Cathepsin G Cleavage of PAR4 Generates a Novel Tethered Ligand That Induces Platelet Activation

Atherosclerotic vessel injury induces recruitment of both platelets and neutrophils where multiple proteases induce platelet activation and aggregation. Platelets contain two protease activated receptors, PAR1 and PAR4, the cleavage of which results in exposure of a new amino terminus to serve as a tethered ligand. Released neutrophil cathepsin G (CatG) has been shown to be a physiologic modulator of platelet thrombus formation in mice. CatG activates PAR4 and not PAR1, presumably because CatG cleaves PAR1 by removing its tethered ligand. However, neither the CatG biochemical cleavage of PAR4 nor the resulting tethered ligands have been reported. The goals of the current study are to (1) identify the CatG-PAR4 cleavage sites and resulting tethered ligands and (2) determine how CatG-stimulated PAR4 signaling is altered by the PAR4 Ala120Thr variant. We synthesized two portions of the PAR4 extracellular N-terminus: amino acids Asp38-Ser58 (PAR4-B) and Asp57-Arg78 (PAR4-C) and exposed each peptide to purified CatG. Mass spectrometry identified two major cleavage sites for PAR4-B: the previously documented CatG and thrombin site Arg47-Gly48 and a novel Cys54-Ala55. Analysis of PAR4-C digestion yielded an additional three novel CatG cleavage sites, two major: Arg68-Ala69 and Leu71-Leu72, and one minor: Leu70-Leu71. Neither concentration or time of exposure appeared to alter the CatG cleavage sites. To assess functionality, we generated peptides based on the novel cleavage sites produced by CatG cleavage. Human washed platelets were treated with each peptide, and platelet activation was assessed by PAC-1 binding. As expected, the known tethered ligand sequence GYPGQV showed a statistically significant increase in PAC-1 binding (p=.02) compared to resting platelets. Three of the remaining four novel peptides generated no significant change in PAC-1 binding compared to baseline. However, peptide 3, representing novel tethered ligand ALLLGW, induced a substantial increase (462%) in PAC-1 binding compared to resting platelets. To assess the effect of the PAR4Ala120Thr variant on CatG-stimulated platelet reactivity, human washed platelets were collected from donors homozygous for Ala120 or Thr120 and stimulated with CatG. Platelets expressing the Thr120 variant displayed a significant increase in PAC-1 compared to Ala120 platelets (54%; p=.004). Addition of a CatG inhibitor caused a significant decrease in platelet activation triggered by CatG for both groups (Thr: -81%, p=.000001; Ala: -78%, p=.00017), and abrogated the significant increase in platelet activation displayed by Thr120 platelets (p=.99). To further examine the relationship between the PAR4 Ala120Thr variant and CatG, platelets from each variant were subjected to increasing amounts of CatG. Platelet activation was measured by PAC-1 binding and P-selectin expression. Both PAC-1 binding and P-selectin expression were significantly increased in platelets from Thr120 donors compared to Ala120 platelets (PAC-1, p=.026; P-selectin, p=.025). Overall, our study identified a previously unidentified CatG cleavage site in PAR4, which produced a novel tethered ligand capable of activating platelets. Because cleavage resulting in the ALLLGW ligand is downstream of the thrombin cleavage site, CatG may modulate in vivo thrombin-induced signaling in platelets or other cell types. Our findings also indicate the hyperactive response of 120Thr platelets is not dependent on a specific PAR4 protease. These new insights into PAR4 biology may provide targets for future antithrombotic therapies. Disclosures No relevant conflicts of interest to declare.

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STIM1 Deficiency Results In Impaired Platelet Procoagulant Activity and Protection From Arterial Thrombosis

Blood ◽

10.1182/blood.v116.21.485.485 ◽

2010 ◽

Vol 116 (21) ◽

pp. 485-485

Author(s):

Firdos Ahmad ◽

Lucia Stefanini ◽

Timothy Daniel Ouellette ◽

Teshell K Greene ◽

Stefan Feske ◽

...

