Production of the Generalized Shwartzman Reaction in Rabbits by Ancrod (''Arvin'') Infusion and Endotoxin Injection

S ummary . The effect of the coagulant fraction from Agkistrodon rhodostoma venom (Arvin = ancrod) on the generation of microthrombi was studied in rabbits. Ancrod infusion caused hypofibrinogenaemia and induced the formation of circulating fibrin monomer complexes, but microthrombi were not detected in the glomerular capillaries. If, however, endotoxin was injected into ancrod-infused rabbits, 73% of the animals showed microclots in the glomerular capillaries typical of the generalized Shwartzman reaction. These microclots exhibited antigenic characteristics of fibrinogen derivatives. Injection of endotoxin only did not produce glomerular capillary thrombosis and the mean fibrinogen levels did not decrease significantly, although fibrin monomer complexes could be detected in all the rabbits. Generation of microthrombosis by ancrod infusion and endotoxin injection could not be prevented by treating the animals with heparin. Thus endotoxin mediated the generation of microclots in ancrod-infused rabbits by a mechanism different from its procoagulant activity. Heparin treatment interfered neither with the decrease in fibrinogen levels caused by ancrod infusion nor with the fall in the platelet counts caused by endotoxin injection.     

Ancrod causes rapid thrombolysis in patients with acute stroke

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 mu/kg in normal saline administered as a constant-rate 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 re-thrombosis occurred within the 90 day follow-up period. That ancrod acted rapidly was shown by a significant decrease in functional plasminogen activator inhibitor (PA-I) within 60 minutes, and by significant elevations of fibrin(ogen) degradation products (FDP) and D-dimer 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.     

The fibrinolytic response to ancrod therapy: characterization of fibrinogen and fibrin degradation products

Ancrod is a purified coagulant venom which renders blood incoagulable by cleaving fibrinopeptide A (FPA) from fibrinogen, but the mechanism involved in the clearance of fibrin from the circulation is unknown. To investigate the fibrinolytic response to ancrod, and to increase understanding of clearance mechanisms, six patients with peripheral vascular disease causing claudication were infused with ancrod at 2 u/kg over 6 h followed by 2 u/kg at 12 h intervals for 38 h. Venous blood samples were taken at time 0, 3, 6, 25 and 49 h for assay of fibrinogen (Fbg), fibrinopeptide A (FPA), total fibrin(ogen) degradation products (TDP), fibrin degradation products (FbDP), fibrinogen degradation products (FgDP), cross-linked fibrin degradation products (XL-FDP), tissue plasminogen activator (tPA), urinary type plasminogen activator (u-PA), plasminogen, α₂ antiplasmin (α₂AP) and plasminogen activator inhibitor-1 (PAI-1). Fibrinogen (median and range) was 2.3 (1.4–3.90) g/l at time 0 and thereafter was undetectable. FPA rose from 2.5 (1.8–3.6) to 600 and 188 pmol/l at 3 h and 6 h and remained elevated. TDP, FbDP and FgDP increased greatly following ancrod while there was no evidence of XL-FDP. The surprising increase in FgDP during defibrination suggests either that fibrinogen is digested following its incorporation into circulating fibrin protofibrils or that some of the fibrin subunits in the photofibril retain one of the two fibrinopeptide A's. tPA and uPA remained unchanged. Plasminogen fell from 125 (100–155)% to 79 (40–118)% at 49 h and α₂AP fell from 91 (75–107)% to 24 (10–35)% at 49 h. The level of PAI-1 was depressed during defibrination, with the exception of the 6 h data. The results demonstrate that ancrod removes FPA from fibrinogen to produce non-cross-linked (soluble) fibrin. This is cleared from the circulation without evidence of an increase in the circulating activities of the plasminogen activators, tPA or UK, but with evidence of plasminogen activation and consumption.     

Comparison of polymerization of ancrod and thrombin fibrin monomers.

The polymerization of thrombin and ancrod fibrin monomers was studied with a standardized technique that evaluated turbidity changes and protein incorporation into the clot. Ancrod fibrin monomers were found to polymerize more slowly and form less turbid clots (at identical protein concentrations). Changes in ionic strength and pH influences ancrod fibrin monomer polymerization to a greater extent than thrombin fibrin monomer polymerization. Benzyltriethylammonium chloride was shown to be a potent inhibitor of fibrin monomer polymerization, with a greater inhibitory effect on ancrod fibrin monomers than on thrombin fibrin monomers. The differences between ancrod and thrombin fibrin may play a role in the infrequent thrombotic complications reported with ancrod therapy.     

