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The effects of ancrod anticoagulation on renal function in sheep
THROMBOSIS RESEARCH Printed in the United States
Vol. 8, pp. 673-682, 1976 Pergamon Press, Inc.
THE EFFECTS OF ANCROD ANTICOAGULATION ON RENAL FUNCTION IN SHEEP R. T. Olivet, M. P. Kaye, and F. G. Knox Cardiovascular Surgical Research Laboratory and Department of Physiology and Biophysics Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901
(Received 11.11.1975; in revised form 10.4.1976. Accepted by Editor A.L. Copley) ABSTRACT An alternative to heparinization for systemic anticoagulation is the use of ancrod, an agent that renders blood incoagulable by removing circulating fibrinogen. Ancrod-induced factors that might alter renal function include changes in blood pressure, blood viscosity, plasma oncotic pressure, and the possibility of embolization of fibrin polymers to the renal vasculature. The effect of ancrod anticoagulation on renal function was studied in sheep by measuring inulin, E-aminohippurate, and osmolar clearances before and after defibrinogenation with ancrod. No adverse effects on renal function were demonstrated. INTRODUCTION For many years, heparin has served as the principal systemic anticoagulant in the treatment of various thrombotic disorders and, more recently, in extracorporeal cardiopulmonary bypass. However, in prolonged extracorporeal circulation, difficulty in maintaining an appropriate therapeutic heparin blood level, variations in patient response to heparin, and the propensity of hypoxic patients on heparin therapy to sustain intracerebral hemorrhages make heparin a less than ideal anticoagulant for use in this setting. An alternative anticoagulant is the purified coagulant fraction of the venom of the Malayan pit viper (Agkistrodon rhodostoma). The generic name adopted by the World Health Organization for this agent is ancrod. In Great Britain, it is known as Arvin (Berks, Ltd., London). In this country, it has been studied as Venacil* (Abbott-38414, Abbott Laboratories, North Chicago, IL). Ancrod is a glycoprotein with a molecular *Kindly supplied by Dr. J. F. Donahoe, Abbott Laboratories, North Chicago, Illinois.
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Weight of 30,000 to 40,000 (1). Its mechanism of action involves the conversion of fibrinogen to fibrin by splitting fibrinopeptide A from the alpha chain of the fibrinogen molecule, while leaving the beta and gamma chains unchanged (2). The resulting non-cross-linked fibrin polymers are highly susceptible to breakdown by the fibrinolytic enzymes and removal by the reticuloendothelial system (3). The hypofibrinogenemia produced by treatment with ancrod is associated with a protracted anticoagulant state but with minimal effects on other hemostatic factors such as Factors V and VIII and platelet number (4-6). A variable effect on platelet aggregation has been reported (6,7). Predictable decreases in plasma fibrinogen and plasminogen levels occur, the latter probably reflecting activation of fibrinolytic enzyme systems. Clinically, ancrod has been used during the past decade to treat deep vein thrombosis, priapism, sickle-cell crisis, and central retinal vein thrombosis, with satisfactory results and few adverse side effects. In 1973, the use of ancrod anticoagulation in prolonged extracorporeal membrane oxygenation in lambs was first reported (8). It was suggested that ancrod may be superior to heparin in this setting because of ease of control and the paucity of hemorrhagic complications. Other studies have demonstrated that blood and plasma viscosities decrease dramatically in hypofibrinogenemia and suggest theoretic possibilities of ancrod therapy to improve flow properties of blood in diseases with low flow rates (9). Should ancrod anticoagulation become a more widely used therapeutic modality, it would be important to have demonstrated any adverse effects on renal function. Although no adverse renal effects have been reported, few specific renal function studies in animals or humans treated with ancrod have been carried out. Ancrod-induced factors that might alter renal function include changes in blood pressure, blood viscosity, plasma osmotic pressure, and the possibility of embolization of fibrin polymers to the renal vasculature. Brentjens and associates (10) observed pyelonephritis-like renal lesions in rabbits as a late effect of diffuse intravascular coagulation induced by thrombin infusion. This renal damage was not associated with abnormalities in urinalysis, serum creatinine, or blood pressure. A mechanism postulated for this damage involves saturation of the reticuloendothelial system by phagocytosis of circulating fibrin polymers, which in turn permits fibrin polymers to lodge in the glomerular capillaries and produce renal ischemia. Because of this observation, Vreeken and van Aken (11) cautioned that even a short episode of defibrination may, under suitable circumstances, cause renal damage, not necessarily seen as an abnormality of the urine. To ascertain whether therapeutic defibrination would cause renal damage, Fedor and associates (12) measured renal blood flow and performed renal clearance studies in dogs treated with ancrod. Little or no effect on renal blood flow or renal hemodynamics was detected. METHODS The renal function of five adult female sheep of mixed breed and weighing from 40 to 55 kg was studied. Each of the
