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CARDIOVASCULAR ANESTHESIA

The Effects of a Polymerized Bovine-Derived Hemoglobin Solution in a Rabbit Model of Arterial Thrombosis and Bleeding

Emmanuel Marret, MD^*, Philippe Bonnin, MD PhD, Elisabeth Mazoyer, MD, Bruno Riou, MD PhD^*,, Ted Jacobs, MD^||, Pierre Coriat, MD^*, and Charles-Marc Samama, MD PhD^¶

Departments of *Anesthesiology and Critical Care and Emergency Medicine and Surgery, Hôpital Pitié-Salpêtrière, Paris, France; Department of Functional Investigations and Laboratory of Hematology, Hôpital Lariboisière, Paris, France; ||Biopure Corporation, Cambridge, Massachusetts; and ¶Department of Anesthesiology, Hôpital Avicenne, Bobigny, France

Address correspondence and reprint requests to Charles-Marc Samama, MD, PhD, Département d’Anesthésie-Réanimation, Hôpital Avicenne-125, route de Stalingrad, 93009 Bobigny cedex, France. Address e-mail to cmsamama{at}invivo.edu

	Abstract

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Hemoglobin-based oxygen carriers (HBOCs) have been developed primarily for their oxygenating function and possible use as an alternative to red blood cells during surgery or after major trauma. However, their effect on hemostasis has not been studied extensively. We compared the effects on hemostasis of bovine-derived hemoglobin solution (HBOC-201) with gelatin solution and saline infusion in an experimental model of arterial thrombosis and bleeding. After anesthesia, the Folts model was constructed in 30 rabbits. The common carotid artery was exposed, and a 60% stenosis was induced. A compression injury of the artery was then produced, which triggered a series of cyclic episodes of thrombosis (cyclic flow reductions [CFRs]). After the number of baseline CFRs was counted, animals were assigned randomly to one of three groups (n = 10 each): saline (control), gelatin, or HBOC-201 solution. The effect of studied solutions was observed by recording the number of CFRs during another period and was compared with that of saline. Ear immersion bleeding time was recorded after each CFR period. Gelatin and HBOC-201 had similar effects, manifested by significantly decreased CFRs (from median of 7 to 1 and 6 to 1, respectively) and significantly lengthened bleeding time (from 88 to 98 s and 81 to 102 s, respectively; P < 0.05). Saline infusion had no significant effect on CFRs or bleeding time. HBOC-201 and gelatin had similar effects marked by a reduction in the arterial thrombosis rate and increased bleeding time in rabbits.

IMPLICATIONS: In a rabbit thrombosis and hemorrhagic model, a polymerized bovine-derived hemoglobin solution and a gelatin solution infusion decreased arterial thrombosis and lengthened bleeding time.

	Introduction

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Blood transfusion remains a major public health concern as a result of bacterial, virologic, and immunologic risks and a decline in blood donations (1). Multiple approaches, such as erythropoietin, autologous blood donation, prophylactic treatment with antifibrinolytic drugs, intraoperative and postoperative cell saving, and the use of crystalloids or other intravascular volume expanders, may decrease the need for blood transfusion (2).

Interest in the development of a safe and effective substitute for red blood cells in the form of hemoglobin-based oxygen carriers (HBOC), which do not require typing and cross-matching, has increased substantially in the past decade (1,3). A number of products are under investigation. Because a major clinical indication for HBOCs is the treatment of acute surgical or traumatic blood loss, in which it is desirable to have normal hemostasis without hypercoagulability, there is also continued interest in the effects of HBOCs on coagulation variables. The role of platelet activation by one HBOC was investigated in a rat carotid model, in which Olsen et al. (4) showed that human-derived diaspirin cross-linked hemoglobin increased platelet deposition on injured arterial vessels. However, other preparations of hemoglobin solution have had no effects or have had inhibitory effects on platelet aggregation in vitro (5,6). There have been no reported adverse clinical effects of bovine-derived hemoglobin solution (HBOC-201) related to coagulation, but HBOC-201 solution has not been studied in an animal model of thrombosis. Moreover, interactions between HBOC-201 and hemostasis have been studied only in vitro and on coagulation testing (7,8). In addition, gelatin-based solutions containing hydrolyzed bovine-derived collagen have had wide clinical use in Europe and are generally regarded as safe plasma expanders in the treatment of acute blood loss; they have not been associated with clinical perioperative coagulation problems (9). However, several in vitro studies have indicated that gelatin-based solutions might affect platelet adhesion and clot stability (10–12).

