formed within platelets was postulated to inhibit platelet activation and aggregation. We have assessed the role of eNOS in platelet aggregation in vitro and in vivo by comparison of WT and eNOS-/- mice. Aggregometer studies revealed that collagen over a concentration range of 0.36–10 µg aggregated WT and eNOS-/- platelets to the same extent (10 µg: WT 86.7±4.7%, eNOS-/- 91±12%, n=6). Collagen treatment did not result in a significant increase in cGMP formation and VASP phosphorylation. Thrombin-induced P-selectin surface expression was unchanged in eNOS-/- platelets. In line with these findings no eNOS protein was detectable within the platelets of WT mice. In vivo, bleeding time after tail tip resection tended to be shorter in eNOS/- mice (WT: 116±35 s; eNOS-/- 109±37 s, n.s). Similarly, time to occlusion of the A.carotis after focal induction of thrombosis was 501±76 s (WT) and 457±95 s (eNOS-/-) (n.s.). These data demonstrate that eNOS-deficiency minimally affects platelet aggregation and is not associated with accelerated arterial thrombosis in vivo. Thus, in the mouse endothelial NO synthase does not play a major role in the autocrine modulation of platelet function and in thrombosis of conduit vessels in vivo.

Role of Thromboxane A2 as a Mediator of Platelet-Activating-Factor-Induced Aggregation of Human Platelets

1989 ◽  
Vol 77 (1) ◽  
pp. 99-103 ◽  
Author(s):  
R. K. McCulloch ◽  
J. Summers ◽  
R. Vandongen ◽  
I. L. Rouse
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activating Factor ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Minor Role ◽  
A Minor ◽  
Induced Aggregation

1. At present it is unclear whether platelet-activating-factor (PAF)-induced aggregation is mediated by thromboxane. To obtain further information about this event we have compared the affects of aspirin on platelet aggregation and secretion induced by PAF and collagen. 2. Collagen and PAF induced aggregation and secretion in human platelets in a dose-related manner. 3. Aspirin inhibited the magnitude of both platelet aggregation and secretion induced by PAF and collagen, but the degree of inhibition was much greater for collagen. 4. Aspirin strongly inhibited the aggregation rate of collagen-induced platelet aggregation, but had no measurable effect on the rate of PAF-induced aggregation. 5. Inconsistencies reported in previous studies of the effect of aspirin on PAF-induced platelet aggregation may be explained, in part, by the doses of PAF used and the method of inactivating cyclo-oxygenase (in vitro compared with in vivo). 6. Our results suggest that the initial events of PAF-induced aggregation are independent of thromboxane A2 formation and that thromboxane A2 plays only a minor role in the later phase of PAF-induced aggregation.

Comparison of the effect of coagulation and platelet function impairments on various mouse bleeding models

2014 ◽  
Vol 112 (08) ◽  
pp. 412-418 ◽  
Author(s):  
Nima Vaezzadeh ◽  
Ran Ni ◽  
Paul Y. Kim ◽  
Jeffrey I. Weitz ◽  
Peter L. Gross
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Saphenous Vein ◽  
Ex Vivo ◽  
Arterial Injury ◽  
Inhibitory Antibody ◽  
Study Objective ◽  
Tail Tip