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five ewes underwent the same 2-day experimental protocol: day 1, time control; day 2, the effect of ancrod. Day 1 After having had overnight access to food and water, each ewe was briefly anesthetized by inhalation of ether. During anesthesia, the left carotid artery and left jugular vein were cannulated with polyethylene tubing filled with heparinized saline and occluded by three-way stopcocks. A 20-F balloontipped catheter was inserted transurethrally into the bladder. Each ewe was administered a single intramuscular injection of benzathine penicillin G suspension (1,200,OOO units). As the ewe recovered from the anesthetic, she was tethered in a large rectangular box, open at the top and at one end, with enough freedom to stand or to lie at will. (Each ewe was standing during all pressure measurements and plasma or urine collections.) Free access to water was allowed, but not to food. The arterial neckline was connected to a strain gauge (Statham P23Db), and arterial blood pressure was read directly from a calibrated meter zeroed at right atria1 level. The venous neckline was connected to a constant infusion pump (Harvard) for the administration of drugs and fluids. The urinary catheter was attached to a closed drainage bag. A priming dose of inulin and E-aminohippurate (PAH) was infused rapidly into the jugular vein. The priming dose consisted of approximately 0.8 ml/kg of a 10% inulin in saline solution to which was added 2.25 ml of a 20% solution of PAH. After the priming dose was given, a sustaining solution of inulin-PAH was infused at a constant rate of 1.15 ml/min for the next 90 minutes. The sustaining solution was prepared by adding 10.8 ml of 20% PAH to 3 ml/kg of a 10% inulin solution and sufficient saline to make a final volume of 300 ml. After the first 45 minutes of inulin-PAH infusion, the urine output was collected quantitatively for three consecutive 15-minute periods. From these collections, urine flow was calculated and specimens were saved for measurement of urine osmolality and concentrations of inulin and PAH. Seven and one-half minutes into each 15-minute collection period, a 5-ml sample of blood was drawn from the carotid artery into a heparinized syringe. The hematocrit level of each blood sample was determined, and the blood was centrifuged. The supernatant plasma was aspirated and saved for determination of gross available fibrinogen, inulin concentration, PAH concentration, plasma protein level, and plasma osmolality. Available fibrinogen was detected by a technique described by White et al. (13). This involves introduction of 1 ml of plasma into 5 ml of a standard ancrod solution (1 unit/ml) and observing whether a clot forms. The formation of a clot indicates free fibrinogen. Just before each blood collection, the arterial systolic and diastolic blood pressures were recorded. After the first 45-minute collection period, the inulin-PAH infusion was discontinued and a 5-hour infusion of 500 ml of saline was begun. Occasional small increments of heparinized saline were flushed through the arterial cannula to maintain its patency. At the end of the 5 hours, saline infusion was discontinued, and a complete repetition of the earlier go-minute period of inulin-PAH prime and sustaining solution infusion (with the same
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schedule of blood and urine collection and pressure measurement) was carried out. At the end of the collection period, arterial and venous cannulae were filled with heparin and their stopcocks dead-ended. The urinary catheter was left in place but disconnected from the drainage system. Once again, access to food and water was allowed overnight. Day 2 On the following morning, the necklines were flushed and reconnected, as was the urinary catheter. No anesthetic was required. Infusions, collections, and measurements were carried out exactly as on the previous day, with the exception of the S-hour saline infusion. In its place, a S-hour infusion of ancrod (1 unit/kg in 500 ml of saline) was administered. After that, a "booster dose" of ancrod (1 unit/kg in 25 ml of saline) was infused over a lo-minute period. At the end of the final collection period, the urinary catheter and vascular cannulae were removed. The carotid artery and jugular vein were ligated. To provide histologic documentation of possible embolization of fibrin polymers, particularly to the renal vasculature in ancrod-treated sheep, two sheep were defibrinogenated with ancrod, sacrificed, and immediately examined for gross and microscopic evidence of fibrin embolization. One ewe was given ancrod in a dosage and rate of administration identical to those used on day 2 of the experimental protocol. The second ewe was given a corresponding dose of ancrod, but the drug was infused over a lo-minute period rather than infused over the recommended slow (S-hour) period of administration. Each ewe was sacrificed at the end of treatment by the intravenous administration of a large dose of sodium pentobarbital. Immediate complete postmortem examinations were performed. Calculations Mean blood pressure was calculated from the relationship: Mean pressure (mm Hg) = diastolic pressure + systolic pressure - diastolic pressure. 3 Plasma osmotic pressure (JT)was calculated from the measured plasma protein concentration (c) according to the equation derived by Landis and Pappenheimer (14): u (mm Hg) = 2.1 c Cg/dl) + 0.16 c2 + 0.009 c3. Glomerular filtration rate (GFR) was taken to be equal to the inulin clearance (CI,). Thus: GFR (ml/min) = GIN Cml/min) = 'INv I 'IN in which UIN and PAN are the urine (mg/ml) and plasma (mg/ml) inulin concentrate ns, respectively, and V is the urine flow rate (ml/min).