Numerous animal models of arterial thrombosis have been proposed for experimental investigation. The Folts model of platelet arterial thrombosis has gained popularity because it can be reproduced. This model was designed to closely mimic the acute coronary syndromes observed in humans (13,14). Endothelial damage induces platelet accretion and gradual formation of a platelet plug in stenosed arteries. A platelet thrombus embolizes and is then formed again in periodic cyclic flow reductions (CFRs) (Fig. 1). First described in the canine coronary artery, the Folts model was then applied to other arteries, especially the carotid, and in many different species, such as baboons, pigs, and rabbits.

View larger version (18K): [in this window] [in a new window]   Figure 1. Arterial flow recordings. The vessel was injured and stenosed before spontaneous cyclic flow reductions occurred and corresponded to thrombosis, followed by spontaneous embolization (release) and recurrence of all phenomena. No time scale is provided because the time is shorter on the right portion of the figure, to facilitate the reader’s understanding of the thrombosis process. However, the injury and stenosis required <30 s, and every cyclic flow reduction required approximately 3 or 4 min in the control group.

	Methods

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Care of the rabbits and the study were performed in accordance with the regulations of the official edict of the French Ministry of Agriculture. This involved two parts: the application of the Folts model of the carotid arteries of the rabbit and a bleeding model involving liver and spleen sections and ear immersion bleeding time.

This prospective study included 30 male New Zealand rabbits that weighed 2.8 ± 0.2 kg. Anesthesia was induced with IV pentobarbital and was maintained with sodium pentobarbital (10 mg/kg) as required. Tracheotomy and mechanical ventilation were performed (respiratory rate, 45 breaths/min; tidal volume, 20–30 mL). Arterial blood was sampled to adjust ventilatory variables to maintain arterial carbon dioxide partial pressure (PaCO₂) between 30 and 40 mm Hg, and arterial oxygen partial pressure (PaO₂) at >70 mm Hg. Body temperature was recorded continuously by using a rectal probe and was maintained at approximately 38°C with an electric blanket and a warming table. A femoral artery catheter was placed. A blood pressure transducer was connected to the femoral artery, was calibrated, and allowed blood pressure to be recorded continuously. The electric activity of the heart was recorded with an electrocardiogram with five hypodermic electrodes.

The right carotid artery was exposed and carefully isolated over approximately a 2-cm length. A 1.5-mm-diameter precalibrated electromagnetic circular flowprobe (Skalar Instruments, Delft, The Netherlands) was placed around the right common carotid artery on the distal part of the exposed segment and connected to a flowmeter (Model MDL 1401; Skalar Instruments). Zero calibration was obtained directly by occluding the artery with a cotton-tipped swab.

After 10 min of stabilization (baseline flow), a 60% stenosis in the vessel diameter of the right common carotid artery was produced by placing a vascular clamp around the artery in the proximal part of the 2-cm exposed segment. This degree of stenosis was obtained when baseline flow just began to decline (15). It was released after 10 min. An arterial injury with deendothelialization was induced by gently cross-clamping the middle of the exposed segment of the artery three consecutives times within an elapsed period of 10 s. This was accomplished with a Mayo-Hegar needle holder forceps (Harvard Instruments) with three ratchet clicks closed. Thereafter, the 60% stenosis was reinstituted around the carotid injury. This triggered a series of CFRs characterized by repetitive decreases in blood flow, followed by an abrupt spontaneous return of flow to the original levels (Fig. 1). Beginning with the first CFR, the thrombosis lysis process was observed for 20 min (baseline; CFR1). The number of CFRs during this 20-min observation period was noted. If no CFR was observed during this period, arterial injury was repeated at the same place, and a new 20-min period of observation was allowed. If no CFR occurred during this period, injury was induced on the contralateral carotid artery. This first period was common to all animals.

After the first 20-min period (CFR1), animals were randomly allocated to one of the three treatment groups (Fig. 2):

1. Saline group (control): 8 mL/kg of normal saline (Na 154 mmol/L; osmolarity, 308 mOsm/kg) was infused IV for 5 min.
   
2. Gelatin group (GEL): 8 mL/kg of gelatin solution (Plasmion®; Fresenius, France) was infused IV for 5 min. Plasmion® composition was Na, 150 mmol/L, K, 5 mmol/L; Cl, 100 mmol/L; and lactate, 30 mmol/L (osmolarity, 295 mOsm/kg).
   
