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Cathepsin G-dependent modulation of platelet thrombus formation in vivo by blood neutrophils.

Faraday N, Schunke K, Saleem S, Fu J, Wang B, Zhang J, Morrell C, Dore S - PLoS ONE (2013)

Bottom Line: Tail bleeding time in the mouse was prolonged by a cathepsin G inhibitor and in cathepsin G knockout mice, and formation of neutrophil-platelet conjugates in blood that was shed from transected tails was reduced in the absence of cathepsin G.In the presence of elevated blood neutrophil counts, the anti-thrombotic effect of cathepsin G inhibition was greater than that of aspirin and additive to it when administered in combination.In a vaso-occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, reduced histologic brain injury, and improved neurobehavioral outcome.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology/Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America. nfaraday@jhmi.edu

ABSTRACT
Neutrophils are consistently associated with arterial thrombotic morbidity in human clinical studies but the causal basis for this association is unclear. We tested the hypothesis that neutrophils modulate platelet activation and thrombus formation in vivo in a cathepsin G-dependent manner. Neutrophils enhanced aggregation of human platelets in vitro in dose-dependent fashion and this effect was diminished by pharmacologic inhibition of cathepsin G activity and knockdown of cathepsin G expression. Tail bleeding time in the mouse was prolonged by a cathepsin G inhibitor and in cathepsin G knockout mice, and formation of neutrophil-platelet conjugates in blood that was shed from transected tails was reduced in the absence of cathepsin G. Bleeding time was highly correlated with blood neutrophil count in wildtype but not cathepsin G deficient mice. In the presence of elevated blood neutrophil counts, the anti-thrombotic effect of cathepsin G inhibition was greater than that of aspirin and additive to it when administered in combination. Both pharmacologic inhibition of cathepsin G and its congenital absence prolonged the time for platelet thrombus to form in ferric chloride-injured mouse mesenteric arterioles. In a vaso-occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, reduced histologic brain injury, and improved neurobehavioral outcome. These experiments demonstrate that neutrophil cathepsin G is a physiologic modulator of platelet thrombus formation in vivo and has potential as a target for novel anti-thrombotic therapies.

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Congenital deficiency of cathepsin G improves cerebral blood flow (CBF) and reduces brain injury in an ischemic stroke model.The stroke model was as described for fig, 6. Data are mean ± SEM; N = 6 each group, *P<0.05 vs. control, **P<0.01 vs. control. A. CBF of wildtype (WT) or cathepsin G deficient (CatG−/−) mice. x-axis represents time in relation to insertion of the filament; immed1 = insertion, immed2 = withdrawal. B and C. Representative TTC-stained brain slices from WT (B) or CatG−/− (C) mice. D. Cerebral infarct volumes of WT and CatG−/− mice. E. Neurobehavioral deficit scores of WT or CatG−/− mice.
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pone-0071447-g007: Congenital deficiency of cathepsin G improves cerebral blood flow (CBF) and reduces brain injury in an ischemic stroke model.The stroke model was as described for fig, 6. Data are mean ± SEM; N = 6 each group, *P<0.05 vs. control, **P<0.01 vs. control. A. CBF of wildtype (WT) or cathepsin G deficient (CatG−/−) mice. x-axis represents time in relation to insertion of the filament; immed1 = insertion, immed2 = withdrawal. B and C. Representative TTC-stained brain slices from WT (B) or CatG−/− (C) mice. D. Cerebral infarct volumes of WT and CatG−/− mice. E. Neurobehavioral deficit scores of WT or CatG−/− mice.

Mentions: The impact of cathepsin G on CBF and ischemic brain injury was examined using a previously described mouse stroke model [22], [23] with modification. Vaso-occlusive stroke was induced by inserting a collagen-coated filament into the MCA, rather than an uncoated filament, to more closely simulate a vascular thrombotic injury model. In preliminary experiments, insertion of a collagen-coated filament into the MCA for 60 minutes caused a greater reduction in CBF after filament withdrawal (36±9% vs. 84±4% of baseline CBF for collagen-coated vs. uncoated, respectively, P<0.05) and greater cerebral infarct volume (corrected infarct volumes of 34±6% vs. 15±3% for collagen-coated vs. uncoated, respectively, P<0.05) than a standard uncoated nylon filament. Intravenous injection of cathepsin G inhibitor I prior to filament insertion markedly improved MCA blood flow after withdrawal of the filament, consistent with improved vascular patency (fig. 6A). Compared with vehicle treated mice, mice treated with cathepsin G inhibitor I had markedly reduced cerebral infarct volumes (fig. 6B–D) and improved neurobehavioral deficit scores (fig. 6E) 48 hours after injury. Experiments comparing CatG−/− to WT mice showed very similar results: congenital deficiency of cathepsin G was associated with a marked improvement in CBF after filament withdrawal (fig. 7A), reduced cerebral infarct volume (fig. 7B–D), and improved neurobehavioral deficit score (fig. 7E) compared to WT animals.