Keyword(s):

Platelet Activation ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Critical Role ◽

Phosphatidyl Serine ◽

Flow Chamber ◽

Procoagulant Activity ◽

Static Conditions

Abstract Abstract 485 Platelet activation is a central event in thrombosis and hemostasis. We recently demonstrated that most aspects of platelet activation depend on synergistic signaling by two signaling modules: 1) Ca2+/CalDAG-GEFI/Rap1 and 2) PKC/P2Y12/Rap1. The intracellular Ca2+ concentration of platelets is regulated by Ca2+ release from the endoplasmic reticulum (ER) and store-operated calcium entry (SOCE) through the plasma membrane. Stromal interaction molecule 1 (STIM1) was recently identified as the ER Ca2+ sensor that couples Ca2+ store release to SOCE. In this study, we compared the activation response of platelets lacking STIM1−/− or CalDAG-GEFI−/−, both in vitro and in vivo. To specifically investigate Ca2+-dependent platelet activation, some of the experiments were performed in the presence of inhibitors to P2Y12. The murine Stim1 gene was deleted in the megakaryocyte/platelet lineage by breeding Stim flox/flox mice with PF4-Cre mice (STIM1fl/fl). STIM1fl/fl platelets showed markedly reduced SOCE in response to agonist stimulation. aIIbβ3 activation in STIM1fl/fl platelets was significantly reduced in the presence but not in the absence of the P2Y12 inhibitor, 2-MesAMP. In contrast, aIIbb3 activation was completely inhibited in 2-MesAMP-treated CalDAG-GEFI−/− platelets. Deficiency in STIM1, and to a lesser extent in CalDAG-GEFI, reduced phosphatidyl serine (PS) exposure in platelets stimulated under static conditions. PS exposure was completely abolished in both STIM1fl/fl and CalDAG-GEFI−/− platelets stimulated in the presence of 2-MesAMP. To test the ability of platelets to form thrombi under conditions of arterial shear stress, we performed flow chamber experiments with anticoagulated blood perfused over a collagen surface. Thrombus formation was abolished in CalDAG-GEFI−/− blood and WT blood treated with 2-MesAMP. In contrast, STIM1fl/fl platelets were indistinguishable from WT platelets in their ability to form thrombi. STIM1fl/fl platelets, however, were impaired in their ability to express PS when adhering to collagen under flow. Consistently, when subjected to a laser injury thrombosis model, STIM1fl/fl mice showed delayed and reduced fibrin generation, resulting in the formation of unstable thrombi. In conclusion, our studies indicate a critical role of STIM1 in SOCE and platelet procoagulant activity, but not in CalDAG-GEFI mediated activation of aIIbb3 integrin. Disclosures: No relevant conflicts of interest to declare.

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Premature Platelet Activation and Resistance to P2Y12 Inhibitors in Rasa3 Mutant Mice

Blood ◽

10.1182/blood.v124.21.91.91 ◽

2014 ◽

Vol 124 (21) ◽

pp. 91-91

Author(s):

Wolfgang Bergmeier ◽

David S Paul ◽

Lucia Stefanini ◽

Raymond F. Robledo ◽

E. Ricky Chan ◽

...

Keyword(s):

Platelet Activation ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Small Gtpase ◽

Nucleotide Exchange ◽

P2y12 Inhibitors ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor

Abstract The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the guanine nucleotide exchange factor CalDAG-GEFI and an unknown regulator operating downstream of the ADP receptor, P2Y12, the target of antithrombotic therapy. Here we provide evidence that the GTPase-activating protein, RASA3, is a critical inhibitor of platelet activation and the missing link in the P2Y12/RAP1 signaling pathway. Genetic inactivation of Rasa3 led to premature activation and markedly reduced lifespan of circulating platelets in mice (t1/2=14 hrs vs. 55 hrs in controls). The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Importantly, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Thus, constitutively active RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling. At sites of vascular injury, P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation. Our findings provide critical mechanistic insights for the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders. Disclosures No relevant conflicts of interest to declare.

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Phosphatidylinositol-3,4-Bisphosphate-Akt Signaling Pathway Promotes Platelet Activation

Blood ◽

10.1182/blood-2018-99-115066 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1131-1131

Author(s):

Jasna Marjanovic ◽

Brad Rumancik ◽

Luke Weber ◽

Felix Wangmang ◽

Dane Fickes ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Glycogen Synthase ◽