SummaryHaemostatic impairments are studied in vivo using one of several murine bleeding models. However it is not known whether these models are equally appropriate for assessing coagulation or platelet function defects. It was our study objective to assess the performance of arterial, venous and combined arterial and venous murine bleeding models towards impaired coagulation or platelet function. Unfractionated heparin (UFH) or αIIbβ3 inhibitory antibody (Leo.H4) were administered to mice, and their effects on bleeding in saphenous vein, artery, and tail tip transection models were quantified and correlated with their effects on plasma clotting and ADP-induced platelet aggregation, respectively. All models exhibited similar sensitivity with UFH (EC50 dose = 0.19, 0.13 and 0.07 U/g, respectively) (95% CI = 0.14 – 0.27, 0.08 – 0.20, and 0.03 – 0.16 U/g, respectively). Maximal inhibition of ex vivo plasma clotting could be achieved with UFH doses as low as 0.03 U/g. In contrast, the saphenous vein bleeding model was less sensitive to αIIbβ3 inhibition (EC50 = 6.9 µg/ml) than tail transection or saphenous artery bleeding models (EC50 = 0.12 and 0.37 µg/ml, respectively) (95% CI = 2.4 – 20, 0.05 – 0.33, and 0.06 – 2.2 µg/ml, respectively). The EC50 of Leo.H4 for ADP-induced platelet aggregation in vitro (8.0 µg/ml) was at least 20-fold higher than that of the tail and arterial, but not the venous bleeding model. In conclusion, venous, arterial and tail bleeding models are similarly affected by impaired coagulation, while platelet function defects have a greater influence in models incorporating arterial injury.

scholarly journals NADPH Oxidases Are Required for Full Platelet Activation In Vitro and Thrombosis In Vivo but Dispensable for Plasma Coagulation and Hemostasis

2020 ◽  
Author(s):  
Dina Vara ◽  
Reiner K. Mailer ◽  
Anuradha Tarafdar ◽  
Nina Wolska ◽  
Marco Heestermans ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Single Gene ◽  
Coagulation Cascade ◽  
Intracellular Cgmp ◽  
Antithrombotic Effects ◽  
Tail Tip ◽  
Synthase Inhibitor

Objective: Using 3KO (triple NOX [NADPH oxidase] knockout) mice (ie, NOX1 −/− /NOX2 −/− /NOX4 −/− ), we aimed to clarify the role of this family of enzymes in the regulation of platelets in vitro and hemostasis in vivo. Approach and Results: 3KO mice displayed significantly reduced platelet superoxide radical generation, which was associated with impaired platelet aggregation, adhesion, and thrombus formation in response to the key agonists collagen and thrombin. A comparison with single-gene knockouts suggested that the phenotype of 3KO platelets is the combination of the effects of the genetic deletion of NOX1 and NOX2, while NOX4 does not show any significant function in platelet regulation. 3KO platelets displayed significantly higher levels of cGMP—a negative platelet regulator that activates PKG (protein kinase G). The inhibition of PKG substantially but only partially rescued the defective phenotype of 3KO platelets, which are responsive to both collagen and thrombin in the presence of the PKG inhibitors KT5823 or Rp-8-pCPT-cGMPs, but not in the presence of the NOS (NO synthase) inhibitor L-NG-monomethyl arginine. In vivo, triple NOX deficiency protected against ferric chloride–driven carotid artery thrombosis and experimental pulmonary embolism, while hemostasis tested in a tail-tip transection assay was not affected. Procoagulatory activity of platelets (ie, phosphatidylserine surface exposure) and the coagulation cascade in platelet-free plasma were normal. Conclusions: This study indicates that inhibiting NOXs has strong antithrombotic effects partially caused by increased intracellular cGMP but spares hemostasis. NOXs are, therefore, pharmacotherapeutic targets to develop new antithrombotic drugs without bleeding side effects.

scholarly journals In Vitro and In Vivo Biology of Recombinant Adenovirus Vectors with E1, E1/E2A, or E1/E4 Deleted

1998 ◽  
Vol 72 (3) ◽  
pp. 2022-2032 ◽  
Author(s):  
M. Lusky ◽  
M. Christ ◽  
K. Rittner ◽  
A. Dieterle ◽  
D. Dreyer ◽  
...  
Keyword(s):  
Recombinant Adenovirus ◽  
Virus Genome ◽  
Minor Role ◽  
Immunodeficient Mice ◽  
Viral Genomes ◽  
Viral Genes ◽  
Adenovirus Vectors ◽  
A Minor

ABSTRACT Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.

