Enhancement of platelet deposition by cross-linked hemoglobin in a rat carotid endarterectomy model

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 (alpha alpha Hb) could also increase platelet deposition at sites of subintimal injury. Methods and Results Male Sprague-Dawley rats were infused with alpha alpha Hb (0.88 g/kg, n=8) or with the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 30 mg/kg, n=7) before undergoing microsurgical carotid endarterectomy. In-111-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+/-0.7x10(6)/mm(2). Platelet deposition was significantly increased above controls in rats that received alpha alpha Hb (13.2+/-0.9x10(6)/mm(2), P=.0004) and in rats infused with L-NMMA (13.9+/-1.0x10(6)/mm(2), P=.0002). The increase was prevented by infusion of L-arginine (150 mg/kg) immediately after alpha alpha Hb or L-NMMA. To determine whether aspirin (ASA) blocked the increased deposition induced by alpha alpha Hb, rats received oral ASA (10 mg/kg) 18 hours before endarterectomy. Platelet deposition in animals receiving ASA alone was 6.4+/-0.9x10(6)/mm(2) (n=8). This was significantly increased to 10.8+/-0.8x10(6)/mm(2) (P=.002) for the ASA-treated group that received alpha alpha Hb at the time of endarterectomy (n=8). The prolonged bleeding times induced by ASA were unaffected by the infusion of alpha alpha Hb. Conclusions These data suggest that in a rat endarterectomy model, alpha alpha Hb increases platelet deposition at sites of subintimal injury by binding NO. Increased deposition induced by alpha alpha Hb can be prevented by administration of L-aginine but not by pretreatment with aspirin.