Thrombus Formation ◽

Type I ◽

Dependent Manner ◽

Wild Type ◽

Phosphorylated Akt

Abstract Phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) is a messenger that accumulates in platelets in a phosphoinositide 3-kinase and platelet aggregation-dependent manner. PtdIns(3,4)P2 is broken down by inositol polyphosphate 4-phosphatases, type I (INPP4A) and type II (INPP4B). These enzymes do not catalyze hydrolysis of phosphoinositides other than PtdIns(3,4)P2, and therefore provide unique means for studying the role of this lipid in platelet activation. We have found that the dominant isoform of 4-phosphatases expressed in platelets is INPP4A and we have generated radiation chimera mice with the deficiency in INPP4A restricted to hematopoietic cell lineage. Compared to wild type platelets, agonist-stimulated INPP4A-deficient platelets accumulated higher levels of PtdIns(3,4)P2. An increase in platelet aggregation in INPP4A-deficient platelets was observed with all tested agonists. To study platelet function in vivo, we performed carotid artery injury mouse thrombosis model experiments. Time to occlusion was dramatically reduced in mice with INPP4A deficiency. These data support the hypothesis that by regulating PtdIns(3,4)P2 levels, INPP4A downregulates platelet aggregation and thrombus formation. To investigate mechanisms mediating INPP4A-dependent signals, we compared levels of phosphorylated Akt and phosphorylated glycogen synthase kinase (GSK) in wild type and INPP4A-deficient platelets in response to agonist stimulation. An increase in phospho-Akt levels was observed in INPP4A-deficient platelets, suggesting that in addition to its well-characterized regulator, PtdIns(3,4,5)P3, PtdIns(3,4)P2 can promote Akt activation. Interestingly, this was not accompanied by a significant increase in phospho-GSK levels, suggesting a possible novel mechanism involved in platelet aggregation. Disclosures No relevant conflicts of interest to declare.

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Agonist-Induced Binding of the Regulatory Scaffold Protein Spinophilin to Protein Phosphatase-1 In Platelets

Blood ◽

10.1182/blood.v116.21.2029.2029 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2029-2029

Author(s):

Andrew Sinnamon ◽

Peisong Ma ◽

Lawrence F. Brass

Keyword(s):

Platelet Activation ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Tyrosine Phosphatase ◽

Critical Role ◽

Scaffold Protein ◽

Western Blots ◽

Co Precipitation

Abstract Abstract 2029 Platelet regulation plays a critical role in hemostasis. Underactivation can result in failure to stop bleeding, whereas inappropriate platelet activation can cause thrombus formation. The 130-kDa scaffold protein spinophilin (SPL) has recently been shown to play a role in preventing platelet overactivation by forming a complex with the proteins RGS10, RGS18, and the tyrosine phosphatase SHP-1. This complex dissociates when platelet are activated by thrombin or thromboxane A2 and evidence from spinophilin knockout mice suggests that this regulates platelet activation in vitro and in vivo. Spinophilin was originally isolated as a binding partner for the serine/threonine phosphatase, PP-1, in neurons. Here we asked whether PP-1 forms a complex with spinophilin in human platelets and, if so, whether the complex is affected by platelet activation. The approaches that we used to answer this question included Western blotting with antibodies to PP-1 and spinophilin, and co-precipitation studies looking for an association between spinophilin and PP-1. The results of the Western blots confirm the presence of PP-1 in platelets. The initial co-precipitation studies show that little, if any, PP-1 is associated with spinophilin in resting platelets, but there is a time-dependent increase in the SPL/PP-1 complex when platelets are activated with the PAR1 (thrombin receptor) activating peptide, SFLLRN. Thus it appears that within approximately the same time frame that the SPL/RGS/SHP-1 complex is decaying in activated platelets, the SPL/PP-1 complex is forming. Targets for PP1 have not been fully identified in platelets, but it is known that spinophilin localizes to the plasma membrane upon platelet activation. Since spinophilin is thought to direct PP1 targeting in neurons, it is reasonable to propose that it may be directing PP1 to targets in platelets in a similar manner. The studies described in this abstract were supported in part by a 2010 ASH Trainee Research Award to Andrew Sinnamon, who is a first year medical student at the University of Pennsylvania. Disclosures: No relevant conflicts of interest to declare.

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Racial Differences In Thrombin-Induced Human Platelet PAR4 Reactivity

Blood ◽

10.1182/blood.v122.21.1054.1054 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1054-1054

Author(s):

Leonard C. Edelstein ◽

Lukas M Simon ◽

Raul Teruel Montoya ◽

Michael Holinstat ◽

Edward Chen ◽

...