C57BL/6JOlaHsd Mice with Tandem Deletion of the Multimerin 1 and Alpha-Synuclein Genes Have Impaired Platelet Function in Vivo and in Vitro That Can Be Corrected by Multimerin 1

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3926-3926 ◽  
Author(s):  
Subia Tasneem ◽  
Adili Reheman ◽  
Heyu Ni ◽  
Catherine P.M. Hayward
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Knockout Mice ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Alpha Synuclein ◽  
Type I ◽  
Significant Difference

Abstract Studies of mice with genetic deficiencies have provided important insights on the functions of many proteins in thrombosis and hemostasis. Recently, a strain of mice (C57BL/6JOlaHsd, an inbred strain of C57BL/6J) has been identified to have a spontaneous, tandem deletion of the multimerin 1 and α-synuclein genes, which are also adjacent genes on human chromosome 4q22. Multimerin 1 is an adhesive protein found in platelets and endothelial cells while α-synuclein is a protein found in the brain and in blood that is implicated in neurodegenerative diseases and exocytosis. In vitro, multimerin 1 supports platelet adhesion while α-synuclein inhibits α-granule release. We postulated that the loss of multimerin 1 and α-synuclein would alter platelet function and that recombinant human multimerin 1 might correct some of these abnormalities. We compared platelet adhesion, aggregation and thrombus formation in vitro and in vivo in C57BL/6JOlaHsd and C57BL/6 mice. Thrombus formation was studied by using the ferric-chloride injured mesenteric arteriole thrombosis model under intravital microscopy. We found that platelet adhesion, aggregation and thrombus formation in C57BL/6JOlaHsd were significantly impaired in comparison to control, C57BL/6 mice. The number of single platelets, deposited 3–5 minutes after injury, was significantly decreased in C57BL/6JOlaHsd mice (P <0.05, platelets/min: C57BL/6 = 157 ± 15, n=16; C57BL/6JOlaHsd = 77 ± 13, n=17). Moreover, thrombus formation in these mice was significantly delayed. Thrombi in C57BL/6JOlaHsd were unstable and easily dissolved, which resulted in significant delays (P<0.001) in vessel occlusion (mean occlusion times: C57BL/6 = 15.6 ± 1.2 min, n=16; C57BL/6JOlaHsd = 31.9 ± 2.1 min, n=17). We further tested platelet function in these mice by ADP and thrombin induced platelet aggregation using platelet rich plasma and gel-filtered platelets, respectively. Although no significant differences were seen with ADP aggregation, thrombin-induced platelet aggregation was significantly impaired in C57BL/6JOlaHsd mice. Platelet adhesion to type I collagen (evaluated using microcapillary chambers, perfused at 1500 s−1 with whole blood) was also impaired in C57BL/6JOlaHsd mice. However, platelets from C57BL/6JOlaHsd mice showed a normal pattern of agonist-induced release of α-granule P-selectin. Multimerin 1 corrected the in vitro aggregation and adhesion defects of C57BL/6JOlaHsd platelets. Furthermore, the transfusion of multimerin 1 into C57BL/6JOlaHsd mice corrected the impaired platelet deposition and thrombus formation in vivo. No significant difference was found in tail bleeding time between the two groups of mice. As α-synuclein knockout mice have a shortened time to thrombus formation (Circulation2007;116:II_76), the effects of multimerin 1 on impaired platelet function in C57BL/6JOlaHsd mice provide supportive evidence that multimerin 1 contributes to platelet adhesion and thrombus formation at the site of vessel injury. The findings suggest multimerin 1 knockout mice will be useful to explore platelet function. The first two authors and participating laboratories contributed equally to this study.

