The Effect of Immunodepletion of Antithrombin III on the Response of Rabbits to Russell's Viper Venom–Induced Activation of Factor X
Many years ago it was shown that an infusion of tissue factor (TF) into rabbits causing only limited consumption of factor X and prothrombin resulted in extensive consumption of fibrinogen. More recently it was shown that an injection of a concentration of the factor X–activating fraction of Russell's viper venom (RVV-X) depleting rabbits of factor X resulted in only minimal consumption of both plasma prothrombin and fibrinogen. We report here experiments in which rabbits depleted of antithrombin III (ATIII) to different degrees were infused over 4 hours with a concentration of RVV-X, causing consumption of about 60% of plasma factor X. Similar minimal mean falls in plasma prothrombin and fibrinogen levels were observed in control rabbits given nonimmune goat IgG and in rabbits immunodepleted with goat anti-rabbit ATIII IgG to about 40% of normal plasma ATIII activity. However, if rabbits were immunodepleted to about 10% to 20% of normal plasma ATIII, then mean consumption of prothrombin was increased modestly and, more impressively, mean consumption of plasma fibrinogen was increased markedly. Whereas limited amounts of thrombin generated on the surface of phospholipid vesicles by factor VIIa/TF can trigger extensive intravascular coagulation in rabbits with normal plasma ATIII levels, limited amounts of thrombin generated by reactions triggered by factor Xa formed in fluid phase did so only after plasma ATIII levels were markedly depleted. A possible reason for this difference is discussed.
- Received July 18, 1996.
- Revision received October 17, 1996.
In earlier work from this laboratory, immunodepletion of TFPI was found to sensitize rabbits to disseminated intravascular coagulation induced by an infusion of a low dose of TF1 or by an intravenous dose of endotoxin2 that was essentially without effect in nonimmunodepleted rabbits. However, it was not possible to conclude from these studies that the protective effect of TFPI stemmed solely from inhibition of factor VIIa/TF catalytic activity, since in the process of such inhibition, TFPI also neutralized stoichiometric amounts of factor Xa. Therefore, a further study was carried out in which it was established that TFPI-depleted rabbits were not sensitized to disseminated intravascular coagulation initiated in the absence of TF by factor Xa and phospholipid.3
In a preliminary experiment to select an appropriate dose of the factor X activator from RVV-X for use in this further study, we confirmed an observation reported earlier by Aronson.4 An intravenous injection of normal rabbits with an amount of the factor X activator from RVV-X that depleted rabbit plasma factor X failed to cause an appreciable consumption of fibrinogen. This was the converse of our earlier experience, in which infusing normal rabbits with concentrations of tissue factor that caused much less of a fall in plasma factor X resulted in extensive intravascular coagulation, with a marked fall in plasma fibrinogen.5 6
ATIII, accelerated by vascular wall glycosaminoglycans, is thought to be the primary plasma proteinase inhibitor regulating the in vivo activity of both factor Xa and thrombin.7 We thought it possible that the above-cited contrasting observations might stem from an enhanced ability of ATIII to prevent intravascular clotting when factor Xa was generated in the fluid phase by RVV-X instead of on a surface by VIIa/TF complexes. Therefore, we have carried out the experiments described here to measure the effect on the ability of factor Xa generated by RVV-X to induce disseminated intravascular clotting in rabbits subjected to different degrees of immunodepletion of ATIII.
Two batches of pooled rabbit plasma, one prepared from five and the other from six healthy young rabbits, were used as reference plasma that was arbitrarily assigned a value of 1 U/mL (activity) or 100% (antigen) for all assays. Rabbit brain thromboplastin for factor X and prothrombin assays, Tris, and BSA were from Sigma Chemical Co. Rabbit brain thromboplastin for the TFPI assay was purified rabbit brain tissue incorporated into phosphatidyl choline/phosphatidyl serine vesicles by the use of octylglucopyranoside as detergent. Rabbit factor X–deficient plasma was prepared by immunoaffinity chromatography as described earlier.8 Rabbit prothrombin-deficient plasma was prepared by immunochromatography against a goat anti-rabbit prothrombin IgG. A barium-adsorbed rabbit plasma was prepared as described previously6 from rabbit plasma obtained from a rabbit that had received an intravenous injection of endotoxin to elevate the fibrinogen level.8 Human factor Xa was prepared by activating factor X with insolubilized RVV-X9 and stored in 50% glycerol as a stock reagent containing 100 μg of factor Xa per milliliter. Recombinant human factor VIIa was from Novo/Nordisk. Bovine thrombin and human fibrinogen reference plasma were from Baxter Diagnostics. Chromozyme X was from Boehringer Mannheim. Immulon 4 ELISA plates were from Dynatech, Vectastain ABC staining kit was from Vector Laboratories, and peroxidase substrate kit ABTS was from Bio-Rad. Biotin-N-hydroxysuccinimide ester was from Pierce, and horseradish peroxidase was from Calbiochem.