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Effective renal plasma flow (ERPF) was taken to equal the PAH clearance (C ) calculated from the concentrations of PAH in urine (UPAH, mg/ml?%d plasma (PpAH, mg/ml) and the urine flow rate: 'PAHV ERPF (ml/min) = CpAH (ml/min) = -* 'PAH Renal blood flow (RHF) was calculated from the effective renal plasma flow and the hematocrit level, assuming an extraction ratio of 0.8: RHF (ml/min) = o 8 Ty .
Hi_)'
) was calculated from the measured ~~~:~fi~:~~r~2c~l~~~~M(P~SM) and urine (UOSM) and the urine flow rate: = 'OSM'. COSM W/min 1 'OSM
RFSULTS Results of the study are summarized in Table 1 and represented graphically in Figures 1 through 5. Fibrinogen was present in every plasma sample tested for available fibrinogen before the administration of ancrod. Fibrinogen was absent in every sample tested after ancrod treatment. Defibrinogenation by ancrod seems completely reproducible. Decreases in hematocrit level on the second day (Fig. 1) were seen and were attributable to blood sampling (total of 60 to 72 ml from each ewe over 2-day period), changes in state of hydration, or to a direct effect of the ancrod (day 2 only).
Before saline
After saline
Before ancrod
After ancrod
FIG. 1 Effect of ancrod and saline on hematocrit level of five sheep. Small consistent decreases are seen over the 2-day period. Mean values are indicated by open circles in all figures.
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TABLE 1 Effects* of Ancrod Anticoagulation on Blood and on Renal Function of Sheep Measurement or calculation
Before
Saline After
Before
Ancrod After
Gross available fibrinogen Hematocrit (%) Mean blood pres-
Present
Present
Present
Absent
3U4
3M5
29*4
25*4
77*8
82*8
87*4
84*4
(g/al) Plasma oncotic
7.65*0.45
7.71io.49
7.62kO.39
7.58i0.55
pressure (mm Hg) Glomerular fil-
29.854.3
30.2*2.9
29.5rt2.4
29.453.2
151*29
146*16
141*15
82=134
996*203
935*115
1,017*139
1,692&259
1,714*253
sure (mm Hg) Plasma protein
tration rate (ml/min) Effective renal
166i8
plasma flow (ml/min) Renal blood flow (ml/min) Osmolar clearance (ml/min)
1,536*292 7.73i3.22
1,80M374 6.0M1.19
4.84i1.00
4.30a0.50
*Mean SE (n=5).
Mean blood pressure (Fig. 2) was essentially unchanged on either day of the experiment. As anticipated, plasma protein concentrations (Fig. 3) were not lowered appreciably by the removal of fibrinogen. The contribution of fibrinogen to the total plasma protein is very small compared to that of albumin and the other plasma proteins. This is also reflected, in the negligible change in calculated plasma osmotic pressure after defibrinogenation (Table 1). Glomerular filtration rate (Fig. 4), effective renal plasma flow (Fig. 5), and renal blood flow (Table 1) showed more variability, especially on the control day. No consistent changes were apparent after defibrinogenation. Likewise, osmolar clearance (Table 1) showed no definite trend.
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Before saline
After sol ine
Before ancrod
679
After ancrod
FIG. 2 Effect of ancrod and saline on mean blood pressure of five sheep. No significant changes are noted.
Before saline
After saline
Before ancrod
After ancrod
FIG. 3 Effect of ancrod and saline on plasma protein level of five sheep. No consistent decreases are seen.
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Before saline
After saline
Before ancrod
After ancrod
FIG. 4 Effect of ancrod and saline on glomerular filtration rate of five,sheep. Note inconsistent small increases.
F \
s k p:
k lu
1000
1 Before saline
After saline
Before ancrod
After ancrod
FIG. 5 Effect of ancrod and saline on effective renal plasma flow of five sheep. No consistent trend is noted. All five ewes tolerated the surgical manipulations and drug treatments well and survived in apparent good health. Approximately 1 month after being used in the study, one of the ewes died of causes apparently unrelated to the experiment. Autopsy revealed one normal kidney and one nonfunctional hydronephrotic kidney secondary to long-standing complete unilateral ureteral obstruction by a stone.
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IN SHEEP
681
Doth ewes that were treated with ancrod for histologic studies tolerated the drug administration. The ewe that received aIiCrGd at the recommended dose rate showed no discernible clinical effects. The ewe that received the rapidly administered ancrod, however, showed moderate respiratory distress; the distress seemed to stabilize and not progress until the time of elective sacrifice. Postmortem examination revealed no pathologic changes in either ewe --changes that could be attributable to ancrod treatment, except for scattered petechial hemorrhages in the lungs and subcutaneous tissues of the rapidly treated ewe. No fibrin deposition could be demonstrated microscopically in the renal or pulmonary vasculature of either ewe.