Plasminogen activator inhibitor: a regulator of ancrod-induced fibrin deposition in rabbits

Plasma levels of a fast-acting plasminogen activator inhibitor (PAI), which neutralizes both tissue plasminogen activator (t-PA) and urokinase, are markedly increased in endotoxin-treated rabbits. The ability of this inhibitor to prevent the fibrinolysis that occurs after a thrombogenic stimulus was investigated in a rabbit model. Normal and endotoxin-treated male New Zealand rabbits were infused with ancrod, an enzyme that causes noncrosslinked fibrin formation in vivo. Ancrod stimulated t-PA activity by 90% in normal rabbits and caused hypofibrinogenemia but did not increase PAI levels or induce fibrin deposition in target organs. Rabbits injected with endotoxin (10 micrograms/kg) showed an increase in PAI from less than 1 to 32 U/mL 4 hours later. When ancrod was infused at this time, 90% of the rabbits developed renal fibrin thrombi. Fibrin deposition was recorded in 40% of the rabbits that received a lower dose of endotoxin (1.0 microgram/kg) and had a PAI level of 14 U/ml at the time of ancrod infusion 4 hours later. Fibrin deposition did not occur in the endotoxin-treated rabbits that received normal saline. These data suggest that high levels of PAI inhibit fibrinolysis in vivo, thereby promoting fibrin clot deposition following a thrombogenic stimulus.     

Progress of fibrinolysis during tumor necrosis factor infusions in humans. Concomitant increase in tissue-type plasminogen activator, plasminogen activator inhibitor type-1, and fibrin(ogen) degradation products.

Several investigators have reported that tumor necrosis factor (TNF) can alter the production of plasminogen activator type-1 (PAI-1) and plasminogen activators (PAs) by endothelial cells in vitro. We have examined the in vivo effects of recombinant human TNF administration on fibrinolysis as assessed by parameters in plasma during a 24-hour period of continuous TNF infusion to 17 cancer patients with active disease. The plasma levels of PAI activity increased sevenfold after 3 and 24 hours of TNF infusion. This was the result of an increase of PAI-1 antigen; PAI-2 antigen was not detectable. Plasma concentrations of tissue-type PA (t-PA) antigen increased twofold to fivefold after 3 and 24 hours of TNF infusion, whereas urokinase-type PA antigen levels in plasma remained unaltered. After 3 hours of TNF infusion the plasma levels of alpha 2-antiplasmin were slightly decreased, 5% on average, suggesting that fibrinolysis continued. After 24 hours of TNF infusion a highly significant increase in fibrin- plus fibrinogen-degradation products, and separately of fibrin degradation products and fibrinogen degradation products, was found. This indicates that fibrinolysis persisted, at least partly, in the presence of high levels of PAI activity. Whereas PAI-1 production increased, t-PA production by human endothelial cells in vitro remains unaltered or even decreases on TNF addition. It has been shown previously that TNF infusion in our patients results in thrombin and fibrin generation. Therefore, it is possible that thrombin, not TNF, is the actual stimulus for t-PA production in our patients. We speculate that fibrin is formed during TNF infusions and that plasmin is generated by t-PA action immediately on the initial formation of (soluble) fibrin molecules. Such a process may explain the generation of degradation products of both fibrin and fibrinogen during infusion of TNF in patients.     

SDS Polyacrylamide Gel Characterization of Serum FDP Produced in Response to Ancrod and Streptokinase/Plasminogen Infusions in Man

S ummary . The serum FDP produced in response to defibrination with ancrod and to thrombolytic therapy with intermittent streptokinase/plasminogen infusion have been characterized using a method of solid phase immuno-precipitation followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Using this method it is possible to distinguish between the plasmin degradation products of fibrinogen and factor XIIIa induced crosslinked fibrin. During ancrod administration, fragments of similar mobility to fibrinogen fragments X, Y, D and E are present in serum samples taken 24 h after the initiation of treatment, while subsequent samples contain mainly fragments with the same mobility as fibrinogen fiagment D. The intermittent nature of the streptokinase/plasminogen infusions produces fibrinogen fragments X, Y, D and E in the serum 1 h after each daily streptokinase/plasminogen administration. An early clearance of fragment E from the circulation of patients receiving either ancrod or streptokinase/plasminogen infusions accords with the results of other investigations. The presence of the D dimer fragment, which is produced only by plasmin lysis of factor XIIIa crosslinked fibrin, could not be conclusively confirmed in either group of patients.     