A Locally Administered Novel Heparin Complex Attenuates Collagen-Induced Platelet Activation and Thrombosis In Vivo,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3361-3361
Author(s):  
Riitta Lassila ◽  
Annukka Jouppila ◽  
Ulla M Marzec ◽  
Stephen R Hanson
Keyword(s):  
Blood Flow ◽  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Ptfe Graft ◽  
Thrombin Time ◽  
Thrombosis Model ◽  
Baboon Model ◽  
Induced Aggregation

Abstract Abstract 3361 We have developed a semi-synthetic antithrombotic heparin complex, APL001, to mimic mast cell-derived natural heparin proteoglycans (HepPG). HepPG attenuate platelet-collagen interactions under blood flow by inhibiting VWF- and GPIIb/IIIa -mediated platelet aggregation. In addition, rat-derived HepPG arrest platelet thrombus growth on collagen surfaces or at vascular injury sites, both in vitro and in vivo (Lassila et al.ATVB 1997, Kauhanen et al. ATVB 2000, Olsson et al. Thromb Haemost 2002). Our objective was to study the inhibitory capacity of APL001 for preventing human platelet aggregation in vitro and acute thrombosis in a baboon model in vivo. The effects of unfractionated heparin (UFH) and APL001 were compared in relevant coagulation assays (APTT, PT, thrombin time, anti-FXa activity, fibrinogen, FVIII:C and VWF activity (VWF:RCo) and antigen). Additionally, agonist-induced (collagen, ristocetin and ADP) platelet aggregation in citrate or hirudin-anticoagulated whole blood (Multiplate®) (n=10 healthy subjects), and platelet function analysis (PFA100®) in citrated platelet rich plasma (PRP) were assessed. In a well-established baboon thrombosis model a collagen-coated PTFE graft (length 2 cm, lumen 4 mm) was placed in an arterio-venous shunt. Prior to blood contact the thrombogenic surface was treated for 10 min with UFH or APL001 (both at 4 mg/mL). Thrombus formation was initiated by exposing the surface to blood flow (100 mL/min, shear rate 265−1), and the deposition of 111-In-labeled platelets and of fibrin was quantified continuously over 1h. Fibrin thrombus accumulation was assessed from the incorporation of circulating 125-I-fibrinogen. In the heparin-relevant coagulation tests APL001 was comparable or 20–30% more potent than UFH while FVIII, fibrinogen and VWF variables remained unaltered. In contrast to UFH, APL001 (300 μg/mL) consistently inhibited collagen- and ristocetin-induced platelet aggregation, whereas UFH had only a modest effect in comparison with PBS control (Table). ADP-induced aggregation was unaffected. Comparable results were observed in the PRP aggregation assay. PFA100 testing also demonstrated inhibitory effects. In the in vivo thrombosis model (n=4) APL001 reduced platelet deposition on collagen (vs. the results with UFH) by 34% (p=0.01), while platelet accumulation in distal propagated thrombus was reduced by 61% (p=0.16). APL001-treated surfaces accumulated 45% less fibrin than the UFH-treated surfaces (p=0.008). In conclusion, when compared with UFH APL001 inhibited both collagen- and ristocetin-induced platelet aggregation in human blood, while anticoagulant properties were comparable. In the absence of systemic antithrombotic drugs, exposure of APL001 to a highly thrombogenic collagen surface arrested thrombus formation in an in vivo baboon model. This finding suggests that locally administered APL001 alone, due to its dual antiplatelet and anticoagulant effects, may limit the growth and size of thrombus and thereby prevent subsequent thrombo-occlusion.TableAnticoagulantInhibition-% of platelet aggregation ± SDConc. 300 μg/mLnColl (3.2 μg/mL)Ristocetin (0.77 mg/mL)ADP (6.4 μM)CitrateAPL0011033 ± 1543 ± 166 ± 24UFH1011 ± 1323 ± 153 ± 7p value0.0030.0100.700HirudinAPL0011032 ± 1043 ± 178 ± 10UFH108 ± 1116 ± 166 ± 9p value0.0000.0020.600 Disclosures: Lassila: Aplagon: Chief Scientific Advisor.