DISCUSSION Since the same five sheep served as both the time controls on day 1 and the treatment group on day 2, it would have been ideal to alternate the sequence in which saline or ancrod was infused. However, alternation was precluded by the fact that, although saline infusion seems to leave no after effects, recovery from ancrod treatment is slow. After cessation of treatment, about 72 hours are required for fibrinogen levels to increase to a range adequate for good clotting, and as much as 2 or 3 weeks may be required for levels to return to a completely normal range. An alternative approach could have been to use different sheep for the controls and treatment group. However, analysis of the results reveals no particular trend that might be attributable to the sequence of treatment. Histologic examination of renal tissue for pathologic changes such as vascular fibrin polymer embolization was negative, even when ancrod was deliberately infused very rapidly. However, in the experimental group, a 5-hour period of infusion of the initial defibrinogenating dose of ancrod was used tc allow the removal of fibrin without risk of overloading the fibrinolytic enzyme and reticuloendothelial systems. This rate of infusion has been shown to be safe in lambs from the standpoint of significant fibrin embolization (8). Analysis of the data in this experiment leads to the Except for a consistent small decrease following conclusions. in hematocrit level in the ancrod-treated group, none of the other measured or calculated parameters demonstrated any Application of the "sign test," that is, significant trend. comparing the number increasing with the number decreasing, revealed no definite trends. Because of this, no more formal statistical analyses were considered appropriate. For the same reason, there seemed to be no rationale for carrying out the measurements in a larger number of animals. Within the limitations of the experimental design of this study and the small number of observations, the study offers some reassurance as to the safety of systemic ancrod anticoagulation in regard to renal function. The study tends to confirm in sheep the findings of Fedor and associates (12) in dogs.
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REFERENCES 1.
ESNOUF, M.P. and TUNNAH, G.W. The isolation and properties of the thrombin-like activity from Ancistrodon rhodostoma venom. Br. J. Haematol. 13, 581, 1967.
2.
HOLLEMAN, W.H. and COEN, L.J. Characterization of peptides released from human fibrinogen by Arvin. Biochim. Biophys. Acta. 200, 587, 1970.
3.
KWAAN, H.C. and BARLOW, G.H. The mechanism of action of a coagulant fraction of Malayan pit viper venom, Arvin, and of Reptilase. Thromb. Diath. Haemorrh. Suppl. 45, 63, 1971.
4.
BELL, W.R., PITNEY, W.R., and GOODWIN, J.F. Therapeutic defibrination in the treatment of thrombotic disease. Lancet. 1, 490, 1968.
5.
RODRIGUEZ-ERDMAN, F. and CARPENTER, C.B. Immunoelectrophoretic studies with Arvin (abstract). Fed. Proc. 29, 624, 1970.
6.
SHARP, A.A., WARREN, B.A., PAXTON, A.M., and ALLINGTON, M.J. Anticoagulant therapy with a purified fraction of Malayan pit viper venom. Lancet. 1, 493, 1968.
7.
PRENTICE, C.R.M., TURPIE, A.G.G., HASSANEIN, A.A., McNICOL, G.P., and DOUGLAS, A.S. Changes in platelet behaviour during arvin therapy. Lancet. 1, 644, 1969.
8.
B;KY, B., BELL, W., MATTHEWS, P., REIDY, T., and WHITE, J.J. Arvin anticoagulation for prolonged extracorporeal membrane oxygenation. Trans. Am. Sot. Artif. Intern. Organs. 19, 56, 1973.
9.
Influence of Arvin on the flow properties of Biorheology. 10, 453, 1973.
EHRLY, .A.M.
blood. 10.
BRENTJENS, J.R.H., VREEKEN, J., FELTKAMP-VROOM, T., and HELDER, A.W. Pyelonephritis-like lesions as a late effect of diffuse intravascular coagulation. Acta Med. Stand. 183, 203, 1968.
Il.
VREEKEN, J., and VAN AKEN, W.G. A new approach to anticoagulant therapy? (Letter to the editor.) Lancet. 1, 653, 1968.
12.
FEDOR, E.J., FRONDYK, H.D., WIEMELER, L.H., and HWANG, K. Effect of ABBOTT-38414 (ancrod) on renal blood flow and clearance, coronary sinus flow and PO and femoral arterial blood flow (abstract). Fed. Proc. 38, 424, 1971.
13.
WHITE, J.J., BUKY, B., KEHRFR, B., REIDY, T., MATTHEWS, P., and BELL, W. Simplified control of ancrod anticoagulation for prolonged extracorporeal bypass. Surg. Forum. 25, ,4.l't3) 1974. and PAPPENHEIMER, J.R. Exchange of substances LANDIS, E.M. through the capillary Walls. In: Handbook of Physiology, Section 2: Circulation. W.F. Hamilton and P. Dow (Eds.). Washington, D.C.: American Physiological Society, 1963, vol. 2, p. 961.
