Ancrod Causes Rapid Thrombolysis in Patients with Acute Stroke VICTOR E. POLLAK, MO, PIA GLAS-GREENWALT, MO, NAND K. WADHWA, MD, STEVEN A. MYRE, PHARMD
ABSTRACT: Clot lysis is desirable in patients with thrombi in arteries and arterioles by a safe rapidly-acting thrombolytic agent. Ancrod cleaves fibrinogen; the resulting circulating ancrod-fibrin stimulates fibrinolysis. Ancrod action and effect were studied in 20 patients with acute developing stroke in a double-blind, placebo-controlled study. Patients were randomly assigned to one of two treatment groups, and received either normal saline or ancrod 0.5 u/kg in normal saline administered as a constantrate intravenous infusion over 6 hours. Subsequent doses of ancrod (or saline placebo) were determined daily thereafter for a total treatment period of 7 days. Neither bleeding nor rethrombosis occurred within the 90 day followup period. That ancrod acted rapidly was shown by a significant decrease in functional plasminogen activator inhibitor (P A-I) within 60 minutes, and by significant elevations of fibrin(ogen) degradation products (FDP) and Ddimer within 3 and 4 hours. The biological effect of fibrinolysis in ancrod infused patients was demonstrated by a greater improvement in stroke score when compared to those infused with saline. KEY INDEXING TERMS: Acute From the ~i~ision of Nephrology and Hypertension, the Department of Medlcme, the Department of Neurology, and the Division of Clinical Pharmacy, University of Cincinnati Medical Center Cin~M~O~ , Supported in part by grants from the Orphan Drug Program, Food and Drug Administration (FD-R-000295J, Research and Educational Funds of Dialysis Clinic, Inc. , and the Monarch Foundation Cincinnati, Ohio. ' The authors thank Judith Spilker, RN, Davis Stroop, Donna Kinnemey~r, Lawrence Healey, Mary Ann Miller, and Robert Eberley for theIr help. The support of Knoll Pharmaceuticals and Dr. Juan Guerero is gratefully acknowledged. Richard Hart, American Diagnostica Inc., kindly supplied ELISA kits for assay of D-dimer and electroimmunodilfusion plates for measurement of Protein C. Dr. Wa~hwa 's present address is the Division of Nephrology and Hypertenswn, Department of Medicine, State University of New York at Stony Brook, Stony Brook, New York, 11794. Reprint requests: Victor E. Pollak, MD, Division of Nephrology and Hypertension, Department of Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio, 45267-0585. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
CHARLES P. OLINGER, MO,
stroke; Fibrinolysis; Ancrod; Tissue Plasminogen Activator; Control Trial. [Am J Med Sci 1990; 299(5):319-325.]
A
drug effective in treating recent acute and lifethreatening thrombosis should be safe and act very rapidly. Heparin is associated with significant bleeding and may have little therapeutic effect. Streptokinase and urokinase can produce rapid clot dissolution, l but cause systemic plasminemia by activating the fibrinolysis system throughout the vasculature; their administration may be associated with a significant incidence of bleeding and re-thrombosis. 2 Tissue plasminogen activator (t-PA) binds to plasminogen associated with fibrin in clot, and activates plasminogen to plasmin which results locally in digestion of fibrin. 3 Recent clinical trials of recombinant t-PA preparations, given in pharmacologic doses, show that exogenously administered t-PA results in rapid clot lysis,4 but may also be followed by re-thrombosis or associated with bleeding due to systemic activation of fibrinolysis. Safety and efficacy of a thrombolytic agent is particularly important in treating cerebral vascular thrombosis. Ancrod, an active principle of the Malayan pit viper (Agkistrodon rhodostoma) venom, cleaves fibrinogen in a thrombin-like fashion and leads to circulation of soluble non-crosslinked "ancrod-fibrin," which appears to stimulate endogenous t-P A release from vessel walls. 5- 7 Fibrinolysis, long inferred from indirect evidence,S occurs relatively early after ancrod is given. 6 ,7,9 Its administration is rarely associated with significant bleeding. 5 - 7,9.lo The initial ancrod dose should be given slowly over several hours because a bolus results in rapid breakdown of a large circulating fibrinogen pool and could overwhelm the reticuloendothelial system, resulting in clots in vessels in glomeruli and lungs. 5 ,6,IO,12.13 Perhaps as a consequence, it was assumed that ancrod could not induce fibrinolysis sufficiently rapidly to treat acute thrombosis in the circulatory bed of a vital organ. During a pilot double-blind randomized control 319
Rapid Thrombolysis
study of ancrod in acute developing stroke,14 we examined serially the effects of ancrod and placebo on the fibrinolytic system. Fibrinolysis occurred rapidly without bleeding. The clinical data demonstrated that effective fibrinolysis during ancrod administration was associated with a favorable clinical response. Material and Methods Patients. Twenty adults, aged 35-80 years, were
studied13 using a protocol and informed consent form approved by the University of Cincinnati Institutional Review Board. Each had had the sudden onset of a focal or multi focal neurologic deficit lasting >30 minutes, persisting to the time of admission, and distinguished from an episode of generalized ischemia and from a transient ischemic attack. Excluded were those who were comatose, had known vasculitis, recent acute myocardial infarction, severe hypertension, renal or hepatic failure, and coagulation abnormalities. Those treated received no other drugs known to affect coagulation, thrombolysis, and platelet aggregation. Cerebral hemorrhage, hemorrhagic infarction, and intracranial mass were excluded by computed tomography. Ancrod or Placebo Infusion. Patients were randomly allocated to receive ancrod, 0.5 units/kg body weight in 250 ml normal saline, or saline placebo, by constant-rate intravenous infusion over 6 hours. As there was no information on the ancrod dose needed to achieve a predetermined fibrinogen level, the dose of ancrod used was guided by previous experience in lupus nephritis,6,7 and was titrated to keep the plasma fibrinogen level at about 1.0 gil. A second infusion was given on the first day when the 6 hour fibrinogen measurement (fibrinogen > 1.80 gil) suggested it was needed to reduce the fibrinogen to the desired level (1.0 gil). For the next 6 days, ancrod or placebo was given intravenously once or twice daily in 50 ml normal saline over 30 minutes, titrated to maintain plasma fibrinogen at approximately 1.0 gil. Coagulation and Fibrinolysis Studies. Blood was drawn from an antecubital vein directly into a plastic syringe using an 18 gauge needle with minimal venous occlusion and free flow. On day 1, blood was taken prior to the start of, at 0.5, 1, 2, 3, 4, 5, and 6 hours during the infusion, and at 12 and 24 hours following the start of the infusion. On days 2-7, one or, in patients who received two infusions, two blood sample(s) were drawn. The final blood sample was drawn on day 8, about 24 hours after the last infusion. Fibrinogen was measured by the method of Clauss,15 plasminogen spectrophotometrically using