Novel Small Molecule Inhibitors Of The Gas6/TAM Signaling Pathway Inhibit Platelet Aggregation In Vitro and Protect Mice From Arterial and Venous Thrombosis In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2296-2296
Author(s):  
Gilbert Acevedo ◽  
Brian R. Branchford ◽  
Christine Brzezinski ◽  
Susan Sather ◽  
Gary Brodsky ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Venous Thrombosis ◽  
Thrombus Formation ◽  
Patent Application ◽  
Mean Values ◽  
Inhibition Of Platelet Aggregation ◽  
Mean Time ◽  
Dose Dependent

Abstract Background Growth Arrest Specific gene 6 (Gas6) is a ligand for the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases found on the surface of platelets. Previous studies have shown that stimulation of these receptors results in amplification of platelet activation and thrombus stabilization via activation of phosphatidylinositol-3-kinase (PI3K) and Akt, leading to phosphorylation of the β3 integrin. Previous work (from our lab and others) demonstrated that inhibition of the Gas6/TAM pathway results in impaired platelet aggregation, reduced aggregate stability, and decreased platelet spreading. Additionally, knockout mice deficient in the receptor or ligand are protected from venous and arterial thrombosis, but retain normal tail bleeding times. Here, we describe development and characterization of novel Mer-selective small molecule inhibitors (SMIs) for thrombosis applications. Objectives To determine if Mer-selective SMIs can inhibit platelet aggregation and protect mice from thrombosis using in vitro and in vivo models Methods We used aggregometry and in vivo murine models of arterial and venous thrombosis to compare two Mer-selective SMIs (UNC Mer TKI1 and UNC Mer TKI2) and determine the most effective inhibitor of platelet aggregation and thrombus formation. The inhibitory effect of two doses (1µM and 5 µM) of the compounds were determined using standard light-transmission aggregometry after a 30 minute incubation with washed human platelets at 37 ¢ªC and compared to platelets treated with vehicle control or with a TKI control (UNC TKI Null), a SMI with similar structure but minimal anti-TAM activity. Both collagen/epinephrine-induced systemic venous thrombosis and FeCl3-induced carotid artery injury models were used to determine effects on thrombosis mediated by UNC TKIs. Wild type C57Bl/6 mice were treated with one of the two inhibitors and compared to mice treated with vehicle control. Mean values +/- SEM are shown and statistical significance (p<0.05) was determined using the student’s paired t-test. Results UNC Mer TKI1 exhibited more potent inhibition of platelet aggregation in vitro relative to UNC Mer TKI2, although both compounds mediated dose-dependent effects. At a concentration of 1uM, the maximum percent aggregation in UNC Mer TKI1-treated samples (n=7) was significantly greater than samples treated with UNC TKI Null (n=7), 20% DMSO vehicle (n=7), or UNC TKI2 (n=7), with mean values of 69 +/- 2.2%, 76.7 +/-1.8% (p<0.01), 76.9 +/- 2.1% (p=0.001), and 77 +/- 1.8% (p<0.001), respectively. At a concentration of 5 µM, UNC Mer TKI1-treated samples (n=7) exhibited a mean maximum percent aggregation of 23.7 +/- 2.4% compared to 50.4 +/- 4.8% for samples treated with UNC Mer TKI2 (n=7, p<0.001). UNC Mer TKIs also mediated protection from thrombus formation in mice. Following FeCl3 injury to the carotid artery, vehicle-treated mice (n=11) developed stable vessel occlusions with a mean time of 6.77 +/- 0.25 min. In contrast, stable occlusion occurred at a mean time of 46.6 +/- 7.72 min (n=9, p=0.001) for UNC Mer TKI1-treated mice. Survival times following venous injection of collagen and epinephrine were also significantly increased in mice treated with either UNC Mer TKI relative to the UNC TKI Null or vehicle controls. Mice pre-treated with UNC Mer TKI1 (n=9, p=0.04 compared to vehicle alone) or UNC Mer TKI2 (n=9, p=0.03 compared to vehicle alone) survived for 19.84 +/- 4.4 and 21.25 +/- 4.65 minutes, respectively. In contrast, mice given UNC TKI Null (n=3) or vehicle (n=21), only survived for 3.21 +/- 2.4 min and 3.09 +/- 0.22 minutes, respectively. Conclusion UNC Mer TKIs mediate dose-dependent inhibition of platelet aggregation and protect mice from arterial and venous thrombosis. Their pronounced activity compared to an inactive scaffold protein with minimal anti-TAM activity suggest that Gas6/TAM pathway inhibition is the mechanism of action for these novel compounds. UNC Mer TKI1 has more potent anti-thrombotic properties than UNC Mer TKI2. Disclosures: Branchford: University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Sather:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. DeRyckere:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Zhang:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Liu:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Earp:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Wang:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Frye:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Graham:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Di Paola:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties.