14.
Vol. 8, pp. 673-682, 1976 Pergamon Press, Inc.
THE EFFECTS OF ANCROD ANTICOAGULATION ON RENAL FUNCTION IN SHEEP R. T. Olivet, M. P. Kaye, and F. G. Knox Cardiovascular Surgical Research Laboratory and Department of Physiology and Biophysics Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901
(Received 11.11.1975; in revised form 10.4.1976. Accepted by Editor A.L. Copley) ABSTRACT An alternative to heparinization for systemic anticoagulation is the use of ancrod, an agent that renders blood incoagulable by removing circulating fibrinogen. Ancrod-induced factors that might alter renal function include changes in blood pressure, blood viscosity, plasma oncotic pressure, and the possibility of embolization of fibrin polymers to the renal vasculature. The effect of ancrod anticoagulation on renal function was studied in sheep by measuring inulin, E-aminohippurate, and osmolar clearances before and after defibrinogenation with ancrod. No adverse effects on renal function were demonstrated. INTRODUCTION For many years, heparin has served as the principal systemic anticoagulant in the treatment of various thrombotic disorders and, more recently, in extracorporeal cardiopulmonary bypass. However, in prolonged extracorporeal circulation, difficulty in maintaining an appropriate therapeutic heparin blood level, variations in patient response to heparin, and the propensity of hypoxic patients on heparin therapy to sustain intracerebral hemorrhages make heparin a less than ideal anticoagulant for use in this setting. An alternative anticoagulant is the purified coagulant fraction of the venom of the Malayan pit viper (Agkistrodon rhodostoma). The generic name adopted by the World Health Organization for this agent is ancrod. In Great Britain, it is known as Arvin (Berks, Ltd., London). In this country, it has been studied as Venacil* (Abbott-38414, Abbott Laboratories, North Chicago, IL). Ancrod is a glycoprotein with a molecular *Kindly supplied by Dr. J. F. Donahoe, Abbott Laboratories, North Chicago, Illinois.
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Weight of 30,000 to 40,000 (1). Its mechanism of action involves the conversion of fibrinogen to fibrin by splitting fibrinopeptide A from the alpha chain of the fibrinogen molecule, while leaving the beta and gamma chains unchanged (2). The resulting non-cross-linked fibrin polymers are highly susceptible to breakdown by the fibrinolytic enzymes and removal by the reticuloendothelial system (3). The hypofibrinogenemia produced by treatment with ancrod is associated with a protracted anticoagulant state but with minimal effects on other hemostatic factors such as Factors V and VIII and platelet number (4-6). A variable effect on platelet aggregation has been reported (6,7). Predictable decreases in plasma fibrinogen and plasminogen levels occur, the latter probably reflecting activation of fibrinolytic enzyme systems. Clinically, ancrod has been used during the past decade to treat deep vein thrombosis, priapism, sickle-cell crisis, and central retinal vein thrombosis, with satisfactory results and few adverse side effects. In 1973, the use of ancrod anticoagulation in prolonged extracorporeal membrane oxygenation in lambs was first reported (8). It was suggested that ancrod may be superior to heparin in this setting because of ease of control and the paucity of hemorrhagic complications. Other studies have demonstrated that blood and plasma viscosities decrease dramatically in hypofibrinogenemia and suggest theoretic possibilities of ancrod therapy to improve flow properties of blood in diseases with low flow rates (9). Should ancrod anticoagulation become a more widely used therapeutic modality, it would be important to have demonstrated any adverse effects on renal function. Although no adverse renal effects have been reported, few specific renal function studies in animals or humans treated with ancrod have been carried out. Ancrod-induced factors that might alter renal function include changes in blood pressure, blood viscosity, plasma osmotic pressure, and the possibility of embolization of fibrin polymers to the renal vasculature. Brentjens and associates (10) observed pyelonephritis-like renal lesions in rabbits as a late effect of diffuse intravascular coagulation induced by thrombin infusion. This renal damage was not associated with abnormalities in urinalysis, serum creatinine, or blood pressure. A mechanism postulated for this damage involves saturation of the reticuloendothelial system by phagocytosis of circulating fibrin polymers, which in turn permits fibrin polymers to lodge in the glomerular capillaries and produce renal ischemia. Because of this observation, Vreeken and van Aken (11) cautioned that even a short episode of defibrination may, under suitable circumstances, cause renal damage, not necessarily seen as an abnormality of the urine. To ascertain whether therapeutic defibrination would cause renal damage, Fedor and associates (12) measured renal blood flow and performed renal clearance studies in dogs treated with ancrod. Little or no effect on renal blood flow or renal hemodynamics was detected. METHODS The renal function of five adult female sheep of mixed breed and weighing from 40 to 55 kg was studied. Each of the
v01.a,N0.5