the chromogenic substrate S-2251. 16 Total fibrinolytic activity, t-PA activity, and t-PA inhibitor (PAl) activity were measured in plasminogen-rich fibrin plates by methods described previously.17 PA-I activity was also determined by addition of purified t-P A (500-1000 iu/l) to plasma followed by euglobulin fractionation and measurement of residual t-PA activity.lB,19 Alpha2-antiplasmin was measured in plasminogen-poor fibrin plates, and also using the chromogenic substrate S-2251Y T-PA antigen was quantified by an enzyme linked immunosorbent assay (ELISA), using a highly purified preparation oft-PA from melanoma 6/20 cell cultures as standard. IS Fibrin/fibrinogen degradation products (FDP) were assayed with the ThromboWellco test,20 D-dimer using a monoclonal antibody in an ELISA assay system. 21 Protein C was measured by the principle of quantitative electroimmunodiffusion. IB ,22 Statistical Methods. All proteins measured are known to be normally distributed after logarithmic transformation. Data are therefore expressed as geometric means, with values for -1 SD and for +1 SD in parentheses. Differences between pre-infusion values and individual time points post-infusion were compared with the Student t-test for paired samples. Results
Mean changes in the first 24 hours and throughout the 7 days of ancrod and placebo administration are summarized in Figures 1 and 2. Pretreatment FDP levels were normal «16 Itg/ml) in 18/20 subjects. In two, both in the placebo group, the pretreatment FDP was elevated (64 Itg/ml), indicating that spontaneous fibrinolysis had occurred. * Data on the placebo controIs are represented in two groups, those from patients who received ancrod in a single group. Fibrinogen. In patients who received ancrod, the fibrinogen decreased from a pre-infusion geometric mean level of 3.8 (-1 SD 3.09, +1 SD 4.68) gil to 0.98 (0.51,1.86) gil at the end of the 6 hour infusion (Figure 1); intermediate 4- and 5-hour values of 2.29 (1. 78, 2.75) and 1.41 (0.72,2.75) gil were respectively close to and below the lower limit of normal (2.0 gil). Twelve and 24 hours values were 0.44 (0.20, 0.93) and 0.47 (0.29,0.76) gil respectively. Six patients received • Three months later, a diagnosis of inoperable adenocarcinoma, probably of the pancreas was made in one patient. In retrospect, it must have been present when she entered the study. The second patient del'eloped fever thought to be due to and treated with antibiotics for bacterial pneumonia on the second day of infusion. Liver function tests were also abnormal.
Figul"e 1. Fibrinolysis measurements (geometric means) during the first 24 hours of ancrod administration to 10 patients (e - e). Spontaneous fibrinolysis was present prior to the start of infusion in two controls (~-.-.-.~) but not in the other eight (0-- --0). The normal ranges are represented by the horizontal lines. Functional assays were used to measure t-PA, PA-I, and alpha2-AP activities.
320
May 1990 Volume 299 Number 5
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Rapid Thrombolysis
a single ancrod infusion in the first 24 hours; their geometric mean 6, 12, and 24 hour fibrinogen values were 0.93, 0.43, and 0.55 gil. In the four who received two infusions, the values were 1.03, 0.44, and 0.36 gil; retrospective analysis suggests that the target fibrinogen level (1.0 gil) would have been reached within 16 hours of the start of a single infusion of 0.5 units/kg. On days 2-8, the mean fibrinogen was between 0.67 and 1.10 gil, and varied little (Figure 2). The 6 day geometric mean fibrinogen levels were similar in patients who received one (1.14 gil) or two (1.22 gil) ancrod infusions daily. Fibrinogen level rose by t.he time of discharge; 14, 30, and 90 days later, the mean levels were similar to those pretreatment. In controls, the mean pre-infusion fibrinogen of 3.3 (2.70, 4.04) gil changed little until days 3-4, when the mean values increased to slightly above the upper limit of normal (4.0 gil). At 6, 12, and 24 hours, the fibrinogen levels were respectively 2.84 (2.51, 3.21), 3.01 (2.45, 3.71), and 3.08 (2.54, 3.67) gil. FOP and D-Dlmer. In patients treated with ancrod, the FOP level rose from a geometric mean of 0.003 gl I to 0.10, 0.23, 0.69 and 0.66 gil by 3, 4, 5, and 6 hours (Figure 1). At 12 hours, it was still 0.66 gil, but by 24 hours had decreased to 0.35 gil. O-dimer was measured in nine patients, but not at all time points. The levels had increased in all five measured at 3 hours, and in all nine with values at 4 hours. Thus, there was clear proof that fibrinolysis had occurred by 3-4 hours. In 7/9 patients, FOP and O-dimer levels were elevated significantly before the fibrinogen had decreased below the lower limit of normal. Both FOP and D-dimer decreased slowly from 24 hours onward, but were still elevated at the end of treatment (Figure 2). In eight placebo controls, all FOP and O-dimer 2.S ' .0
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322
levels were normal; in the two with spontaneous pretreatment fibrinolysis, both FOP and O-dimer were elevated throughout. TFA, t-PA and PA-I Activity Levels. Total fibrinolytic activity (TF A) changed little. Functional t-P A, within normal limits initially, varied inconsistently both in the first 24 hours and throughout the 7 days of ancrod administration. Values decreased in the first and second hour in the placebo controls, but were within normal limits thereafter. PA-I activity, elevated in 6/10 initially, decreased significantly (p < 0.01) from a pre-infusion mean of 1.52 (0.94, 2.44) to 1.01 (0.57, 1.76) I.U. within 60 minutes ofthe start of the ancrod infusion. Thus, within 1-3 hours of the start of, and thereafter throughout ancrod treatment (Figure 1), PA-I levels were significantly lower than both pre-treatment and placebo controls. There was a slight decrease in PA-I activity levels by day 2 in the eight placebo controls, but they subsequently increased to above the upper limits of normal, and remained there throughout (Figure 2). In the two with spontaneous fibrinolysis, initial and all other PA-I activity levels were in the lower range of normal. Alpha-2-AP Activity, Plasminoge.n, and Protein C. In patients treated with ancrod, the change in plasma alpha-2-AP activity measured by the fibrin plate method was very small, but the means were slightly lower than in controls. A small but greater decrease was noted in alpha-2-AP measured in three patients with the chromogenic substrate S-2251. Plasminogen decreased by about 5% within 30-60 minutes of the start of infusion, and remained low (about 50 percent of initial values) throughout. No significant change occurred in the controls. Protein C levels varied slightly but with no directional change after ancrod administration. The clinical findings will be reported in detail elsewhere. The Scandinavian stroke score (normal 46) of ancrod treated patients improved continuously over 90 days. In mild strokes (score 42-44), ancrod and placebo groups improved (to a score of 44-46). In moderate and severe strokes (mean score 32.5, range 14-40), improvement in the ancrod treated group was three times greater. Taken overall, the stroke score improved by 20% or more in five ancrod treated and in one placebo control. In the ancrod treated patients, there was an inverse linear correlation (r = -0.74, p = 0.013) between the 6 hour change in PA-I and the 90 day change in stroke score; there was no correlation in the placebo controls (r = -0.3, p = 0.3). Discussion Ancrod Administration Results In Rapid Fibrinolysis.