The role of granulocytes in the activation of intravascular coagulation and the precipitation of soluble fibrin by endotoxin

This study examines the role of neutrophils (PMN) in the pathogenesis of endotoxin-induced microclot formation. It is intended to clarify whether granulocytes are involved in endotoxin-induced activation of intravascular coagulation (generation of soluble fibrin) and/or in endotoxin-induced precipitation of soluble fibrin. Precipitation of soluble fibrin was achieved by injection of endotoxin into ancrod- infused rabbits with circulating soluble fibrin (first model). Activation of intravascular coagulation was elicited by two intravenous injections of endotoxin into rabbits (second model). Seventy-two and ninety-six hours after injection of nitrogen mustard, leukopenic rabbits had PMN counts between 0 and 50 cells per mul. Neutropenia did not prevent the occurrence of glomerular microclots after infusion of ancrod and injection of endotoxin (first model). Neutropenia influenced neither the decrease in mean fibrinogen concentrations nor the drop in mean platelet counts after ancrod and endotoxin administration. In contrast to the first model, neutropenia prevented the occurrence of glomerular microclots and of circulating soluble fibrin after two injections of endotoxin (second model). It did not, however, protect rabbits from the decrease in mean platelet counts after endotoxin administration. These data indicate that granulocytes are involved in endotoxin-induced activation of intravascular coagulation and the production of soluble fibrin but are not essential to endotoxin-induced precipitation of soluble fibrin.     

The resistance of fibrinogen and soluble fibrin monomer in blood to degradation by a potent plasminogen activator derived from cadaver limbs.

The effect of a cadaver-derived vascular plasminogen activator (VA) on the degradation of fibrinogen, soluble fibrin monomer, and fibrin was studied and compared with the effect of equivalent fibrinolytic potencies of streptokinase (SK), urokinase (UK), and plasmin. The proteolytic activity of the three activators and plasmin was determined by a standard fibrin plate assay and was expressed in CTA units from a UK reference curve. Fibrinogen degradation was measured by clottable protein determinations and by an electrophoretic technique sensitive to small changes in the molecular weight of fibrinogen. When VA was incubated in plasma, no degradation of fibrinogen occurred, whereas rapid fibrinolysis took place after the plasma was clotted. By contrast, equivalent potencies of SK, UK, and plasmin caused extensive fibrinogenolysis. Since the plasmin added and that formed by the three activators had equivalent fibrinolytic activity, the failure of VA to induce fibrinogen degradation was attributed to antiactivators rather than antiplasmins. VA activity in plasma was consumed by clotting, whereas the antiactivator activity remained in the serum, suggesting dissociation of the VA-antiactivator complex on the fibrin clot. Fibrinogen and its soluble derivatives resisted degradation by VA in plasma because a solid phase appeared necessary for the complex to dissociate. The findings indicated that the degradation of fibrinogen or soluble fibrin in blood as a result of plasminogen activation by VA was unlikely to occur due to a large excess of antiactivator activity. Alternative pathways for their catabolism are discussed.     

Role of plasminogen activator inhibitor-1 in promoting fibrin deposition in rabbits infused with ancrod or thrombin

The role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well established. The present study compared the efficacy of thrombin or ancrod, a venom-derived enzyme that clots fibrinogen, to induce fibrin formation in rabbits with elevated PAI-1 levels. One set of male New Zealand rabbits received intravenous endotoxin to increase endogenous PAI-1 activity followed by a 1-hour infusion of ancrod or thrombin; another set of normal rabbits received intravenous human recombinant PAI-1 (rPAI-1) during an infusion of ancrod or thrombin. Thirty minutes after the end of the infusion, renal fibrin deposition was assessed by histopathology. Animals receiving endotoxin, rPAI-1, ancrod, or thrombin alone did not develop renal thrombi. All endotoxin-treated rabbits developed fibrin deposition when infused with ancrod (n = 4) or thrombin (n = 6). Fibrin deposition occurred in 7 of 7 rabbits receiving both rPAI-1 and ancrod and in only 1 of 6 receiving rPAI-1 and thrombin (P < .01). In vitro, thrombin but not ancrod was inactivated by normal rabbit plasma and by purified antithrombin III or thrombomodulin. The data indicate that elevated levels of PAI-1 promote fibrin deposition in rabbits infused with ancrod but not with thrombin. In endotoxin-treated rabbits, fibrin deposition that occurs with thrombin infusion may be caused by decreased inhibition of procoagulant activity and not increased PAI-1 activity.     