Novel 12-LOX Inhibitor ML355 Attenuates Platelet Reactivity and Impairs Thrombus Growth, Stability and Vessel Occlusion In Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3442-3442 ◽  
Author(s):  
Reheman Adili ◽  
Theodore R Holman ◽  
Michael Holinstat
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Platelet Reactivity ◽  
Thrombus Formation ◽  
Vessel Occlusion ◽  
Growth Stability

Abstract Background: Adequate platelet reactivity is required for platelet adhesion and aggregation at the site of vascular injury to maintain hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi, the predominate underlying cause of myocardial infarction and stroke. While current anti-platelet treatments limit platelet function, they often result in an increased risk of bleeding. 12-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated by our lab and others to regulate PAR4 and GPVI-mediated platelet reactivity suggesting a role of 12-LOX in regulation of vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Aims: To determine how 12-LOX regulates thrombus formation in vivo and whether platelet 12-LOX is an effective target for anti-platelet therapeutics, wild-type (WT) or 12-LOX deficient (12-LOX-/-) mice were treated with or without the 12-LOX inhibitor, ML355, and were assessed for inhibitory effects on platelet activation in vitro, ex-vivo and in vivo. Methods: The effect of the novel 12-LOX inhibitor ML355 on human platelet function was assessed in vitro by platelet aggregometry, ex vivo by perfusion chamber. In vivo thrombus formation and vessel occlusion in small and large vessels were studied in 12-LOX-/-, WT mice and mice treated with ML355 using intravital microscopy using the FeCl3 injury models. Results: Using in vitro platelet aggregation assays, ML355 dose dependently inhibited thrombin, PAR1-AP, and PAR4-AP-induced aggregation in washed human platelets. Interestingly, the negative regulatory effects of ML355 inhibition of 12-LOX can be overcome by high concentration of thrombin. Additionally, ML355 was able to attenuate ADP-induced platelet aggregation both in platelet-rich-plasma and whole blood. In ex vivo flow chamber assays, platelet adhesion and thrombus formation on collagen-coated surfaces at high shear was attenuated in both mouse and human whole blood after incubation with ML355. Further, platelet aggregation and thrombus growth in 12-LOX-/- mice was impaired in FeCl3-induced mesenteric or carotid artery thrombosis models. Thrombi in 12-LOX-/- mice were unstable and frequently form emboli, which resulted in impaired vessel occlusion or reopening. Additionally, thrombus formation and vessel occlusion was impaired in ML355 treated WT mice. Conclusions: The highly selective 12-LOX inhibitor ML355 inhibits platelets aggregation induced by various platelet agonists and ML355 inhibition of platelet function is not agonist specific. Platelet function at high shear in ex vivo conditions in both mice and human was attenuated in the presence of ML355. Thrombus growth, stability, and vessel occlusion was impaired in mice deficient for 12-LOX. Finally, the highly selective 12-LOX inhibitor ML355 attenuates thrombus formation and prevents vessel occlusion in vivo. Our data strongly indicates 12- LOX is an important determinant of platelet reactivity and inhibition of platelet 12-LOX may represent a new target for anti-platelet therapeutics. Disclosures No relevant conflicts of interest to declare.

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