3. Hemoglobin solution group (HBOC-201): 8 mL/kg of HBOC-201 (Hemopure®; Biopure Corp., Cambridge, MA) was infused IV for 5 min. HBOC-201 solution composition was Na, 140 mmol/L; polymerized bovine-derived hemoglobin, 12–14 g/dL; and methemoglobin, <10%. Osmolarity was 300 mOsm/kg; colloid oncotic pressure (COP) was 25 mm Hg at 13 g/dL; average molecular weight was 250 kDa; P₅₀ (partial pressure of oxygen at which hemoglobin solution is 50% saturated) was 38 mm Hg; endotoxin was < 0.5 EU/mL; and viscosity was 1.3 centipoise at 37°C.
   

View larger version (28K): [in this window] [in a new window]   Figure 2. The thrombosis and bleeding experiment. The three solutions’ effects (saline, gelatin, and bovine-derived hemoglobin solution [HBOC-201]) were assessed by comparing the rate of cyclic flow reduction (CFR) and the ear immersion bleeding time before and after an 8 mL/kg infusion (representing a top-loading infusion of approximately 10% of estimated blood volume). At the end of the study, liver and spleen bleeding were measured. EBT = ear bleeding time.

The CFRs were then recorded during the second period (CFR2).

Two points of measurements were defined: T1 at the end of CFR1 and T2 at the end of CFR2 (Fig. 2). The period between CFR1 and CFR2 (15 min) was identical in the three groups. All the CFR recordings were reviewed blindly by an independent observer (author PB).

The ear immersion bleeding time was measured at the beginning of and after each CFR period. The ear was cleaned and shaved on the external side. A small incision (5 mm x 1 mm deep) was made, and the ear was placed in a beaker containing 20 mL of saline solution maintained at 38°C. Bleeding time was measured until the trickle caused by the incision stopped, as previously reported (16–18).

At the end of the experiment, a xyphopubic laparotomy was performed. The spleen and liver were isolated. These incisions were standardized and were similar in all animals. The spleen was transected at the free border from the lower pole to the mid level. For the liver, a 2-cm section parallel to the plane of the ligament falciform was made between the right and left halves. Bleeding was recorded until it stopped spontaneously. Swabs, placed close to the spleen and the liver before the transection, were weighed to estimate blood loss. The total amount of blood loss (spleen and liver bleeding) was estimated just after the laparotomy and 10 min later, as previously reported (16,17).

Blood samples were obtained at T0, T1, and T2. Blood gases (1 mL per blood sample) were analyzed at T0, T1, and T2. Hematocrit levels were measured, and arterial blood samples were collected on EDTA for platelet counts (5 mL per blood sample) and on 3.8% trisodium citrated tubes (5 mL per blood sample) for prothrombin time, fibrinogen, and platelet aggregation measurements.

Ex vivo platelet aggregation analysis (Pack 4; Platelet Aggregation Chromogenic Kinetic System; Helena Laboratories, Beaumont, TX) was performed on platelet-rich plasma (PRP) and calibrated with platelet-poor plasma. PRP was obtained by centrifuging whole blood at 200g for 10 min at 37°C. Platelet-poor plasma was prepared from the same blood sample by centrifuging blood at 1500g for 20 min. Platelets were counted in PRP to check the homogeneity of the samples. Platelet aggregation was induced by arachidonic acid 5 mg/mL. The increase in light transmission was recorded for 4 min after the aggregating agent (agonist) was added. Aggregation induced by the agonist in PRP was evaluated by measuring light transmission in stimulated PRP, assuming that light transmission was 100% in platelet-poor plasma and 0% in nonstimulated PRP. The maximal intensity of platelet aggregation was defined as the maximal increase in light transmission, and the velocity of platelet aggregation (slope of the curve) was defined as the speed of the increase in light transmission after the aggregating agent was added.

For platelet cyclic guanosine monophosphate (cGMP) measurements, PRP was obtained by centrifugation of whole blood at 200g for 10 min at 37°C, and platelets were counted in each sample. One-milliliter samples of PRP were then centrifuged at 2500g for an additional minute to obtain a platelet pellet. An aliquot of 1 mL of 6% trichloroacetic acid was added to each platelet pellet and shaken for 1 min. Samples were then centrifuged at 9000g for 15 min, and then the aqueous phase was stored at -20°C (19). Supernatant fractions were extracted 4 times with 5 volumes of water-saturated diethyl ether, lyophilized, and assayed for cGMP content by enzyme immunoassay (Amersham, Les Ullis, France). The assay was based on the competition between unlabeled cGMP and a fixed quantity of peroxidase-labeled cGMP for a limited number of binding sites on a cGMP-specific antibody. Platelets were measured with a nonacetylation method; the standard curves ranged from 50 to 12,800 fmol per well. Platelet cGMP was expressed as picomoles per 10⁸ platelets.