Interference of Lysed Fibrin with Coagulation: a Quantifying Approach Using Isotopic Techniques

S ummary . Coagulation of plasma fibrinogen in the presence of fibrin degradation products was studied in rabbits treated with ancrod (Arvin), Two techniques were employed: the first, based on trace-labelling of plasma samples with labelled fibrinogen in vitro , was to measure the degree to which solubilized fibrin inhibited coagulation; in the second, two differently labelled fibrinogens were used, one injected intravenously and the other added in vitro , by which estimation of fibrinogen in serum relative to the amount of circulating breakdown products was attempted.
The first technique is considered to provide reliable information about the extent of defective coagulation, and hence the proportion of masked fibrinogen present in serum. Because of the difficulty in obtaining resolution between fibrinogen and its breakdown products in clots, the second technique is thought to be more suitable for comparative studies rather than for measurements in absolute terms.
High doses of hydrocortisone administered to rabbits for several days prior to ancrod significantly reduced the speed of the dissolution of microclots. The data obtained are consistent with the hypothesis that the hormone interfered with fibrinolysis at the plasminogen activator level.     

Kinetic characterization of a saturable pathway for rapid clearance of circulating fibrin monomer

The mechanism of clearance of circulating fibrin monomer was investigated in rabbits through (1) study of decay in plasma concentrations of 125I-labeled monomers with variant fibrinopeptide content and (2) concurrent analysis of decay of the monomers relative to coinjected 131I-fibrinogen. Under the conditions employed, essentially all of the fibrin became distributed in a soluble form in plasma and decayed independently of the coinjected fibrinogen. Among the species of fibrin studied, monomer lacking fibrinopeptide A alone (alpha-fibrin) underwent very rapid clearance by a saturable mechanism that was not evident in relatively sluggish clearance of monomer lacking either fibrinopeptide B alone (beta-fibrin) or both fibrinopeptides A and B (alpha beta-fibrin). Decay of alpha-fibrin conformed with a kinetic mechanism involving first-order permeation of the fibrin into extravascular space at a rate equivalent to that of permeation of fibrinogen; unlike fibrinogen, however, the alpha-fibrin underwent immediate absorption in parallel with permeation (t1/2 = 2.6 hours) at doses below an apparent saturating level of 3 mg/kg. At doses near the absorptive limit, the uptake accompanying permeation diminished as in a second-order kinetic mechanism, and at very high doses the plasma decay of the alpha-fibrin approached that of fibrinogen. The beta- and alpha beta-fibrins also permeated extravascular space in parallel with fibrinogen, but absorption proceeded sluggishly (t1/2 = 11 and 16 hours, respectively) at low doses and did not change with increasing dose. The uniquely rapid and saturable clearance of alpha-fibrin is suggested to involve uptake through the fibrin aggregation site that is blocked by fibrinopeptide A in fibrinogen and beta-fibrin and by tight binding to fibrinogen in soluble complexes formed by alpha beta-fibrin. A corollary of this hypothesis is that rapid uptake depends on dissociability of fibrin complexes for access to the aggregation site, a mechanism that is just the converse of uptake through aggregation.     

Detection of soluble intermediates of the fibrinogen-fibrin conversion using erythrocytes coated with fibrin monomers

The presence of minimal amounts of fibrinogen-fibrin intermediates in human plasma was visualized by an agglutination reaction of glutaraldehyde-treated human erythrocytes coated with purified fibrin monomers. A degree of monomer coating was established which produced erythrocytes not agglutinated by normal plasma but by plasma containing minimal amounts of soluble complexes of fibrinogen with fibrin monomers. Under standardized conditions of coating, erythrocyte concentration, temperature, pH, and incubation time, the agglutination time varied with the ratio of soluble fibrin to fibrinogen in plasma. The test was sensitive down to a soluble fibrin concentration of 0.675% of the plasma fibrinogen concentration. Early fibrinogen and fibrin degradation products (FDP) in the plasma led to a prolongation of the agglutination time at a concentration of more than 16 mg/100 ml. Late FDP in a concentration of 100 mg/100 ml did not convert a positive test to negative. The test was not affected by heparin and protamine at concentrations of up to 12.5 and 50 NIH units/ml, respectively.     