Keyword(s):

Racial Differences ◽

Platelet Activation ◽

Platelet Function ◽

Racial Difference ◽

Conflicts Of Interest ◽

Platelet Reactivity ◽

Wild Type Mouse ◽

Induced Aggregation ◽

Megakaryocytic Cell

Abstract Compared to white patients, black patients have worse outcomes after acute coronary events, but there is a paucity of literature considering racial differences in platelet function. Thrombin is an especially potent in vivo platelet agonist, and no work has considered racial differences in thrombin-induced platelet aggregation. Using PAR1- and PAR4- activation peptides (APs), we recently reported that platelets from healthy black subjects (n = 70) demonstrated greater aggregation to the PAR4-AP than platelets from white subjects (n = 84) (p = 5.15 x 10-8). There was no racial difference to PAR1-AP, ADP or CRP. The goal of the current study was to determine if this racial difference in PAR4-AP-mediated platelet reactivity was also observed with thrombin and to investigate responsible molecular and genetic mechanism(s). A detailed dose-response study (4 blacks and 3 whites) revealed that when thrombin signaling was restricted to PAR4 by inhibiting PAR1 with BMS-200261, platelets from black subjects aggregated faster than platelets from white subjects at low concentrations of thrombin. No PAR1-AP-induced aggregation occurred in the presence of BMS-200261. A subsequent replication study (an additional 5 blacks and 5 whites) again showed platelets from black subjects aggregated faster than white subjects in the absence of PAR1 signaling (p = 3.56x10-5). DNA from all 154 subjects was genotyped for 5 million SNPs (HumanOmni5 array) and principal component analysis revealed that the genotypes segregated into two distinct groups that correlated perfectly with subject self-identified race. Gene expression profiling on leukocyte-depleted platelets from all 154 subjects revealed numerous differentially expressed (DE) RNAs associated with both race and PAR4 reactivity. The gene encoding phosphatidylcholine transfer protein (PC-TP), PCTP, showed the strongest correlation with race (p = 10-23; q = 10-20) and with PAR4 reactivity (p = 3.4x10-8; q = 3.5x10-4). PC-TP protein was higher in platelets from blacks (p = 3.8x10-6) and levels correlated with reactivity to PAR4-AP (r = 0.249, p = 0.002). Pctp has been knocked out in mice, but we found that wild type mouse platelets express little or no Pctp protein, consistent with mouse platelet RNA data from Rowley et al (Blood 2011). However, a specific PC-TP inhibitor resulted in a reduced aggregation response to PAR4-AP, but not PAR1-AP. Transfection of a siRNA against PCTP reduced both PCTP mRNA and PC-TP protein levels, and inhibited Ca+ release in a megakaryocytic cell line, Meg-01 in response to PAR4-AP but not PAR1-AP. A racial difference in platelet Ca+ release in response to PAR4-AP treatment was also observed (p = 0.02). Platelet microRNA (miRNA) profiling from all 154 subjects also revealed numerous DE miRNAs associated with both race and PAR4 reactivity. Target prediction analysis indicated that miR-376c is a candidate for regulating PCTP expression. qRT-PCR of all 154 subjects indicated that miR-376c levels are expressed higher in platelets from whites (p = 1.47 x10-4; q = 1.38 x10-3), and are inversely correlated with PCTP mRNA (r = -0.214; p = 0.008), PC-TP protein (r = -0.211; p = 0.009) and PAR4 reactivity (r = -0.161; p = 0.049). Transfection of megakaryocytic cell lines or cord blood CD34+ derived megakaryocytes with the pre-miR-376c precursor or LNA-miR-376c inhibitor resulted in decreased (P<0.01) or increased PCTP (p = 0.0003), respectively. Co-transfection of the miRNA precursor or inhibitor with a luciferase vector containing the PCTP 3’UTR indicated the regulation was dependent on the predicted miR-376c target site. In summary, we have uncovered a racial difference in thrombin-induced PAR4 platelet activation. This finding has potential clinical significance because PAR4 is the primary means by which thrombin activates platelets in the presence of vorapaxar (a PAR1 inhibitor in clinical trials), and the risks and benefits of vorapaxar by race are unknown. This racial difference in platelet activation is mediated, in part, by PC-TP, a novel protein in platelet biology. Our data also supports racial differences in miRNA expression, one of which (miR-376c) regulates PC-TP expression. These results indicate a genomic contribution to platelet function that differs by race, emphasize a need to consider race effects when developing anti-thrombotic drugs and raise the possibility that PC-TP inhibition might be a useful anti-thrombotic strategy. Disclosures: No relevant conflicts of interest to declare.