Activation of fibrinolysis while ancrod was given is clearly shown by the changes in PA-I activity, FOP, and O-dimer levels. Clinical improvement, documented with the Scandinavian stroke score, demonstrated clear biologic effects.14 May 1990 Volume 299 Number 5
Pollak et al
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Over the 8 day period, the behavior of measured fibrinolysis components was similar to that in patients with lupus nephritis defined as fibrinolysis responders by the rise in plasma FDP levels to > 1 mg/mI. 6.7.9 Normalization of elevated PA-I activity levels had occurred in these patients when the first measurements were made at 24 hours. In the present study, FDP and D-dimer levels were first elevated significantly by 3 hours, while the PA-I activity level had decreased impressively by 1 hour (Figures 1 and 3). When these components were examined serially, the first clearly detectable change at 1-2 hours was in PA-I activity. This was 1-2 hours before definite changes in FDP and D-dimer (Figure 3) and when the change in fibrinogen was small and inconstant from patient to patient (Figure 4). When ancrod splits fibrinogen, a non cross-linked "ancrod-fibrin" circulates. This in turn stimulates endothelial release of t-PA which activates plasminogen associated with fibrin in clot. A certain proportion oft-PA released in excess binds to its inhibitor (PA-I) in the circulation, leading to decreased PA-I activity. The decrease in PA-I activity within 1 hour ofthe start of ancrod administration is consistent with rapid release of endogenous t-PA. T-PA activation of plasminogen to plasmin at the site of clot leads to FDP and D-dimer release. Free plasmin cannot ordinarily be measured in plasma, as plasmin released after action on clot is bound rapidly to alpha2-antiplasmin. Using the Western blot technique, preliminary observations in THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
two patients demonstrated plasmin-anti-alpha2-AP complexes in the circulation within 60 minutes of starting ancrod infusion. These observations show that ancrod administration leads to very rapid clot dissolution. Dosage of Ancrod. When ancrod was used in control clinical trials in venous thrombosis and peripheral arterial disease, a fixed dose was given and fibrinogen monitored. 13•23 The dose needed to achieve a predetermined fibrinogen level was not determined. In our previous study, ancrod was given once or twice daily to maintain plasma fibrinogen in the range 0.501.0 g/1.6.7.9 The protocol for the present study was based on this experience. In the controls, the fibrinogen on days 4-7 after stroke was higher than on days 1- 3 (Figure 2) . In those receiving ancrod, the relationship between dose and fibrinogen was relatively stable over the 7 day infusion period, but a slightly higher dose was given on days 4-7 than on days 2-3. Retrospective analysis suggests that this was in fact required on days 4-7. Following an initial single 6 hour infusion of 0.5 ~/kg, the predicted single infusion doses of ancrod on days 2-3 and 4-7 are listed in Table 1. This prediction was tested by retrospective analysis of fibrinogen 24 hours after this theoretical dose on six occasions in each of eight patients. Although fibrinogen on days 6 and 7 had risen to 1.95 and 1.94 gil in one patient, prediction was satisfactory on the other 46 occasions. Three patients were given ancrod twice daily. The actual doses were slightly higher than theoretical, but in retrospect these higher doses did not seem to have been
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323
Rapid Thrombolysis
Table 1. Prediction of Doses of Ancrod Required on the Days Following the Initial Infusion
Measured Fibrinogen Level on Prior Day (g/l)
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Dose of Ancrod Predicted to Maintain the Plasma Fibrinogen Level at about 1.0 g/I (units/Kg) Days 2-3
Days 4-7
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needed. Two patients were inadvertently given 3-fold larger initial and subsequent daily infusions without adverse effect. Retrospective analysis suggests that larger doses were unnecessary. Heretofore, we have discussed the ancrod dose required to lower fibrinogen to a pre-determined level. Previous studies have provided strong evidence that ancrod action on clot is indirect, and triggered by circulating "ancrod-fibrin" causing t-PA release from endothelium. The immediate reduction in plasma PA-I activity, even preceding a measurable fall of plasma fibrinogen below the lower limit of normal (Figure 4) and a rise in FDP are strong indicators of early t-PA release. The limited cleavage of plasma fibrinogen to "ancrod-fibrin" during the first hour of ancrod administration may suffice to set the process in motion and induce fibrinolysis. This is followed by conversion of the major portion of the fibrinogen pool to "ancrod-fibrin" and its digestion to FDPs. Once· the plasma fibrinogen is reduced to the target level of 0.50-1.0 gil, its continued conversion on a smaller scale to "ancrod-fibrin" appears to perpetuate fibrinolysis. Safety. In the present study there was no evidence of bleeding or re-thrombosis during ancrod administration. Consistent with published literature lO and our observations in glomerulonephritis with severe hypertension, renal failure and iatrogenic Cushing's syndrome7 •9 •1S this contrasts with experience using thrombolytic therapy with streptokinase or recombinant rt-PA. Re-thrombosis and increased liberation of both fibrinopeptides A and B (FpA, FpB) have been reported with rt-P A administration. 24 - 26 FpA and FpB potentiate platelet aggregation and potently antagonize the platelet inhibiting effect of PGI2 and PGD2 respectively, thus facilitating new thrombus formation. 27 In addition, FpB has many known adverse biologic effects. 2s•29 Exogenously administered t-PAis present in the circulation in pharmacologic quantities. When first given to a patient with a normal or high fibrinogen level, release ofrelatively large amounts of FpA and FpB occurs which may playa role in re-thrombosis. By contrast, ancrod cleaves FpA but not FpB from fibrinogen. When an-
324