Some Effects of Splenectomy in Thalassaemia Major

S ummary . The fibrin digestion products which appear in serum of patients having treatment with ancrod have been compared with derivatives of human fibrinogen produced by plasmin by means of immunoelectrophoresis, immunodiffusion and gel filtration techniques. In ancrod-treated patients the large molecular weight derivatives X and Y, as well as fragments D and E were seen 4 hr after the start of therapy. By 24 hr the main serum component was fragment D with low concentrations of large molecular weight derivatives still present, but fragment E had been cleared from the circulation. The identity of these fragments to the known products of fibrinogen proteolysis by plasmin has been confirmed. It is suggested that the initial degradation of fibrin micro-clots produced in the circulation during ancrod infusion is mediated by the fibrinolytic enzyme system.     

Ancrod decreases the frequency of cyclic flow variations and causes thrombolysis following acute coronary thrombosis

The potential use of ancrod, a purified isolate from the venom of the Malaysian pit viper, Agkistrodon rhodostoma, in decreasing the frequency of cyclic flow variations in severely stenosed canine coronary arteries and causing thrombolysis of an acute coronary thrombus induced by a copper coil was evaluated. Open-chest, anesthetized dogs were used. Ancrod was given intravenously (8 U/kg) over 1 hour and caused a significant reduction in the frequency of cyclic flow variations (5.8 +/- 0.7 to 3.6 +/- 0.8 cyclic flow variations per 30 minutes, p less than 0.05), whereas control animals failed to decrease the frequency of their cyclic flow variations over the same time period (5.3 +/- 0.3 to 5.0 +/- 0.4 cyclic flow variations per 30-minute period). Twenty-seven dogs had a coronary thrombus induced by a copper coil positioned directly in a major coronary artery; of these, four died of ventricular fibrillation prior to treatment, eight received an infusion of saline and showed no thrombolysis over 5 hours, and three died of ventricular fibrillation during the initial part of an intravenous infusion of ancrod. The remaining 12 dogs received ancrod intravenously (16 U/kg); six demonstrated lysis of the coronary thrombus (mean time to lysis, 65 +/- 20 minutes). The concentrations of ancrod used in these studies produced a severe decrease in systemic fibrinogen concentration and a significant decrease in the inhibitor of plasminogen activator levels. Thus, ancrod decreases the frequency of cyclic flow variations in stenosed canine coronary arteries and may cause coronary thrombolysis in approximately 50% of animals within 65 +/- 20 minutes of its intravenous administration.     

Validity of enzyme-linked immunosorbent assays of cross-linked fibrin degradation products as a measure of clot lysis

Concentrations of cross-linked fibrin degradation products (XL-FDPs) in plasma, measured by enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies (MAbs) raised against fragment D-dimer of cross-linked fibrin, increase when patients are given fibrinolytic agents. Whether XL-FDPs derive from circulating cross-linked fibrin polymers in plasma, compared with clot-associated fibrin, has been questioned because increases in XL-FDP are measured by some assays after fibrinolysis in vitro in the absence of clot. We characterized the source of XL-FDP immunoreactivity in plasma of patients with acute myocardial infarction and ischemic heart disease and the response to plasminogen activation in vitro induced by pharmacological concentrations of tissue-type plasminogen activator (t-PA) and streptokinase. XL-FDPs were measured with two different ELISA. One, "pan-specific tag ELISA," was based on a capture MAb specific for XL-FDP and a tag MAb that recognizes an epitope exposed in the fragment D region of both fibrin and fibrinogen, whereas the other, "fibrin-specific tag ELISA," was based on a capture and tag MAbs both specific for fibrin. After plasminogen activation was induced in vitro in plasma from patients with myocardial infarction, increased concentrations of XL-FDP were measured by the pan-specific tag ELISA; however, concentrations measured with the fibrin-specific tag ELISA were not increased. To determine the mechanism for this discrepancy, plasma was subjected to immunoadsorption with a MAb specific for fragment D-dimer before and after in vitro activation of the fibrinolytic system and immunoblotting with a fragment D-dimer-specific MAb and with the pan-specific MAb. Increased concentrations of fragment D-dimer, as well as fibrinogen fragment D at high concentrations, were recognized by the specific MAb. Non-cross-linked fragments were also shown by immunoblotting with the pan-specific MAb to coprecipitate with cross-linked fibrin fragments. This suggested the increased concentrations of XL-FDP measured by the pan-specific tag ELISA after in vitro activation of the fibrinolytic system were due to detection of non-cross-linked fibrinogen fragments. However, fibrin fragment D-dimer concentrations were found to increase in plasma of 15 patients given t-PA for acute myocardial infarction. We conclude fragment D-dimer in plasma of patients during thrombolysis does not originate from circulating soluble cross-linked fibrin but rather is a marker of solid-phase fibrin dissolution, which may be quantitated with assays based on capture and tag antibodies that do not detect fibrinogen or its degradation products.     