Cathepsin G-dependent modulation of platelet thrombus formation in vivo by blood neutrophils.

Faraday N, Schunke K, Saleem S, Fu J, Wang B, Zhang J, Morrell C, Dore S - PLoS ONE (2013)

Congenital deficiency of cathepsin G improves cerebral blood flow (CBF) and reduces brain injury in an ischemic stroke model.The stroke model was as described for fig, 6. Data are mean ± SEM; N = 6 each group, *P<0.05 vs. control, **P<0.01 vs. control. A. CBF of wildtype (WT) or cathepsin G deficient (CatG−/−) mice. x-axis represents time in relation to insertion of the filament; immed1 = insertion, immed2 = withdrawal. B and C. Representative TTC-stained brain slices from WT (B) or CatG−/− (C) mice. D. Cerebral infarct volumes of WT and CatG−/− mice. E. Neurobehavioral deficit scores of WT or CatG−/− mice.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3733958&req=5

pone-0071447-g007: Congenital deficiency of cathepsin G improves cerebral blood flow (CBF) and reduces brain injury in an ischemic stroke model.The stroke model was as described for fig, 6. Data are mean ± SEM; N = 6 each group, *P<0.05 vs. control, **P<0.01 vs. control. A. CBF of wildtype (WT) or cathepsin G deficient (CatG−/−) mice. x-axis represents time in relation to insertion of the filament; immed1 = insertion, immed2 = withdrawal. B and C. Representative TTC-stained brain slices from WT (B) or CatG−/− (C) mice. D. Cerebral infarct volumes of WT and CatG−/− mice. E. Neurobehavioral deficit scores of WT or CatG−/− mice.
Mentions: The impact of cathepsin G on CBF and ischemic brain injury was examined using a previously described mouse stroke model [22], [23] with modification. Vaso-occlusive stroke was induced by inserting a collagen-coated filament into the MCA, rather than an uncoated filament, to more closely simulate a vascular thrombotic injury model. In preliminary experiments, insertion of a collagen-coated filament into the MCA for 60 minutes caused a greater reduction in CBF after filament withdrawal (36±9% vs. 84±4% of baseline CBF for collagen-coated vs. uncoated, respectively, P<0.05) and greater cerebral infarct volume (corrected infarct volumes of 34±6% vs. 15±3% for collagen-coated vs. uncoated, respectively, P<0.05) than a standard uncoated nylon filament. Intravenous injection of cathepsin G inhibitor I prior to filament insertion markedly improved MCA blood flow after withdrawal of the filament, consistent with improved vascular patency (fig. 6A). Compared with vehicle treated mice, mice treated with cathepsin G inhibitor I had markedly reduced cerebral infarct volumes (fig. 6B–D) and improved neurobehavioral deficit scores (fig. 6E) 48 hours after injury. Experiments comparing CatG−/− to WT mice showed very similar results: congenital deficiency of cathepsin G was associated with a marked improvement in CBF after filament withdrawal (fig. 7A), reduced cerebral infarct volume (fig. 7B–D), and improved neurobehavioral deficit score (fig. 7E) compared to WT animals.

Bottom Line: Tail bleeding time in the mouse was prolonged by a cathepsin G inhibitor and in cathepsin G knockout mice, and formation of neutrophil-platelet conjugates in blood that was shed from transected tails was reduced in the absence of cathepsin G.In the presence of elevated blood neutrophil counts, the anti-thrombotic effect of cathepsin G inhibition was greater than that of aspirin and additive to it when administered in combination.In a vaso-occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, reduced histologic brain injury, and improved neurobehavioral outcome.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology/Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America. nfaraday@jhmi.edu

ABSTRACT
Neutrophils are consistently associated with arterial thrombotic morbidity in human clinical studies but the causal basis for this association is unclear. We tested the hypothesis that neutrophils modulate platelet activation and thrombus formation in vivo in a cathepsin G-dependent manner. Neutrophils enhanced aggregation of human platelets in vitro in dose-dependent fashion and this effect was diminished by pharmacologic inhibition of cathepsin G activity and knockdown of cathepsin G expression. Tail bleeding time in the mouse was prolonged by a cathepsin G inhibitor and in cathepsin G knockout mice, and formation of neutrophil-platelet conjugates in blood that was shed from transected tails was reduced in the absence of cathepsin G. Bleeding time was highly correlated with blood neutrophil count in wildtype but not cathepsin G deficient mice. In the presence of elevated blood neutrophil counts, the anti-thrombotic effect of cathepsin G inhibition was greater than that of aspirin and additive to it when administered in combination. Both pharmacologic inhibition of cathepsin G and its congenital absence prolonged the time for platelet thrombus to form in ferric chloride-injured mouse mesenteric arterioles. In a vaso-occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, reduced histologic brain injury, and improved neurobehavioral outcome. These experiments demonstrate that neutrophil cathepsin G is a physiologic modulator of platelet thrombus formation in vivo and has potential as a target for novel anti-thrombotic therapies.

Show MeSH
Related in: MedlinePlus