crod is first given, two factors appear important in avoiding the re-thrombosis reported with other thrombolytic agents including rt-PA: (1) FPB is not released; and (2) as endogenous and recombinant t- P A probably act identically on fibrinogen and the quantity of t-P A released following ancrod cannot exceed the endothelial capacity and is physiologic, little release of FpA and FpB from fibrinogen occurs. Rethrombosis does not seem to occur during the course of ancrod administration over days and weeks. We have shown previously that spontaneous platelet hyperaggregability prior to ancrod treatment normalized after ancrod was given. 30 After the first 4-6 hours, the amount of FpA and FpB released from fibrinogen must be relatively small because the fibrinogen pool is greatly reduced (Figures 1 and 2). These two effects of ancrod and the physiologic limitation of t-P A availability appear to account, at least in part, for its safety and the rarity of both bleeding and re-thrombosis. References 1. Tiefenbrunn AJ, Sobel BE: The impact of coronary thrombolysis on myocardial infarction. Fibrinolysis 3:1-15,1989. 2. Haber E, Quertermous T, Matsueda GR, Runge MS: Innovative approaches to plasminogen activator therapy. Science 243: 51-56,1989. 3. Kluft C: t-PA in fibrin dissolution and hemostasis, in Kluft C. (ed): Tissue-type Plasminogen Activator (t-PA): Physiological and Clinical Aspects. Boca Raton, Florida, CRC Press, 1988, pp 47-82. 4. Sobel B: Thrombolytic therapy with T-PA, in Kluft C (ed):
Tissue-type Plasminogen Activator (t-PA): Physiological and Clinical Aspects. Boca Raton, Florida, CRC Press, 1988, pp 119-127. 5. Bell WR, Pitney WR, Oakley CM, Goodwin JF: Therapeutic defibrination in the treatment of thrombotic disease. Lancet 1: 490-493,1968. 6. Glas-Greenwalt P, Kant KS, Dosekun AK, Frazier J, Allen C, Pollak VE: Effects of ancrod: Normalization of fibrinolytic enzyme abnormalities in patients with SLE and lupus nephritis. J Lab Clin Med 105:99-107,1985. 7. Kant KS, Pollak VE, Dosekun AK, Glas-Greenwalt P, Weiss MA, Glueck HI: Lupus glomerulonephritis with thrombosis and abnormal fibrinolysis: Effect of ancrod. J Lab Clin Med 105:77-88,1985. 8. Bell WR: Defibrinogenating enzymes, in Colman RW, Hirsh J, Marder VJ, Salzman EW, (ed): Hemostasis and Thrombosis. Philadelphia, J . B. Lippincott, 1982, pp 1013-1027. 9. Kim S, Wadwha N, Kant KS, Pollak VE, Glas-Greenwalt P, Weiss MA, Hong CD: Fibrinolysis in glomerulonephritis treated with aricrod: Renal functional, immunologic, and histopathologic effects. Quart J Med 69:875-895, 1988. 10. Latallo ZS: Retrospective study on complications and adverse effects of treatment with thrombinlike enzymes. Thromb Haemost 50:604-609, 1983. 11. Muller-Berghaus G, Hocke M: Production of the generalized Shwartzman reaction in rabbits by ancrod ('Arvin') infusion and endotoxin injection. BrJ Haemat 25:111-122,1973. 12. Muller-Berghaus G, Mann B: Precipitation of ancrod-induced soluble fibrin by aprotinin and norepinephrine. Thromb Res 2: 305-322, 1973. 13. Sharp AA, Warren BA, Paxton AM, Allington MJ: Anticoagulant therapy with a purified fraction of Malayan pit viper venom. Lancet 1:493-499, 1968. 14. Olinger CP, Brott TG, Barsen WG, Hedges JR, Glas-Greenwalt P, Pollak VE, Spilker J, Eberle R: Use of ancrod in acute May 1990 Volume 299 Number 5
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15. 16. 17. 18.
19.
20. 21.
or progressing ischemic cerebral infarction. Ann Emerg Med 17:1208-1209, 1988. Clauss A: Gerinnungphysiologische Schnell methode zur Bestimmung des Fibrinogens. Acta Haemat 17:237-246, 1957. Friberger P, Knos M, Gustavsson S, Aurell L, Claeson G: Methods of determination of plasmin, antiplasmin, and plasminogen by means of S-2251. Haemostasis 7:138-145,1978. Glas-Greenwalt P, Kant KS, Allen C, Pollak VE: Fibrinolysis in health and disease: Abnormalities in systemic lupus erythematosus. J Lab Clin Med 104:962-976, 1984. Glas-Greenwalt P, Hall JM, Panke T, Kant KS, Allen C, Pollak VE: Fibrinolysis in health and disease: Abnormal levels of plasminogen activator, plasminogen activator inhibitor, and protein C in thrombotic thrombocytopenic purpura. J Lab Clin Med 108:415-422, 1986. Juhan-Vague I, Meerman B, Ailland MF, Collen D: Plasma levels of a specific inhibitor of tissue-type plasminogen activator (and urokinase) in normal and pathological conditions. Thromb Res 33:523-530, 1984. Garvey MB, Black IN: The detection of fibrinogen/fibrin degradation products by means of a new antibody-coated latex particle. J Clin Path 25:680-682, 1972. Whitaker AN, Elms MJ, Masci PP, Bundesen PG, Rylatt DB, Webber AJ, Bunce IH: Measurement of crosslinked fibrin derivatives in plasma: An immunoassay using monoclonal antibodies. J Clin Path 37:882-887,1984.
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
22. Laurell CB: Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem 15:4552,1966. 23. Bell WR, Pitney WR, Goodwin JF: Therapeutic defibrination in the treatment of thrombotic disease. Lancet 1:490-493, 1968. 24. Loscalzo J, Braunwald E: Tissue plasminogen activator. N Engl J Med 319:925-931, 1988. 25. Owen J, Friedman KD, Grossman BA, Wilkins C, Berke AD, Powers ER: Thrombolytic therapy with tissue plasminogen activator or streptokinase induces transient thrombin activity. Blood 72:616-620,1988. 26. Weitz JI, Cruickshank MK, Thong B, Leslie B, Levine MM, Ginsberg J, Eckhardt T: Human tissue-type plasminogen activator releases fibrinopeptides A and B from fibrinogen. J Clin Invest 82:1700-1707,1988. 27. Moore PK, Hussaini I, Bhardwaj R: Effect of fibrinopeptides A and B on human and rat platelet aggregation in vitro. Eur J Pharmacol153:231-238, 1988. 28. Moore PK, Hussaini I, Bhardwaj R: Cardiovascular effects of fibrinopeptide Br J Pharm Pharmacol40:558-561, 1988. 29. Senior RM, Skogen WF, Griffin GL, Wilner GD: Effects of fibrinogen derivatives upon the inflammatory response: Studies with human fibrinopeptide BrJ Clin Invest 77:1014-1019,1988. 30. Pollak VE, Glueck HI, Weiss MA, Lebron-Berges A, Miller MA: Defibrination with ancrod in glomerulonephritis: Effects on clinical and histologic findings and on blood coagulation. Am J Nephrol2:195-207, 1983.
