Altered bioavailability of platelet-derived factor VIII during thrombocytosis reverses phenotypic efficacy in haemophilic mice.

Ectopic delivery of factor VIII (FVIII) to megakaryocytes (Mk) represents a viable approach for localized tenase generation by concentrating the FVIIIa/FIXa enzyme-cofactor complex onto activated platelet membranes. We utilized a core rat platelet factor 4 (PF4) promoter for Mk/platelet-restricted expression of human B-domain-deleted (hBDD) FVIII within the background of a haemophilia A mouse (rPF4/hBDD/FVIII-/-). Platelets from rPF4/hBDD/FVIII(-/-) mice contained approximately 122 mU FVIII:C/1 x 10(9) platelets/ml with no detectable plasmatic FVIII:C, and with no effect on alpha-granule-derived platelet factor V/Va function. Paired tenase assays (+/- thrombin) confirmed that platelet (pt) FVIII (unlike platelet FV) required thrombin cleavage for complete activation. rPF4/hBDD/FVIII(-/-) mice exposed to a thrombocytotic stimulus (thrombopoietin, TPO) demonstrated a statistically-significant 66% reduction in molar ptFVIII activity with a non-significant reduction in total ptFVIII biomass. Decreased molar ptFVIII concentration correlated with loss of phenotypic correction as evaluated using a haemostatic tail-snip assay. Comparative studies using a transgenic mouse expressing human amyloid-beta-precursor protein (hAbetaPP) from the rPF4 promoter confirmed diminished hAbetaPP expression without affecting endogenous alpha-granule PF4, establishing generalizability of these observations. While Mk/platelet-released ptFVIII (unlike pFV) is proteolytically inactive, we also conclude that thrombocytotic stimuli negatively affect ptFVIII bioavailability and phenotypic efficacy, results which correlate best with molar ptFVIII concentration, and not systemically available ptFVIII.

Effects of abciximab on the acute pathology of blood vessels after arterial stenting in nonhuman primates.

OBJECTIVES: This study was designed to assess the effect of abciximab on platelet and leukocyte deposition 60 min after stent insertion in nonhuman primates. BACKGROUND: Although it is well established that abciximab improves both short- and long-term clinical outcomes after stent placement, there have been no studies assessing its effect on early platelet and leukocyte deposition. METHODS: Cynomolgus monkeys were pretreated with aspirin and either saline or a 0.4 mg/kg bolus of abciximab, and then subjected to angioplasty and Palmaz-Schatz stent placement in the common iliac artery or abdominal aorta. After 60 min, animals were euthanized and the stented artery was evaluated by immunohistochemistry and morphometry. RESULTS: Complete occlusion of the stented vessel with a thin fibrin(ogen) meshwork and trapped blood occurred in two saline-treated and two abciximab-treated animals. In the four remaining saline-treated animals, a layer of erythrocytes trapped in a network of fibrin(ogen) was noted close to the vessel wall, and this was covered by a layer of large, irregular platelet thrombi. Leukocytes formed a monolayer on top of the platelets and near stent struts. In the four remaining abciximab-treated animals, the mean erythrocyte area was 65% smaller (p = 0.070), the platelet aggregate area was 89% smaller (p = 0.049) and the luminal area was 59% larger (p = 0.004). A monolayer of leukocytes also formed on top of the platelets and near stent struts. CONCLUSIONS: In control stented blood vessels in this study, platelet thrombi formed not at the vessel wall, but on top of an erythrocyte-rich layer, and platelets recruited leukocytes. Abciximab decreased the size of platelet thrombi, but did not prevent leukocyte recruitment.