Anti-Rabbit ATIII IgG
Anti-rabbit ATIII antiserum was raised in two goats. In one goat a commercial rabbit ATIII (Sigma), and in the second goat a rabbit ATIII prepared in this laboratory by heparin affinity chromatography was administered as antigen by subcutaneous injection in Freund's complete adjuvant. Antiserum prepared from blood withdrawn at approximately monthly intervals was heated at 56°C for 30 minutes and stored frozen. On thawing, the IgG fraction was separated by (NH4)2SO4 fractionation at 40% saturation and affinity-purified by passage through a column prepared by coupling rabbit ATIII to Affi-Gel 15 (Bio-Rad). Bound IgG was eluted with 0.1 mol/L glycine at pH 2.4 in 3-mL fractions in tubes containing 300 μL of 1 mol/L Tris buffer, pH 8.8. On at least two occasions, an aliquot removed at this stage was biotinylated. On Western blotting of normal rabbit plasma, the biotinylated IgG gave a single band at ≈60 000 Mr.
Anti-rabbit ATIII IgG preparations were stored frozen until several batches of antiserum had been processed. The IgG preparations were then thawed and pooled. Aliquots of ≈100 mL were concentrated through use of a stirred cell concentrator and an Amicon YM100 filter (Amicon). As concentration proceeded, ≈100 mL of 10 mmol/L HEPES and 0.15 mol/L NaCl, pH 7.5 (HEPES buffer) was added sequentially at least three times to put the IgG into the vehicle to be used for injection. The preparations were then passed through a sterile 0.22-μm filter unit (Millipore) and collected in two to three aliquots in sterile capped tubes. After subsamples were removed for measurement of anti-ATIII IgG neutralizing activity and (except for the preparation given to the first rabbit) for endotoxin assay (Bio-Whittaker Limulus Amebocyte Lysate), the aliquots were stored frozen until use. Different preparations contained from 12 to 37 mg/mL of IgG in from 8 to 20 mL of HEPES buffer.
ATIII neutralizing activity was measured by incubating 5 μL of normal rabbit plasma for 1 hour at 37°C with 5 μL of dilutions of anti-ATIII IgG in a heparin-containing ATIII assay buffer (0.05 mol/L Tris-HCL and 0.15 mol/L NaCl, pH 7.5 [TBS]; 1 mg/mL BSA; 5 mmol/L EDTA; 0.75 U/mL of Trasylol; and 1 U/mL of heparin). Then, 490 μL of the assay buffer was added and the resultant 1/100 dilution of rabbit plasma assayed for ATIII activity as described below. The ATIII titer of different preparations varied between 1.9 and 2.3 anti-ATIII IU per milligram of IgG, when 1 IU was defined as that amount of anti-rabbit ATIII IgG that neutralized 50% of the ATIII activity in 1 mL of the pooled rabbit reference plasma. Endotoxin contamination varied between 2 and 40 U of endotoxin per milliliter.
Nonimmune Goat IgG
IgG was separated from normal goat serum by treatment with (NH4)2SO4 at 40% saturation followed by DEAE–Affi-Gel blue (Bio-Rad) chromatography and concentrated into HEPES buffer. Two preparations were used in this study. One contained 20 mg/mL IgG and 10 U of contaminating endotoxin per milliliter. The second contained 37 mg/mL of IgG and 48 U of contaminating endotoxin per milliliter. Reduced SDS-polyacrylamide gel electrophoresis of a subsample from one preparation yielded two bands, one at 55 000 Mr and the other at 30 000 Mr.
Anti-rabbit TFPI IgG was separated by DEAE–Affi-Gel blue (Bio-Rad) from antiserum raised in a goat against rabbit TFPI that had been expressed in a baculovirus system. It gave a single band at ≈45 000 Mr on Western blotting of a purified rabbit plasma TFPI preparation.