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Abstract 56: Platelet Specific Knockout of Glucose Transporter 3 Leads to Altered Metabolism and Decreased Platelet Activation

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.56 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Trevor P Fidler ◽

Elizabeth Middleton ◽

Jesse W Rowley ◽

Luc Boudreau ◽

Robert A Campbell ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Plasma Membrane ◽

Glucose Uptake ◽

Platelet Activation ◽

Glucose Transporter ◽

Platelet Reactivity ◽

Thrombus Formation ◽

Mitochondrial Uncoupling ◽

Granule Release

Patients with diabetes display increased thrombosis and platelet activation. In these disorders, the systemic milieu is characterized by multiple metabolic changes including increased glucose concentrations. Preliminary metabolomics analysis of platelets from patients with type 2 diabetes revealed an accumulation of glycolytic and TCA intermediates relative to healthy controls. Therefore we hypothesized that decreasing platelet glucose uptake would limit glycolysis thereby decreasing energy production and platelet reactivity. Platelets import glucose via two glucose transporters GLUT1 and GLUT3. GLUT1 is expressed on the plasma membrane and GLUT3 is expressed predominantly on alpha granule membranes (85%) and to a lesser extent on the plasma membrane (15%). To better understand the consequences of glucose metabolism on platelet function we generated a platelet specific knockout (KO) of GLUT3 using a Pf4 Cre recombinase transgenic mouse crossed to mice that harbor floxed GLUT3 alleles. Platelet glycogen content and glycolytic intermediates were significantly reduced in GLUT3 KO platelets compared to controls, and following mitochondrial uncoupling exhibited reduced glycolysis rates. Interestingly, under these conditions, mitochondrial maximal respiration was increased two-fold, with no change in mitochondrial density, or citric acid cycle intermediates. In vitro , GLUT3 deficient platelets display a 90% reduction of spreading on fibrinogen and collagen matrixes and significant reductions in CD62p surface translocation and GPIIbIIIa activation following stimulation with multiple agonists. Additionally makers of alpha granule release were significantly reduced. In vivo analysis of GLUT3 KO mice using a 10% ferric chloride model of arterial thrombosis and a tail-bleed model indicated no alteration in thrombosis between littermate controls and knockouts. However in a KBx/N model of rheumatoid arthritis GLUT3 KO mice exhibited significantly reduced disease severity. Together, these data indicate that GLUT3-mediated glucose uptake is essential for platelet activation, spreading and alpha granule release. GLUT3 modulates mechanisms that promote rheumatoid arthritis but not those that regulate in vivo thrombus formation.

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Defibrotide Inhibits Platelet Activation by Cathepsin G Released From Stimulated Polymorphonuclear Leukocytes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648519 ◽

1992 ◽

Vol 67 (06) ◽

pp. 660-664 ◽

Cited By ~ 17

Author(s):

Virgilio Evangelista ◽

Paola Piccardoni ◽

Giovanni de Gaetano ◽

Chiara Cerletti

Keyword(s):

Platelet Activation ◽

Polymorphonuclear Leukocytes ◽

Direct Interaction ◽

Cathepsin G ◽

In Vivo Test ◽

Cell Functions ◽

Test Models ◽

Antithrombotic Effects

SummaryDefibrotide is a polydeoxyribonucleotide with antithrombotic effects in experimental animal models. Most of the actions of this drug have been observed in in vivo test models but no effects have been reported in in vitro systems. In this paper we demonstrate that defibrotide interferes with polymorphonuclear leukocyte-induced human platelet activation in vitro. This effect was not related to any direct interaction with polymorphonuclear leukocytes or platelets, but was due to the inhibition of cathepsin G, the main biochemical mediator of this cell-cell cooperation. Since cathepsin G not only induces platelet activation but also affects some endothelial cell functions, the anticathepsin G activity of defibrotide could help to explain the antithrombotic effect of this drug.

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FUNDC2 regulates platelet activation through AKT/GSK-3β/cGMP axis

Cardiovascular Research ◽

10.1093/cvr/cvy311 ◽

2018 ◽

Vol 115 (11) ◽

pp. 1672-1679 ◽

Cited By ~ 1

Author(s):

Qi Ma ◽

Weilin Zhang ◽

Chongzhuo Zhu ◽

Junling Liu ◽

Quan Chen

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Mitochondrial Protein ◽

Dependent Manner ◽

Clot Retraction ◽

Akt Phosphorylation ◽

Gsk 3Β

Abstract Aims AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. Methods and results We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5′-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3β in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3β/cGMP axis also regulates clot retraction of platelet-rich plasma. Conclusion FUNDC2 positively regulates platelet functions via AKT/GSK-3β/cGMP signalling pathways, which provides new insight for platelet-related diseases.

