Enhancement of Platelet Deposition by Cross-Linked Hemoglobin in a Rat Carotid Endarterectomy Model

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Abstract
Background Purified human cross-linked hemoglobin, which is now being used in clinical trials, increases mean arterial pressure through binding of nitric oxide (NO). We postulated that binding of NO by cross-linked hemoglobin (ααHb) could also increase platelet deposition at sites of subintimal injury. Methods and Results Male Sprague-Dawley rats were infused with ααHb (0.88 g/kg, n=8) or with the NO synthase inhibitor N G -monomethyl- l -arginine (L-NMMA, 30 mg/kg, n=7) before undergoing microsurgical carotid endarterectomy. 111 In-labeled platelets were infused after endarterectomy, and platelet deposition was measured 20 minutes later. In control endarterectomized rats (n=8), mean platelet deposition was 7.7±0.7×10 6 /mm 2 . Platelet deposition was significantly increased above controls in rats that received ααHb (13.2±0.9×10 6 /mm 2 , P =.0004) and in rats infused with L-NMMA (13.9±1.0×10 6 /mm 2 , P =.0002). The increase was prevented by infusion of l -arginine (150 mg/kg) immediately after ααHb or L-NMMA. To determine whether aspirin (ASA) blocked the increased deposition induced by ααHb, rats received oral ASA (10 mg/kg) 18 hours before endarterectomy. Platelet deposition in animals receiving ASA alone was 6.4±0.9×10 6 /mm 2 (n=8). This was significantly increased to 10.8±0.8×10 6 /mm 2 ( P =.002) for the ASA-treated group that received ααHb at the time of endarterectomy (n=8). The prolonged bleeding times induced by ASA were unaffected by the infusion of ααHb. Conclusions These data suggest that in a rat endarterectomy model, ααHb increases platelet deposition at sites of subintimal injury by binding NO. Increased deposition induced by ααHb can be prevented by administration of l -arginine but not by pretreatment with aspirin.