325
ABSTRACT: Clot lysis is desirable in patients with thrombi in arteries and arterioles by a safe rapidly-acting thrombolytic agent. Ancrod cleaves fibrinogen; the resulting circulating ancrod-fibrin stimulates fibrinolysis. Ancrod action and effect were studied in 20 patients with acute developing stroke in a double-blind, placebo-controlled study. Patients were randomly assigned to one of two treatment groups, and received either normal saline or ancrod 0.5 u/kg in normal saline administered as a constantrate intravenous infusion over 6 hours. Subsequent doses of ancrod (or saline placebo) were determined daily thereafter for a total treatment period of 7 days. Neither bleeding nor rethrombosis occurred within the 90 day followup period. That ancrod acted rapidly was shown by a significant decrease in functional plasminogen activator inhibitor (P A-I) within 60 minutes, and by significant elevations of fibrin(ogen) degradation products (FDP) and Ddimer within 3 and 4 hours. The biological effect of fibrinolysis in ancrod infused patients was demonstrated by a greater improvement in stroke score when compared to those infused with saline. KEY INDEXING TERMS: Acute From the ~i~ision of Nephrology and Hypertension, the Department of Medlcme, the Department of Neurology, and the Division of Clinical Pharmacy, University of Cincinnati Medical Center Cin~M~O~ , Supported in part by grants from the Orphan Drug Program, Food and Drug Administration (FD-R-000295J, Research and Educational Funds of Dialysis Clinic, Inc. , and the Monarch Foundation Cincinnati, Ohio. ' The authors thank Judith Spilker, RN, Davis Stroop, Donna Kinnemey~r, Lawrence Healey, Mary Ann Miller, and Robert Eberley for theIr help. The support of Knoll Pharmaceuticals and Dr. Juan Guerero is gratefully acknowledged. Richard Hart, American Diagnostica Inc., kindly supplied ELISA kits for assay of D-dimer and electroimmunodilfusion plates for measurement of Protein C. Dr. Wa~hwa 's present address is the Division of Nephrology and Hypertenswn, Department of Medicine, State University of New York at Stony Brook, Stony Brook, New York, 11794. Reprint requests: Victor E. Pollak, MD, Division of Nephrology and Hypertension, Department of Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio, 45267-0585. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
CHARLES P. OLINGER, MO,
stroke; Fibrinolysis; Ancrod; Tissue Plasminogen Activator; Control Trial. [Am J Med Sci 1990; 299(5):319-325.]
A
drug effective in treating recent acute and lifethreatening thrombosis should be safe and act very rapidly. Heparin is associated with significant bleeding and may have little therapeutic effect. Streptokinase and urokinase can produce rapid clot dissolution, l but cause systemic plasminemia by activating the fibrinolysis system throughout the vasculature; their administration may be associated with a significant incidence of bleeding and re-thrombosis. 2 Tissue plasminogen activator (t-PA) binds to plasminogen associated with fibrin in clot, and activates plasminogen to plasmin which results locally in digestion of fibrin. 3 Recent clinical trials of recombinant t-PA preparations, given in pharmacologic doses, show that exogenously administered t-PA results in rapid clot lysis,4 but may also be followed by re-thrombosis or associated with bleeding due to systemic activation of fibrinolysis. Safety and efficacy of a thrombolytic agent is particularly important in treating cerebral vascular thrombosis. Ancrod, an active principle of the Malayan pit viper (Agkistrodon rhodostoma) venom, cleaves fibrinogen in a thrombin-like fashion and leads to circulation of soluble non-crosslinked "ancrod-fibrin," which appears to stimulate endogenous t-P A release from vessel walls. 5- 7 Fibrinolysis, long inferred from indirect evidence,S occurs relatively early after ancrod is given. 6 ,7,9 Its administration is rarely associated with significant bleeding. 5 - 7,9.lo The initial ancrod dose should be given slowly over several hours because a bolus results in rapid breakdown of a large circulating fibrinogen pool and could overwhelm the reticuloendothelial system, resulting in clots in vessels in glomeruli and lungs. 5 ,6,IO,12.13 Perhaps as a consequence, it was assumed that ancrod could not induce fibrinolysis sufficiently rapidly to treat acute thrombosis in the circulatory bed of a vital organ. During a pilot double-blind randomized control 319
Rapid Thrombolysis
study of ancrod in acute developing stroke,14 we examined serially the effects of ancrod and placebo on the fibrinolytic system. Fibrinolysis occurred rapidly without bleeding. The clinical data demonstrated that effective fibrinolysis during ancrod administration was associated with a favorable clinical response. Material and Methods Patients. Twenty adults, aged 35-80 years, were
studied13 using a protocol and informed consent form approved by the University of Cincinnati Institutional Review Board. Each had had the sudden onset of a focal or multi focal neurologic deficit lasting >30 minutes, persisting to the time of admission, and distinguished from an episode of generalized ischemia and from a transient ischemic attack. Excluded were those who were comatose, had known vasculitis, recent acute myocardial infarction, severe hypertension, renal or hepatic failure, and coagulation abnormalities. Those treated received no other drugs known to affect coagulation, thrombolysis, and platelet aggregation. Cerebral hemorrhage, hemorrhagic infarction, and intracranial mass were excluded by computed tomography. Ancrod or Placebo Infusion. Patients were randomly allocated to receive ancrod, 0.5 units/kg body weight in 250 ml normal saline, or saline placebo, by constant-rate intravenous infusion over 6 hours. As there was no information on the ancrod dose needed to achieve a predetermined fibrinogen level, the dose of ancrod used was guided by previous experience in lupus nephritis,6,7 and was titrated to keep the plasma fibrinogen level at about 1.0 gil. A second infusion was given on the first day when the 6 hour fibrinogen measurement (fibrinogen > 1.80 gil) suggested it was needed to reduce the fibrinogen to the desired level (1.0 gil). For the next 6 days, ancrod or placebo was given intravenously once or twice daily in 50 ml normal saline over 30 minutes, titrated to maintain plasma fibrinogen at approximately 1.0 gil. Coagulation and Fibrinolysis Studies. Blood was drawn from an antecubital vein directly into a plastic syringe using an 18 gauge needle with minimal venous occlusion and free flow. On day 1, blood was taken prior to the start of, at 0.5, 1, 2, 3, 4, 5, and 6 hours during the infusion, and at 12 and 24 hours following the start of the infusion. On days 2-7, one or, in patients who received two infusions, two blood sample(s) were drawn. The final blood sample was drawn on day 8, about 24 hours after the last infusion. Fibrinogen was measured by the method of Clauss,15 plasminogen spectrophotometrically using