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five ewes underwent the same 2-day experimental protocol: day 1, time control; day 2, the effect of ancrod. Day 1 After having had overnight access to food and water, each ewe was briefly anesthetized by inhalation of ether. During anesthesia, the left carotid artery and left jugular vein were cannulated with polyethylene tubing filled with heparinized saline and occluded by three-way stopcocks. A 20-F balloontipped catheter was inserted transurethrally into the bladder. Each ewe was administered a single intramuscular injection of benzathine penicillin G suspension (1,200,OOO units). As the ewe recovered from the anesthetic, she was tethered in a large rectangular box, open at the top and at one end, with enough freedom to stand or to lie at will. (Each ewe was standing during all pressure measurements and plasma or urine collections.) Free access to water was allowed, but not to food. The arterial neckline was connected to a strain gauge (Statham P23Db), and arterial blood pressure was read directly from a calibrated meter zeroed at right atria1 level. The venous neckline was connected to a constant infusion pump (Harvard) for the administration of drugs and fluids. The urinary catheter was attached to a closed drainage bag. A priming dose of inulin and E-aminohippurate (PAH) was infused rapidly into the jugular vein. The priming dose consisted of approximately 0.8 ml/kg of a 10% inulin in saline solution to which was added 2.25 ml of a 20% solution of PAH. After the priming dose was given, a sustaining solution of inulin-PAH was infused at a constant rate of 1.15 ml/min for the next 90 minutes. The sustaining solution was prepared by adding 10.8 ml of 20% PAH to 3 ml/kg of a 10% inulin solution and sufficient saline to make a final volume of 300 ml. After the first 45 minutes of inulin-PAH infusion, the urine output was collected quantitatively for three consecutive 15-minute periods. From these collections, urine flow was calculated and specimens were saved for measurement of urine osmolality and concentrations of inulin and PAH. Seven and one-half minutes into each 15-minute collection period, a 5-ml sample of blood was drawn from the carotid artery into a heparinized syringe. The hematocrit level of each blood sample was determined, and the blood was centrifuged. The supernatant plasma was aspirated and saved for determination of gross available fibrinogen, inulin concentration, PAH concentration, plasma protein level, and plasma osmolality. Available fibrinogen was detected by a technique described by White et al. (13). This involves introduction of 1 ml of plasma into 5 ml of a standard ancrod solution (1 unit/ml) and observing whether a clot forms. The formation of a clot indicates free fibrinogen. Just before each blood collection, the arterial systolic and diastolic blood pressures were recorded. After the first 45-minute collection period, the inulin-PAH infusion was discontinued and a 5-hour infusion of 500 ml of saline was begun. Occasional small increments of heparinized saline were flushed through the arterial cannula to maintain its patency. At the end of the 5 hours, saline infusion was discontinued, and a complete repetition of the earlier go-minute period of inulin-PAH prime and sustaining solution infusion (with the same
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676
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schedule of blood and urine collection and pressure measurement) was carried out. At the end of the collection period, arterial and venous cannulae were filled with heparin and their stopcocks dead-ended. The urinary catheter was left in place but disconnected from the drainage system. Once again, access to food and water was allowed overnight. Day 2 On the following morning, the necklines were flushed and reconnected, as was the urinary catheter. No anesthetic was required. Infusions, collections, and measurements were carried out exactly as on the previous day, with the exception of the S-hour saline infusion. In its place, a S-hour infusion of ancrod (1 unit/kg in 500 ml of saline) was administered. After that, a "booster dose" of ancrod (1 unit/kg in 25 ml of saline) was infused over a lo-minute period. At the end of the final collection period, the urinary catheter and vascular cannulae were removed. The carotid artery and jugular vein were ligated. To provide histologic documentation of possible embolization of fibrin polymers, particularly to the renal vasculature in ancrod-treated sheep, two sheep were defibrinogenated with ancrod, sacrificed, and immediately examined for gross and microscopic evidence of fibrin embolization. One ewe was given ancrod in a dosage and rate of administration identical to those used on day 2 of the experimental protocol. The second ewe was given a corresponding dose of ancrod, but the drug was infused over a lo-minute period rather than infused over the recommended slow (S-hour) period of administration. Each ewe was sacrificed at the end of treatment by the intravenous administration of a large dose of sodium pentobarbital. Immediate complete postmortem examinations were performed. Calculations Mean blood pressure was calculated from the relationship: Mean pressure (mm Hg) = diastolic pressure + systolic pressure - diastolic pressure. 3 Plasma osmotic pressure (JT)was calculated from the measured plasma protein concentration (c) according to the equation derived by Landis and Pappenheimer (14): u (mm Hg) = 2.1 c Cg/dl) + 0.16 c2 + 0.009 c3. Glomerular filtration rate (GFR) was taken to be equal to the inulin clearance (CI,). Thus: GFR (ml/min) = GIN Cml/min) = 'INv I 'IN in which UIN and PAN are the urine (mg/ml) and plasma (mg/ml) inulin concentrate ns, respectively, and V is the urine flow rate (ml/min).