Fibrin degradation products, fibrin monomer and soluble fibrin in disseminated intravascular coagulation.

Disseminated intravascular coagulation (DIC) is characterized by activation of hemostasis and fibrinolysis resulting in the formation of thrombin and plasmin, and the characteristic effects of these enzymes on plasma fibrinogen can be useful in diagnosis. Thrombin cleaves fibrinopeptides from fibrinogen, forming fibrin monomer that rapidly polymerizes to form a clot. Small amounts can circulate in plasma as "soluble fibrin," which may have a complex composition and include fibrinogen and a variable amount of cross-linking. Plasmic degradation of cross-linked fibrin forms a heterogeneous group of degradation products reactive in assays for D-dimer, and their levels provide a measure of the amount of fibrin formation and lysis. Caution should be exercised in comparing quantitative results using different assays because of problems with standardization and variable reactivity with different molecular forms. Marked elevations of fibrin(ogen) degradation products are a constant finding in experimental animal models of DIC. In human models of DIC resulting from endotoxin infusion, D-dimer is elevated early and high levels persist, reflecting lysis of microvascular fibrin deposits. Elevated levels of D-dimer and soluble fibrin are very sensitive for the diagnosis of DIC, and a normal level has a high negative predictive value. Serial monitoring of soluble fibrin or D-dimer assays may be of value in evaluating the response to therapy and possibly in identifying at-risk patients.     

Regulation by alpha 2-antiplasmin and fibrin of the activation of plasminogen with recombinant staphylokinase in plasma

The effects of alpha 2-antiplasmin and fibrin on the activation of plasminogen by recombinant staphylokinase (STAR) were studied in an effort to elucidate further the molecular basis of the fibrin- specificity of this fibrinolytic agent. In purified systems consisting of 1.5 mumol/L intact or low-M(r) plasminogen and 3 mumol/L alpha 2- antiplasmin, at 37 degrees C and in the absence of fibrin, STAR did not induce plasminogen activation and plasmin-alpha 2-antiplasmin complex (PAP) formation. Addition of a purified fibrin clot (30% vol at a concentration of 3 mg/mL) to mixtures containing intact plasminogen caused approximately 40% plasminogen activation within 2 hours, whereas in mixtures containing low-M(r) plasminogen, no activation was observed. In contrast, 10 nmol/L streptokinase (SK) induced 74% to 100% plasminogen activation within 2 hours in mixtures containing either intact or low-M(r) plasminogen, in both the absence and the presence of fibrin. In citrated human plasma in the absence of fibrin, 30 nmol/L STAR did not induce measurable plasminogen activation and PAP formation (< 1.5% within 2 hours), whereas addition of a plasma clot (12% vol) resulted in complete clot lysis and conversion of 19% +/- 8% of the plasminogen to PAP within 2 hours. Addition of a second plasma clot produced 23% +/- 2% additional plasminogen activation. Equipotent concentrations for plasma clot lysis of SK (100 nmol/L) induced 54% +/- 11% plasminogen activation in the absence and 49% +/- 16% in the presence of fibrin. Addition of 50 mmol/L 6-aminohexanoic acid (6-AHA) abolished the effect of fibrin on plasminogen activation with STAR, but not on activation with SK. In alpha 2-antiplasmin-depleted human plasma in the absence of fibrin, 30 nmol/L STAR did not induce fibrinogen breakdown (> 90% residual fibrinogen after 6 hours), whereas 30 nmol/L preformed plasmin-STAR complex induced extensive fibrinogen degradation (70% within 20 minutes). Thus, in the absence of fibrin, alpha 2- antiplasmin inhibits the activation of plasminogen by STAR, by preventing generation of active plasmin-STAR complex. Fibrin stimulates plasminogen activation by STAR via mechanisms involving the lysine- binding sites of plasminogen, probably by facilitating the generation of plasmin-STAR complex and by delaying its inhibition at the clot surface.     