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Abstract 54: ML355 in Prevention of Thrombosis in vivo with Minimal Effects on Hemostasis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.54 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Reheman Adili ◽

Katherine Mast ◽

Michael Holinstat

Keyword(s):

Ex Vivo ◽

Platelet Reactivity ◽

Thrombus Formation ◽

Flow Chamber ◽

Human Platelets ◽

Mass Spectrometry Analysis ◽

Vessel Occlusion ◽

Spectrometry Analysis ◽

Thrombosis Model

12-lipoxygenase (12-LOX) has been demonstrated to regulate platelet function, hemostasis, and thrombosis ex vivo , supporting a key role for 12-LOX in regulation of in vivo thrombosis. While pharmacologically targeting 12-LOX in vivo has been a challenge to date, the recent development of the 12-LOX selective inhibitor, ML355, as an effective antiplatelet therapeutic in vivo was assessed. ML355 potently inhibited thrombin and other agonist-induced platelet aggregation ex vivo in washed human platelets and inhibited downstream oxylipin production of platelet 12-LOX as confirmed by Mass spectrometry analysis. Ex vivo flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen was attenuated in human whole blood treated with ML355 to a greater extent compared to aspirin. In vivo , PK assessment of ML355 showed reasonable 12-LOX plasma levels 12 hours following administration of ML355. FeCl 3 -induced injury of the mesenteric arterioles resulted in less stable thrombi in 12-LOX -/- mice and ML355-treated WT mice resulting in impairment of vessel occlusion. Additionally, ML355 dose-dependently inhibited laser-induced thrombus formation in the cremaster arteriole thrombosis model in WT, but not in 12-LOX -/- mice. Importantly, hemostatic plug formation and bleeding following treatment with ML355 were not affected in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model. Our data strongly supports 12-LOX as a key determinant of platelet reactivity in vivo and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapeutics.

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Effect Of Selective Inhibition Of Platelet Thromboxane On Platelet-Vessel Wall Interaction In Vivo

10.1055/s-0038-1652581 ◽

1981 ◽

Author(s):

Y C Chen ◽

K K Wu ◽

E R Hall ◽

D L Venton ◽

G C Le Breton

Keyword(s):

Vessel Wall ◽

Platelet Reactivity ◽

Thrombus Formation ◽

Balloon Catheter ◽

Specific Inhibitor ◽

Constant Infusion ◽

Catheter Technique ◽

Platelet Thrombus ◽

Platelet Accumulation

It is well recognized that thromboxane A2(TXA2) plays an important role in platelet reactivity. To determine the role of TXA2 in platelet-vessel wall (P-V) interaction, the effect of 1-benzylimidazole (1-BI), a specific inhibitor of thromboxane synthetase, and 13-azaprostanoic acid (APA), a TXA2 antagonist, on platelet thrombus formation was evaluated in vivo in NZW male rabbits using the autologous indium-111 (111In) labeled platelet technique. Rabbits were treated with intravenous 1-BI or APA or vehicles. After injection of autologous 111In-platelets, de-endothelialization of the abdominal aorta was created by a balloon catheter technique. At 3 hrs, blood samples were obtained and the animals were sacrificed. The aortae were removed and the injured and uninjured segments were dissected. Radioactivity counts and dry weight of the tissues and blood were determined. The vascular radioactivity counts were converted to platelet numbers by using a standard linear calibration curve. As small numbers of platelets adhered to normal vessel wall nonspecifically, this number was subtracted to obtain specific platelet accumulation at the injured sites. 1-BI at 10mg/kg reduced the specific platelet accumulation significantly (n=5, 12.3±S.D.I.5×106 pl/gm tissue; p<0.01) when compared with the controls (n=10, 33.0±5.1×106 pl/gm tissue). Platelet accumulation was further reduced by increasing the dosage to 30mg/kg. By contrast, APA injection (10mg/kg) had no significant effect. However, when APA was given by constant infusion at 250μg/kg/min 1 hr prior to injury, the APA-treated animals had an 80% reduction of platelet accumulation relative to controls. These findings indicate that TXA2 plays an important role in P-V interaction and specific inhibition of TXA2 appears to be efficacious in eliminating platelet thrombus formation.

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