Data are expressed as the mean ± SD, except for discrete variables such as CFRs and spleen and liver bleeding, which are expressed as medians with ranges. Several mean values were compared by using two-way analysis of variance, followed by the Scheffé test. Medians of each time point were compared by using, for the treatment assignment, the Kruskal-Wallis test for independent measures, followed, when significant, by a Mann-Whitney U-test with Bonferroni’s correction. For the comparison of time points in each group, data were analyzed by Wilcoxon’s ranked sum test. Group sizes were calculated to observe a 50% decrease in CFRs (with a power of 90% and with an risk of 0.05). All comparisons were two sided. Probability values <0.05 were required to reject the null hypothesis. Statistical analysis was performed with a computer and StatView SE Graphics.

	Results

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Thirty rabbits were included in this study. No significant difference was observed among the three groups for body weight, blood gas variables (pH, PaCO₂, or PaO₂), heart rate, or temperature before and after infusion of the three solutions (Table 1). A significant increase in mean arterial blood pressure was observed in the HBOC-201 group after infusion of the solution (Table 1). Carotid blood flow (CBF) also increased significantly (58%; P < 0.05) after the infusion of the HBOC-201 solution.

In the control group, the number of CFRs was stable all along the experiment. In the GEL and HBOC-201 groups, CFRs were significantly decreased at T2. This decrease was also statistically significant between the control group and the HBOC-201 group (Table 3).

	Discussion

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HBOC-201 induced a significant arterial antithrombotic effect in a rabbit model of injured carotid arteries. This effect was associated with an alteration of primary hemostasis and a lengthening of ear immersion bleeding time. Of note, a similar magnitude of CFR decrease and ear immersion bleeding time lengthening was also reported with the gelatin solution.

Many experimental models of arterial thrombosis have been designed to study the thrombotic process in vivo. We have been using the animal model developed by Folts et al. (13–15,20), which was originally designed in the dog coronary artery. Because of the good reproducibility in the rabbit, we have been using it as an experimental model of platelet arterial thrombosis (16,17,21). The Folts model is a quantitative model of platelet thrombus formation that involves a stenotic artery with intimal injury. These features promote a cyclic platelet activation and accretion into the injured vessel wall. If the vessel is fixed for ultrastructural examination during the nadir of flow reduction during CFR, the stenotic area of the vessel contains a platelet plug (13).

Many factors, such as hypothermia, acidosis, hypercapnia, and hypoxia, partially inhibit platelet aggregation and reduce the formation and migration of the platelet plug (22–24). In our model, we tried to control most of the extrinsic variables that could interfere with the occurrence of arterial thrombosis and bleeding. Erythrocytes have an established role in the development of arterial thrombosis formation. An adequate hematocrit level is necessary to achieve optimum primary hemostasis, and a decrease from 36% to 23% abolished CFR (17). Throughout the study, hematocrit was sufficient to achieve normal CFR and bleeding in our study. All of the values measured at T1 and T2 were in the normal range and were largely sufficient to sustain normal CFR and normal bleeding time (16,17). We observed comparable CFRs and bleeding time in our control group at T1 and T2 and in previous control groups, although no saline infusion was performed in these two studies (16,17). Therefore, saline had no effects in this bleeding and thrombosis model. We infused a comparable volume of HBOC-201 and gelatin, assuming that their volume expansion properties were very close. Conversely, we chose to infuse only 8 mL/kg of saline because this small volume was not supposed to be responsible for any interactions either in the Folts model or the bleeding model. Like mean blood pressure, carotid blood flow (CBF) increased significantly at T2 in the HBOC-201 group. In our thrombosis model, the degree and the length of the stenosis were continuously fixed because of the use of a metallic vascular clamp that was calibrated at the beginning of the experience. Because CBF increases through a fixed surface area of the vessel, we can assume that the blood flow velocities increase from approximately 50% (CBF [mL/min] = S [mm²] x Vmean [mm/min]), and, consequently, wall shear stress ( = 4 x m x Vmean/r), where S = surface of the artery, V = velocity, m = viscosity, and r = radius, also increases from approximately 50% in this group (HBOC-201). Consequently, the shear stress induced by the CBF was increased in the HBOC-201 group at T2, whereas the number of CFRs decreased. Increased shear forces activate and aggregate platelets. The effects of shear stress on the platelet thrombus were studied by Maalej et al. in their model (25). An increase in shear stress induced an activation of platelets. This can also overcome the antithrombotic effect of aspirin in stenosed dog coronary arteries (26). The decrease in CFRs, despite an increase in CBF, is therefore in favor of a direct inhibition of arterial thrombosis by the infusion of HBOC-201.