Increased immunoreactivity of plasma after fibrinolytic activation in an anti-DD ELISA system. Role of soluble crosslinked fibrin polymers

After addition of a low concentration of thrombin to normal plasma, a progressive and significant increase in crosslinked fibrin polymers was found by sodium dodecyl sulfate agarose gel electrophoresis, reaching 27% of total fibrinogen and fibrin before gel formation. As measured by enzyme-linked immunosorbent assay with a monoclonal antibody specific for an epitope near the gamma gamma crosslink site, increased immunoreactivity of plasma did not occur after adding thrombin despite formation of crosslinked fibrin polymers, which indicates that the antibody does not recognize the epitope in the polymers. Addition of tissue-type plasminogen activator (t-PA) to plasma resulted in a more rapid degradation of fibrin polymers than of fibrinogen, indicating that the fibrin specificity of t-PA is retained with soluble fibrin. Coincident with degradation of plasma crosslinked fibrin polymers, plasma DD immunoreactivity increased 70-fold from 50.3 +/- 4.5 (mean +/- SD) to 3,560 +/- 1,235 ng/ml. The presence of increased crosslinked fibrin polymers produced by adding thrombin to plasma significantly increased maximum immunoreactivity after t-PA-induced degradation to 18,500 +/- 11,780 ng/ml. The increase in DD immunoreactivity was dependent on t-PA concentration; no elevation occurred below 0.01 micrograms/ml, and maximal increases occurred above 100 micrograms/ml. Analysis of gel electrophoretic patterns of thrombin and t-PA-treated plasma samples suggests that the DD reactivity of t-PA-treated plasma is mainly due to degradation of soluble crosslinked fibrin polymers. Our findings indicate that plasmic degradation of soluble fibrin polymers in plasma may be an important source of fragment DD during thrombolytic therapy.     

The interaction of ancrod with human platelets

Ancrod, a serine protease purified from the venom of Agkistrodon rhodostoma, has been used as a therapeutic anticoagulant for a number of indications, including replacement of heparin in patients with heparin-induced thrombocytopenia. Ancrod has similar fibrinolytic activity to thrombin, but ancrod specifically cleaves only the alpha chain of fibrinogen, producing the characteristic fibrinopeptides A, AP and AY. Because ancrod has been used in patients with heparin-induced thrombocytopenia, it is important to ensure that ancrod does not directly affect the platelets and potentially increase the hemostatic effect. The effect of ancrod on platelets has not been well established, and there is not agreement in published studies. Additionally, some of the studies are over 15 years old and pre-date sensitive assays such as glycoprotein analysis. For these reasons, we investigated the interaction of ancrod with human platelets using direct and indirect, functional and biochemical techniques. Incubation of platelets with ancrod alone did not induce platelet aggregation or the release of dense-granule contents. Pre-incubation of platelets with ancrod did not augment or inhibit the maximal aggregation achieved with thrombin, nor did it affect the amount of serotonin release from dense granules caused by activation by thrombin. Studies of ancrod-treated platelets using monoclonal antibody-mediated radioimmunoprecipitation demonstrated that high concentrations of ancrod did not cause measurable cleavage of either the glycoproteins Ib-IX or IIb-IIIa. Incubation of radiolabeled platelets with ancrod-treated plasma also had no effect on the platelet glycoproteins, indicating that ancrod does not indirectly affect the major surface receptors. Direct binding studies using radiolabeled ancrod did not demonstrate specific binding to the platelet surface. Together these studies indicate that ancrod does not directly affect nor bind to platelets in vitro. The hypo-coagulant state and subsequent platelet function defect resulting from the use of ancrod appears to be limited to the removal of fibrinogen from the circulation.