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Effective renal plasma flow (ERPF) was taken to equal the PAH clearance (C ) calculated from the concentrations of PAH in urine (UPAH, mg/ml?%d plasma (PpAH, mg/ml) and the urine flow rate: 'PAHV ERPF (ml/min) = CpAH (ml/min) = -* 'PAH Renal blood flow (RHF) was calculated from the effective renal plasma flow and the hematocrit level, assuming an extraction ratio of 0.8: RHF (ml/min) = o 8 Ty .
Hi_)'
) was calculated from the measured ~~~:~fi~:~~r~2c~l~~~~M(P~SM) and urine (UOSM) and the urine flow rate: = 'OSM'. COSM W/min 1 'OSM
RFSULTS Results of the study are summarized in Table 1 and represented graphically in Figures 1 through 5. Fibrinogen was present in every plasma sample tested for available fibrinogen before the administration of ancrod. Fibrinogen was absent in every sample tested after ancrod treatment. Defibrinogenation by ancrod seems completely reproducible. Decreases in hematocrit level on the second day (Fig. 1) were seen and were attributable to blood sampling (total of 60 to 72 ml from each ewe over 2-day period), changes in state of hydration, or to a direct effect of the ancrod (day 2 only).
Before saline
After saline
Before ancrod
After ancrod
FIG. 1 Effect of ancrod and saline on hematocrit level of five sheep. Small consistent decreases are seen over the 2-day period. Mean values are indicated by open circles in all figures.
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TABLE 1 Effects* of Ancrod Anticoagulation on Blood and on Renal Function of Sheep Measurement or calculation
Before
Saline After
Before
Ancrod After
Gross available fibrinogen Hematocrit (%) Mean blood pres-
Present
Present
Present
Absent
3U4
3M5
29*4
25*4
77*8
82*8
87*4
84*4
(g/al) Plasma oncotic
7.65*0.45
7.71io.49
7.62kO.39
7.58i0.55
pressure (mm Hg) Glomerular fil-
29.854.3
30.2*2.9
29.5rt2.4
29.453.2
151*29
146*16
141*15
82=134
996*203
935*115
1,017*139
1,692&259
1,714*253
sure (mm Hg) Plasma protein
tration rate (ml/min) Effective renal
166i8
plasma flow (ml/min) Renal blood flow (ml/min) Osmolar clearance (ml/min)
1,536*292 7.73i3.22
1,80M374 6.0M1.19
4.84i1.00
4.30a0.50
*Mean SE (n=5).
Mean blood pressure (Fig. 2) was essentially unchanged on either day of the experiment. As anticipated, plasma protein concentrations (Fig. 3) were not lowered appreciably by the removal of fibrinogen. The contribution of fibrinogen to the total plasma protein is very small compared to that of albumin and the other plasma proteins. This is also reflected, in the negligible change in calculated plasma osmotic pressure after defibrinogenation (Table 1). Glomerular filtration rate (Fig. 4), effective renal plasma flow (Fig. 5), and renal blood flow (Table 1) showed more variability, especially on the control day. No consistent changes were apparent after defibrinogenation. Likewise, osmolar clearance (Table 1) showed no definite trend.
Vo1.8,No.5
ANCROD COAGULATION IN SHEEP
Before saline
After sol ine
Before ancrod
679
After ancrod
FIG. 2 Effect of ancrod and saline on mean blood pressure of five sheep. No significant changes are noted.
Before saline
After saline
Before ancrod
After ancrod
FIG. 3 Effect of ancrod and saline on plasma protein level of five sheep. No consistent decreases are seen.
680
Vol.B,No.5
ANCROD COAGULATION IN SHEEP
Before saline
After saline
Before ancrod
After ancrod
FIG. 4 Effect of ancrod and saline on glomerular filtration rate of five,sheep. Note inconsistent small increases.
F \
s k p:
k lu
1000
1 Before saline
After saline
Before ancrod
After ancrod
FIG. 5 Effect of ancrod and saline on effective renal plasma flow of five sheep. No consistent trend is noted. All five ewes tolerated the surgical manipulations and drug treatments well and survived in apparent good health. Approximately 1 month after being used in the study, one of the ewes died of causes apparently unrelated to the experiment. Autopsy revealed one normal kidney and one nonfunctional hydronephrotic kidney secondary to long-standing complete unilateral ureteral obstruction by a stone.
Vo1.8,No.5
ANCROD
COAGULATION
IN SHEEP
681
Doth ewes that were treated with ancrod for histologic studies tolerated the drug administration. The ewe that received aIiCrGd at the recommended dose rate showed no discernible clinical effects. The ewe that received the rapidly administered ancrod, however, showed moderate respiratory distress; the distress seemed to stabilize and not progress until the time of elective sacrifice. Postmortem examination revealed no pathologic changes in either ewe --changes that could be attributable to ancrod treatment, except for scattered petechial hemorrhages in the lungs and subcutaneous tissues of the rapidly treated ewe. No fibrin deposition could be demonstrated microscopically in the renal or pulmonary vasculature of either ewe.