Ear immersion bleeding time is the only available test that globally assesses primary hemostasis in the rabbit. It has been described previously and validated in this model (16,17,27). Microvascular bleeding, reflected by the spleen and wound bleeding, has been also described (16,17). In our study, we did not observe any lengthening of microvascular bleeding among the three groups despite an interesting increase in bleeding time after infusion in the 2 treated groups. These results could be related because of the limited number of rabbits in the 3 groups. Nevertheless, we observed a logical but nonsignificant trend in increased microvascular bleeding in the GEL and HBOC-201 groups compared with the control group. Because the ear bleeding time is a more standard test used in the rabbit (16,17,27), our results suggest that gelatin and HBOC-201 infusion should be considered to impair primary hemostasis in this species.

Gelatin-based solution has been considered as safe, with no influence (9) or moderate effects (2,28,29) on hemostasis. Hemoglobin solutions are promising alternatives to allogenic transfusion without the need for cross-matching (30). These formulations vary widely in their preclinical and clinical effects; this reflects important differences in their chemical composition, because they are derived from a variety of starting materials and are manufactured by using individualized purification and stabilization methods. Our study was essentially observational to compare the effects of two solutions on hemostasis. Despite a relatively moderate level of hemodilution (10%), CFR decreased from a median of 7 to 1 in the GEL group and 6 to 1 in the HBOC-201 group. These results suggested that a near-maximum effect on the Folts model was obtained with a relatively small dose of HBOC-201 and gelatin. Therefore, it is reasonable to speculate that a greater degree of hemodilution (20%–30% or more) would have completely abolished CFR; the interpretation of this expected effect would be confounded by greater variations in platelet count, fibrinogen, or prothrombin time. For these reasons, only one dose of each solution was studied. Moreover, others studies indicate that gelatin-based solution might affect platelet adhesion and clot stability (10,31). In a clot study by scanning electron microscopy, Mardel et al. (31) showed that the fibrin formed a less extensive mesh in the presence of the gelatin-based solution. Tabuchi et al. (10) found that gelatin reduced both the velocity and the maximum amplitude of ristocetin-induced platelet agglutination and the maximum amplitude of agglutination. In addition, in a crossover study including six healthy volunteers, a 1-L infusion of gelatin resulted in a 1.7-fold increase in bleeding time and revealed a substantial decrease in von Willebrand factor antigen, which was associated with an impairment of ristocetin-induced platelet agglutination (12). Several observations suggest that specific hemoglobin solutions can either increase or decrease thrombosis and that these effects are possibly influenced by various factors, such as nitric oxide, the molecular size of hemoglobin, and the cross-linking methodology (4,6,18). Conjugated bovine hemoglobin cross-linked with adenosine significantly inhibited human platelets (6). HBOC-201 is a room-temperature-stable solution with a capacity for prolonged storage (more than two years). In the Folts model, infusion of HBOC-201 resulted in a decrease of arterial thrombosis, a lengthening of ear immersion bleeding time, and an increase in blood pressure associated with an increase in CBF. The decrease in thrombosis is possibly due to several factors, including small variations in platelet count, hematocrit, and platelet aggregation because of the intrinsic propriety of HBOC-201.

In conclusion, the combination of a bleeding model with the Folts model allowed us to demonstrate that neither gelatin nor HBOC-201 solution resulted in increased platelet aggregation or increased thrombus formation. Conversely, both solutions decreased arterial thrombosis and prolonged bleeding time in the rabbit. These are important findings because abnormal platelet activation and hypercoagulability are undesirable events under most clinical conditions.

	Acknowledgments

 
Emmanuel Marret received a Fellowship grant from the Académie Nationale de Médecine for this project. This work was also supported by the Fonds d’Etudes et de Recherche du Corps Médical des Hopitaux de Paris. The HBOC-201 was a gift from Biopure Corporation.

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