the chromogenic substrate S-2251. 16 Total fibrinolytic activity, t-PA activity, and t-PA inhibitor (PAl) activity were measured in plasminogen-rich fibrin plates by methods described previously.17 PA-I activity was also determined by addition of purified t-P A (500-1000 iu/l) to plasma followed by euglobulin fractionation and measurement of residual t-PA activity.lB,19 Alpha2-antiplasmin was measured in plasminogen-poor fibrin plates, and also using the chromogenic substrate S-2251Y T-PA antigen was quantified by an enzyme linked immunosorbent assay (ELISA), using a highly purified preparation oft-PA from melanoma 6/20 cell cultures as standard. IS Fibrin/fibrinogen degradation products (FDP) were assayed with the ThromboWellco test,20 D-dimer using a monoclonal antibody in an ELISA assay system. 21 Protein C was measured by the principle of quantitative electroimmunodiffusion. IB ,22 Statistical Methods. All proteins measured are known to be normally distributed after logarithmic transformation. Data are therefore expressed as geometric means, with values for -1 SD and for +1 SD in parentheses. Differences between pre-infusion values and individual time points post-infusion were compared with the Student t-test for paired samples. Results
Mean changes in the first 24 hours and throughout the 7 days of ancrod and placebo administration are summarized in Figures 1 and 2. Pretreatment FDP levels were normal «16 Itg/ml) in 18/20 subjects. In two, both in the placebo group, the pretreatment FDP was elevated (64 Itg/ml), indicating that spontaneous fibrinolysis had occurred. * Data on the placebo controIs are represented in two groups, those from patients who received ancrod in a single group. Fibrinogen. In patients who received ancrod, the fibrinogen decreased from a pre-infusion geometric mean level of 3.8 (-1 SD 3.09, +1 SD 4.68) gil to 0.98 (0.51,1.86) gil at the end of the 6 hour infusion (Figure 1); intermediate 4- and 5-hour values of 2.29 (1. 78, 2.75) and 1.41 (0.72,2.75) gil were respectively close to and below the lower limit of normal (2.0 gil). Twelve and 24 hours values were 0.44 (0.20, 0.93) and 0.47 (0.29,0.76) gil respectively. Six patients received • Three months later, a diagnosis of inoperable adenocarcinoma, probably of the pancreas was made in one patient. In retrospect, it must have been present when she entered the study. The second patient del'eloped fever thought to be due to and treated with antibiotics for bacterial pneumonia on the second day of infusion. Liver function tests were also abnormal.
Figul"e 1. Fibrinolysis measurements (geometric means) during the first 24 hours of ancrod administration to 10 patients (e - e). Spontaneous fibrinolysis was present prior to the start of infusion in two controls (~-.-.-.~) but not in the other eight (0-- --0). The normal ranges are represented by the horizontal lines. Functional assays were used to measure t-PA, PA-I, and alpha2-AP activities.
320
May 1990 Volume 299 Number 5
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Rapid Thrombolysis
a single ancrod infusion in the first 24 hours; their geometric mean 6, 12, and 24 hour fibrinogen values were 0.93, 0.43, and 0.55 gil. In the four who received two infusions, the values were 1.03, 0.44, and 0.36 gil; retrospective analysis suggests that the target fibrinogen level (1.0 gil) would have been reached within 16 hours of the start of a single infusion of 0.5 units/kg. On days 2-8, the mean fibrinogen was between 0.67 and 1.10 gil, and varied little (Figure 2). The 6 day geometric mean fibrinogen levels were similar in patients who received one (1.14 gil) or two (1.22 gil) ancrod infusions daily. Fibrinogen level rose by t.he time of discharge; 14, 30, and 90 days later, the mean levels were similar to those pretreatment. In controls, the mean pre-infusion fibrinogen of 3.3 (2.70, 4.04) gil changed little until days 3-4, when the mean values increased to slightly above the upper limit of normal (4.0 gil). At 6, 12, and 24 hours, the fibrinogen levels were respectively 2.84 (2.51, 3.21), 3.01 (2.45, 3.71), and 3.08 (2.54, 3.67) gil. FOP and D-Dlmer. In patients treated with ancrod, the FOP level rose from a geometric mean of 0.003 gl I to 0.10, 0.23, 0.69 and 0.66 gil by 3, 4, 5, and 6 hours (Figure 1). At 12 hours, it was still 0.66 gil, but by 24 hours had decreased to 0.35 gil. O-dimer was measured in nine patients, but not at all time points. The levels had increased in all five measured at 3 hours, and in all nine with values at 4 hours. Thus, there was clear proof that fibrinolysis had occurred by 3-4 hours. In 7/9 patients, FOP and O-dimer levels were elevated significantly before the fibrinogen had decreased below the lower limit of normal. Both FOP and D-dimer decreased slowly from 24 hours onward, but were still elevated at the end of treatment (Figure 2). In eight placebo controls, all FOP and O-dimer 2.S ' .0
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Figure 2. Fibrinolysis measurements (geometric means) during and after the 7 days of ancrod administration in 9 patients (e - e). Spontaneous fibrinolysis was present prior to the start of infusion in two controls (~-. - --~) but not in the other eight (0--- -0). The normal ranges are represented by the horizontal lines. A functional assay was used to measure PA-I activity_
322
levels were normal; in the two with spontaneous pretreatment fibrinolysis, both FOP and O-dimer were elevated throughout. TFA, t-PA and PA-I Activity Levels. Total fibrinolytic activity (TF A) changed little. Functional t-P A, within normal limits initially, varied inconsistently both in the first 24 hours and throughout the 7 days of ancrod administration. Values decreased in the first and second hour in the placebo controls, but were within normal limits thereafter. PA-I activity, elevated in 6/10 initially, decreased significantly (p < 0.01) from a pre-infusion mean of 1.52 (0.94, 2.44) to 1.01 (0.57, 1.76) I.U. within 60 minutes ofthe start of the ancrod infusion. Thus, within 1-3 hours of the start of, and thereafter throughout ancrod treatment (Figure 1), PA-I levels were significantly lower than both pre-treatment and placebo controls. There was a slight decrease in PA-I activity levels by day 2 in the eight placebo controls, but they subsequently increased to above the upper limits of normal, and remained there throughout (Figure 2). In the two with spontaneous fibrinolysis, initial and all other PA-I activity levels were in the lower range of normal. Alpha-2-AP Activity, Plasminoge.n, and Protein C. In patients treated with ancrod, the change in plasma alpha-2-AP activity measured by the fibrin plate method was very small, but the means were slightly lower than in controls. A small but greater decrease was noted in alpha-2-AP measured in three patients with the chromogenic substrate S-2251. Plasminogen decreased by about 5% within 30-60 minutes of the start of infusion, and remained low (about 50 percent of initial values) throughout. No significant change occurred in the controls. Protein C levels varied slightly but with no directional change after ancrod administration. The clinical findings will be reported in detail elsewhere. The Scandinavian stroke score (normal 46) of ancrod treated patients improved continuously over 90 days. In mild strokes (score 42-44), ancrod and placebo groups improved (to a score of 44-46). In moderate and severe strokes (mean score 32.5, range 14-40), improvement in the ancrod treated group was three times greater. Taken overall, the stroke score improved by 20% or more in five ancrod treated and in one placebo control. In the ancrod treated patients, there was an inverse linear correlation (r = -0.74, p = 0.013) between the 6 hour change in PA-I and the 90 day change in stroke score; there was no correlation in the placebo controls (r = -0.3, p = 0.3). Discussion Ancrod Administration Results In Rapid Fibrinolysis.