DISCUSSION Since the same five sheep served as both the time controls on day 1 and the treatment group on day 2, it would have been ideal to alternate the sequence in which saline or ancrod was infused. However, alternation was precluded by the fact that, although saline infusion seems to leave no after effects, recovery from ancrod treatment is slow. After cessation of treatment, about 72 hours are required for fibrinogen levels to increase to a range adequate for good clotting, and as much as 2 or 3 weeks may be required for levels to return to a completely normal range. An alternative approach could have been to use different sheep for the controls and treatment group. However, analysis of the results reveals no particular trend that might be attributable to the sequence of treatment. Histologic examination of renal tissue for pathologic changes such as vascular fibrin polymer embolization was negative, even when ancrod was deliberately infused very rapidly. However, in the experimental group, a 5-hour period of infusion of the initial defibrinogenating dose of ancrod was used tc allow the removal of fibrin without risk of overloading the fibrinolytic enzyme and reticuloendothelial systems. This rate of infusion has been shown to be safe in lambs from the standpoint of significant fibrin embolization (8). Analysis of the data in this experiment leads to the Except for a consistent small decrease following conclusions. in hematocrit level in the ancrod-treated group, none of the other measured or calculated parameters demonstrated any Application of the "sign test," that is, significant trend. comparing the number increasing with the number decreasing, revealed no definite trends. Because of this, no more formal statistical analyses were considered appropriate. For the same reason, there seemed to be no rationale for carrying out the measurements in a larger number of animals. Within the limitations of the experimental design of this study and the small number of observations, the study offers some reassurance as to the safety of systemic ancrod anticoagulation in regard to renal function. The study tends to confirm in sheep the findings of Fedor and associates (12) in dogs.
682
ANCROD COAGULATIONIN SHEEP
Vo1.8,No.5
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2.
HOLLEMAN, W.H. and COEN, L.J. Characterization of peptides released from human fibrinogen by Arvin. Biochim. Biophys. Acta. 200, 587, 1970.
3.
KWAAN, H.C. and BARLOW, G.H. The mechanism of action of a coagulant fraction of Malayan pit viper venom, Arvin, and of Reptilase. Thromb. Diath. Haemorrh. Suppl. 45, 63, 1971.
4.
BELL, W.R., PITNEY, W.R., and GOODWIN, J.F. Therapeutic defibrination in the treatment of thrombotic disease. Lancet. 1, 490, 1968.
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RODRIGUEZ-ERDMAN, F. and CARPENTER, C.B. Immunoelectrophoretic studies with Arvin (abstract). Fed. Proc. 29, 624, 1970.
6.
SHARP, A.A., WARREN, B.A., PAXTON, A.M., and ALLINGTON, M.J. Anticoagulant therapy with a purified fraction of Malayan pit viper venom. Lancet. 1, 493, 1968.
7.
PRENTICE, C.R.M., TURPIE, A.G.G., HASSANEIN, A.A., McNICOL, G.P., and DOUGLAS, A.S. Changes in platelet behaviour during arvin therapy. Lancet. 1, 644, 1969.
8.
B;KY, B., BELL, W., MATTHEWS, P., REIDY, T., and WHITE, J.J. Arvin anticoagulation for prolonged extracorporeal membrane oxygenation. Trans. Am. Sot. Artif. Intern. Organs. 19, 56, 1973.
9.
Influence of Arvin on the flow properties of Biorheology. 10, 453, 1973.
EHRLY, .A.M.
blood. 10.
BRENTJENS, J.R.H., VREEKEN, J., FELTKAMP-VROOM, T., and HELDER, A.W. Pyelonephritis-like lesions as a late effect of diffuse intravascular coagulation. Acta Med. Stand. 183, 203, 1968.
Il.
VREEKEN, J., and VAN AKEN, W.G. A new approach to anticoagulant therapy? (Letter to the editor.) Lancet. 1, 653, 1968.
12.
FEDOR, E.J., FRONDYK, H.D., WIEMELER, L.H., and HWANG, K. Effect of ABBOTT-38414 (ancrod) on renal blood flow and clearance, coronary sinus flow and PO and femoral arterial blood flow (abstract). Fed. Proc. 38, 424, 1971.
13.
WHITE, J.J., BUKY, B., KEHRFR, B., REIDY, T., MATTHEWS, P., and BELL, W. Simplified control of ancrod anticoagulation for prolonged extracorporeal bypass. Surg. Forum. 25, ,4.l't3) 1974. and PAPPENHEIMER, J.R. Exchange of substances LANDIS, E.M. through the capillary Walls. In: Handbook of Physiology, Section 2: Circulation. W.F. Hamilton and P. Dow (Eds.). Washington, D.C.: American Physiological Society, 1963, vol. 2, p. 961.
14.