Impact of the neutrophil response to granulocyte colony-stimulating factor on the risk of hemorrhage when used in combination with tissue plasminogen activator during the acute phase of experimental stroke

BACKGROUND: Granulocyte colony-stimulating factor (G-CSF) is a pharmacologic agent inducing neutrophil mobilization and a new candidate for neuroprotection and neuroregeneration in stroke. Its effects when used in combination with tissue plasminogen activator (tPA) were explored during the acute phase of ischemic stroke. METHODS: We used a middle cerebral artery occlusion (MCAO) model of cerebral ischemia, associated with treatment with tPA, in male spontaneously hypertensive rats (SHR). Granulocyte colony-stimulating factor (G-CSF; 60 μg/kg) was injected just before tPA. Neutrophil response in peripheral blood and in the infarct area was quantified in parallel to the infarct volume. Protease matrix metallopeptidase 9 (MMP-9) release from circulating neutrophils was analyzed by immunochemistry and zymography. Vascular reactivity and hemorrhagic volume in the infarct area was also assessed. RESULTS: Twenty four hours after ischemia and tPA, G-CSF administration induced a significant increase of neutrophils in peripheral blood (P <0.05). At 72 hours post-ischemia, G-CSF was significantly associated with an increased risk of hemorrhage in the infarct area (2.5 times more likely; P <0.05) and significant cerebral endothelium-dependent dysfunction. Ex vivo, an increased MMP-9 release from neutrophils after tPA administration correlated to the increased hemorrhagic risk (P <0.05). In parallel, G-CSF administration was associated with a decreased neutrophil infiltration in the infarct area (-50%; P <0.05), with a concomitant significant neuroprotective effect (infarct volume: -40%; P <0.05). CONCLUSIONS: We demonstrate that G-CSF potentiates the risk of hemorrhage in experimental stroke when used in combination with tPA by inducing neutrophilia. This effect is concomitant to an increased MMP-9 release from peripheral neutrophils induced by the tPA treatment. These results highlight the potential hemorrhagic risk of associating G-CSF to thrombolysis during the acute phase of stroke

Modulation de la production de thrombine et fibrino-formation dans l'hémostase

Le facteur X de la coagulation (FX) permet la génération de thrombine responsable de la formation d un réseau de fibrine et de l activation des plaquettes qui libèrent de grande quantité de facteur 4 plaquettaire (PF4). La première partie de ce mémoire concerne la caractérisation d un mutant naturel du FX dont nous avons décrypté le défaut fonctionnel expliquant le phénotype du patient. Nous avons par ailleurs préparé et caractérisé cinq dérivés du FX activables par la thrombine. Ces dérivés rétablissent une amplification de la génération de thrombine dans un plasma d hémophile et pourraient constituer un traitement innovant. La seconde partie de ce mémoire porte sur deux fonctions insoupçonnées du PF4. Nous avons dévoilé qu il limite la génération de thrombine en inhibant l activation du facteur V plasmatique et qu il altère profondément la structure du réseau de fibrine. Ces observations apportent un éclairage nouveau sur la formation et la structure du clou plaquettaire initial.Coagulation factor X (FX) participates in the generation of thrombin, which forms the fibrin network, and activates platelets which release platelet factor 4 (PF4) in large quantities. The first part of this report concerns the characterization of a novel mutant of FX, whose functional defect was deciphered by looking at the phenotype of the patient. We also prepared and characterized five recombinant proteins of thrombin-activable FX. These recombinants restored the amplification of thrombin production in blood plasma of haemophiliacs and could constitute an innovative treatment. For the second part, we investigated the characteristics of PF4 and revealed two unsuspected functions. We found that it prevents the generation of thrombin by inhibiting the activation of the plasma-derived factor V and we demonstrated that PF4 profoundly alters the structure of the fibrin network. These observations provide a new insight on the formation and the structure of the initial platelet clot.PARIS-BIUP (751062107) / SudocSudocFranceF