Activation of fibrinolysis while ancrod was given is clearly shown by the changes in PA-I activity, FOP, and O-dimer levels. Clinical improvement, documented with the Scandinavian stroke score, demonstrated clear biologic effects.14 May 1990 Volume 299 Number 5
Pollak et al
6HA
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Over the 8 day period, the behavior of measured fibrinolysis components was similar to that in patients with lupus nephritis defined as fibrinolysis responders by the rise in plasma FDP levels to > 1 mg/mI. 6.7.9 Normalization of elevated PA-I activity levels had occurred in these patients when the first measurements were made at 24 hours. In the present study, FDP and D-dimer levels were first elevated significantly by 3 hours, while the PA-I activity level had decreased impressively by 1 hour (Figures 1 and 3). When these components were examined serially, the first clearly detectable change at 1-2 hours was in PA-I activity. This was 1-2 hours before definite changes in FDP and D-dimer (Figure 3) and when the change in fibrinogen was small and inconstant from patient to patient (Figure 4). When ancrod splits fibrinogen, a non cross-linked "ancrod-fibrin" circulates. This in turn stimulates endothelial release of t-PA which activates plasminogen associated with fibrin in clot. A certain proportion oft-PA released in excess binds to its inhibitor (PA-I) in the circulation, leading to decreased PA-I activity. The decrease in PA-I activity within 1 hour ofthe start of ancrod administration is consistent with rapid release of endogenous t-PA. T-PA activation of plasminogen to plasmin at the site of clot leads to FDP and D-dimer release. Free plasmin cannot ordinarily be measured in plasma, as plasmin released after action on clot is bound rapidly to alpha2-antiplasmin. Using the Western blot technique, preliminary observations in THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
two patients demonstrated plasmin-anti-alpha2-AP complexes in the circulation within 60 minutes of starting ancrod infusion. These observations show that ancrod administration leads to very rapid clot dissolution. Dosage of Ancrod. When ancrod was used in control clinical trials in venous thrombosis and peripheral arterial disease, a fixed dose was given and fibrinogen monitored. 13•23 The dose needed to achieve a predetermined fibrinogen level was not determined. In our previous study, ancrod was given once or twice daily to maintain plasma fibrinogen in the range 0.501.0 g/1.6.7.9 The protocol for the present study was based on this experience. In the controls, the fibrinogen on days 4-7 after stroke was higher than on days 1- 3 (Figure 2) . In those receiving ancrod, the relationship between dose and fibrinogen was relatively stable over the 7 day infusion period, but a slightly higher dose was given on days 4-7 than on days 2-3. Retrospective analysis suggests that this was in fact required on days 4-7. Following an initial single 6 hour infusion of 0.5 ~/kg, the predicted single infusion doses of ancrod on days 2-3 and 4-7 are listed in Table 1. This prediction was tested by retrospective analysis of fibrinogen 24 hours after this theoretical dose on six occasions in each of eight patients. Although fibrinogen on days 6 and 7 had risen to 1.95 and 1.94 gil in one patient, prediction was satisfactory on the other 46 occasions. Three patients were given ancrod twice daily. The actual doses were slightly higher than theoretical, but in retrospect these higher doses did not seem to have been
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323
Rapid Thrombolysis
Table 1. Prediction of Doses of Ancrod Required on the Days Following the Initial Infusion
Measured Fibrinogen Level on Prior Day (g/l)
1.50
Dose of Ancrod Predicted to Maintain the Plasma Fibrinogen Level at about 1.0 g/I (units/Kg) Days 2-3
Days 4-7
0.15 0.25 0.35
0.18 0.30 0.42
needed. Two patients were inadvertently given 3-fold larger initial and subsequent daily infusions without adverse effect. Retrospective analysis suggests that larger doses were unnecessary. Heretofore, we have discussed the ancrod dose required to lower fibrinogen to a pre-determined level. Previous studies have provided strong evidence that ancrod action on clot is indirect, and triggered by circulating "ancrod-fibrin" causing t-PA release from endothelium. The immediate reduction in plasma PA-I activity, even preceding a measurable fall of plasma fibrinogen below the lower limit of normal (Figure 4) and a rise in FDP are strong indicators of early t-PA release. The limited cleavage of plasma fibrinogen to "ancrod-fibrin" during the first hour of ancrod administration may suffice to set the process in motion and induce fibrinolysis. This is followed by conversion of the major portion of the fibrinogen pool to "ancrod-fibrin" and its digestion to FDPs. Once· the plasma fibrinogen is reduced to the target level of 0.50-1.0 gil, its continued conversion on a smaller scale to "ancrod-fibrin" appears to perpetuate fibrinolysis. Safety. In the present study there was no evidence of bleeding or re-thrombosis during ancrod administration. Consistent with published literature lO and our observations in glomerulonephritis with severe hypertension, renal failure and iatrogenic Cushing's syndrome7 •9 •1S this contrasts with experience using thrombolytic therapy with streptokinase or recombinant rt-PA. Re-thrombosis and increased liberation of both fibrinopeptides A and B (FpA, FpB) have been reported with rt-P A administration. 24 - 26 FpA and FpB potentiate platelet aggregation and potently antagonize the platelet inhibiting effect of PGI2 and PGD2 respectively, thus facilitating new thrombus formation. 27 In addition, FpB has many known adverse biologic effects. 2s•29 Exogenously administered t-PAis present in the circulation in pharmacologic quantities. When first given to a patient with a normal or high fibrinogen level, release ofrelatively large amounts of FpA and FpB occurs which may playa role in re-thrombosis. By contrast, ancrod cleaves FpA but not FpB from fibrinogen. When an-
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crod is first given, two factors appear important in avoiding the re-thrombosis reported with other thrombolytic agents including rt-PA: (1) FPB is not released; and (2) as endogenous and recombinant t- P A probably act identically on fibrinogen and the quantity of t-P A released following ancrod cannot exceed the endothelial capacity and is physiologic, little release of FpA and FpB from fibrinogen occurs. Rethrombosis does not seem to occur during the course of ancrod administration over days and weeks. We have shown previously that spontaneous platelet hyperaggregability prior to ancrod treatment normalized after ancrod was given. 30 After the first 4-6 hours, the amount of FpA and FpB released from fibrinogen must be relatively small because the fibrinogen pool is greatly reduced (Figures 1 and 2). These two effects of ancrod and the physiologic limitation of t-P A availability appear to account, at least in part, for its safety and the rarity of both bleeding and re-thrombosis. References 1. Tiefenbrunn AJ, Sobel BE: The impact of coronary thrombolysis on myocardial infarction. Fibrinolysis 3:1-15,1989. 2. Haber E, Quertermous T, Matsueda GR, Runge MS: Innovative approaches to plasminogen activator therapy. Science 243: 51-56,1989. 3. Kluft C: t-PA in fibrin dissolution and hemostasis, in Kluft C. (ed): Tissue-type Plasminogen Activator (t-PA): Physiological and Clinical Aspects. Boca Raton, Florida, CRC Press, 1988, pp 47-82. 4. Sobel B: Thrombolytic therapy with T-PA, in Kluft C (ed):
Tissue-type Plasminogen Activator (t-PA): Physiological and Clinical Aspects. Boca Raton, Florida, CRC Press, 1988, pp 119-127. 5. Bell WR, Pitney WR, Oakley CM, Goodwin JF: Therapeutic defibrination in the treatment of thrombotic disease. Lancet 1: 490-493,1968. 6. Glas-Greenwalt P, Kant KS, Dosekun AK, Frazier J, Allen C, Pollak VE: Effects of ancrod: Normalization of fibrinolytic enzyme abnormalities in patients with SLE and lupus nephritis. J Lab Clin Med 105:99-107,1985. 7. Kant KS, Pollak VE, Dosekun AK, Glas-Greenwalt P, Weiss MA, Glueck HI: Lupus glomerulonephritis with thrombosis and abnormal fibrinolysis: Effect of ancrod. J Lab Clin Med 105:77-88,1985. 8. Bell WR: Defibrinogenating enzymes, in Colman RW, Hirsh J, Marder VJ, Salzman EW, (ed): Hemostasis and Thrombosis. Philadelphia, J . B. Lippincott, 1982, pp 1013-1027. 9. Kim S, Wadwha N, Kant KS, Pollak VE, Glas-Greenwalt P, Weiss MA, Hong CD: Fibrinolysis in glomerulonephritis treated with aricrod: Renal functional, immunologic, and histopathologic effects. Quart J Med 69:875-895, 1988. 10. Latallo ZS: Retrospective study on complications and adverse effects of treatment with thrombinlike enzymes. Thromb Haemost 50:604-609, 1983. 11. Muller-Berghaus G, Hocke M: Production of the generalized Shwartzman reaction in rabbits by ancrod ('Arvin') infusion and endotoxin injection. BrJ Haemat 25:111-122,1973. 12. Muller-Berghaus G, Mann B: Precipitation of ancrod-induced soluble fibrin by aprotinin and norepinephrine. Thromb Res 2: 305-322, 1973. 13. Sharp AA, Warren BA, Paxton AM, Allington MJ: Anticoagulant therapy with a purified fraction of Malayan pit viper venom. Lancet 1:493-499, 1968. 14. Olinger CP, Brott TG, Barsen WG, Hedges JR, Glas-Greenwalt P, Pollak VE, Spilker J, Eberle R: Use of ancrod in acute May 1990 Volume 299 Number 5
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or progressing ischemic cerebral infarction. Ann Emerg Med 17:1208-1209, 1988. Clauss A: Gerinnungphysiologische Schnell methode zur Bestimmung des Fibrinogens. Acta Haemat 17:237-246, 1957. Friberger P, Knos M, Gustavsson S, Aurell L, Claeson G: Methods of determination of plasmin, antiplasmin, and plasminogen by means of S-2251. Haemostasis 7:138-145,1978. Glas-Greenwalt P, Kant KS, Allen C, Pollak VE: Fibrinolysis in health and disease: Abnormalities in systemic lupus erythematosus. J Lab Clin Med 104:962-976, 1984. Glas-Greenwalt P, Hall JM, Panke T, Kant KS, Allen C, Pollak VE: Fibrinolysis in health and disease: Abnormal levels of plasminogen activator, plasminogen activator inhibitor, and protein C in thrombotic thrombocytopenic purpura. J Lab Clin Med 108:415-422, 1986. Juhan-Vague I, Meerman B, Ailland MF, Collen D: Plasma levels of a specific inhibitor of tissue-type plasminogen activator (and urokinase) in normal and pathological conditions. Thromb Res 33:523-530, 1984. Garvey MB, Black IN: The detection of fibrinogen/fibrin degradation products by means of a new antibody-coated latex particle. J Clin Path 25:680-682, 1972. Whitaker AN, Elms MJ, Masci PP, Bundesen PG, Rylatt DB, Webber AJ, Bunce IH: Measurement of crosslinked fibrin derivatives in plasma: An immunoassay using monoclonal antibodies. J Clin Path 37:882-887,1984.
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22. Laurell CB: Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem 15:4552,1966. 23. Bell WR, Pitney WR, Goodwin JF: Therapeutic defibrination in the treatment of thrombotic disease. Lancet 1:490-493, 1968. 24. Loscalzo J, Braunwald E: Tissue plasminogen activator. N Engl J Med 319:925-931, 1988. 25. Owen J, Friedman KD, Grossman BA, Wilkins C, Berke AD, Powers ER: Thrombolytic therapy with tissue plasminogen activator or streptokinase induces transient thrombin activity. Blood 72:616-620,1988. 26. Weitz JI, Cruickshank MK, Thong B, Leslie B, Levine MM, Ginsberg J, Eckhardt T: Human tissue-type plasminogen activator releases fibrinopeptides A and B from fibrinogen. J Clin Invest 82:1700-1707,1988. 27. Moore PK, Hussaini I, Bhardwaj R: Effect of fibrinopeptides A and B on human and rat platelet aggregation in vitro. Eur J Pharmacol153:231-238, 1988. 28. Moore PK, Hussaini I, Bhardwaj R: Cardiovascular effects of fibrinopeptide Br J Pharm Pharmacol40:558-561, 1988. 29. Senior RM, Skogen WF, Griffin GL, Wilner GD: Effects of fibrinogen derivatives upon the inflammatory response: Studies with human fibrinopeptide BrJ Clin Invest 77:1014-1019,1988. 30. Pollak VE, Glueck HI, Weiss MA, Lebron-Berges A, Miller MA: Defibrination with ancrod in glomerulonephritis: Effects on clinical and histologic findings and on blood coagulation. Am J Nephrol2:195-207, 1983.
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