Anti-Rabbit Factor X IgG
A monoclonal antibody that reacted with both native and activated factor X was prepared and purified as described earlier.8
The factor X activator in RVV-X was purified from crude RVV by gel filtration followed by ion exchange chromatography. Its specific factor X–activating activity was 18 500 U/mg. Details of purification and assay have been described earlier.3
ATIII Activity Assay
ATIII activity was measured as the ability of a rabbit plasma test sample to inhibit human factor Xa. Rabbit plasma was diluted 1/100 in the heparin-containing ATIII assay buffer and 25 μL was pipetted into the well of an ELISA plate. Twenty-five microliters of human factor Xa diluted to 750 ng/mL in the assay buffer was added to the well. After 1 to 2 minutes at room temperature, 50 μL of 1.25 mg/mL Chromozyme X was added and the initial rate of color development recorded continuously with an ELISA reader (Molecular Devices). mOD per minute was converted into percent ATIII activity from a standard curve made with 1/100 to 1/1600 dilutions of the rabbit reference plasma.
ELISA for ATIII Antigen
ELISA plate wells were coated overnight at 4°C with 50 μL of 4 μg/mL goat anti-rabbit ATIII IgG in 0.1 mol/L sodium bicarbonate buffer, pH 9.6. The wells were then blocked with 3% BSA in TBS for 3 hours at 37°C. After washing the wells three times with 300 μL of TBS containing 0.05% Tween 20 (TBS/Tween), 50 μL of a dilution (usually 1/2000) of rabbit plasma test sample in TBS containing 0.1% BSA (TBS/BSA) was added to the wells. After 2 hours at 37°C, the wells were washed three times with 300 μL of TBS/Tween, and 50 μL of biotinylated anti-rabbit ATIII IgG was added. After 2 hours at 37°C, the wells were washed again three times with TBS/Tween, after which 50 μL of Vectastain ABC solution was added for 30 minutes at 37°C. After a further washing of the wells three times with 300 μL of TBS/Tween, 50 μL of ABTS peroxidase substrate was added and the initial rate of color development recorded continuously with an ELISA reader. mOD per minute was converted to percent antigen of the rabbit plasma reference from a standard curve made with 1/500 to 1/40 000 dilutions of the reference plasma.
ELISA for ATIII/Xa Complexes
The enzyme-linked differential-antibody immunosorbent technique of Jesty et al10 was modified as follows. ELISA plate wells were coated with 50 μL of 5 μg/mL anti-rabbit factor X IgG, blocked, and washed as described above. Then the rabbit plasma test sample diluted 1/100 in TBS/BSA was added. After 2 hours, the wells were washed as described above and 50 μL of 1.5 μg/mL biotinylated goat anti-rabbit ATIII IgG was added. The remaining steps of the assay were carried out as described for the ATIII ELISA.
mOD per minute was converted to percent of ATIII/Xa complexes formed when the factor X of a rabbit reference plasma was activated by RVV-X in vitro in a reaction mixture containing 1 mL of the reference plasma, 0.5 μg of RVV-X, 1 U of heparin, and 10 mmol/L CaCl2. After 45 minutes at 37°C, 10 μL of 1 mol/L EDTA was added and the clot removed. The serum was stored frozen for use as a reference standard assigned a value of 100% ATIII-Xa complexes. Dilution curves made with 1/100 to 1/1600 dilutions of the standard were used to convert mOD per minute of test samples to percent ATIII-Xa complexes.
TFPI Activity Assay
TFPI activity was measured in a two-stage capacity assay. In the first stage, a test sample was incubated with factor VIIa, TF, factor Xa, and Ca2+. Residual factor VIIa activity was determined by its ability to activate factor X. The assay has been described in detail.3
ELISA for TFPI Antigen
ELISA plate wells were coated with 50 μL of 25 μg/mL anti-rabbit TFPI IgG, blocked with BSA, and washed with TBS/Tween as described above. Then, 50 μL of test plasma diluted 1/10 in TBS/BSA was added to the wells for 2 hours at 37°C. After washing with TBS/Tween as described above, 50 μL of 25 μg/mL peroxidase-labeled anti-rabbit TFPI IgG was added. After 2 hours at 37°C, the wells were washed again with TBS/Tween and the remaining steps carried out as described for the ATIII ELISA. mOD per minute was converted to percent TFPI antigen from dilution curves made with 1/5 to 1/60 dilutions of the rabbit reference standard.
One-Stage Clotting Assays
Clotting times were determined with an ST4 semiautomated coagulation instrument (Diagnostica Stago). Fibrinogen was determined by the method of Clauss,11 in which 50 μL of 100 U/mL bovine thrombin was added to 100 μL of test plasma diluted 1/10 in Veronal buffer (28.4 mmol/L sodium barbital, 0.125 mol/L NaCl, pH 7.35). Clotting times were converted to fibrinogen concentration from a standard curve prepared with human fibrinogen. Factor X was measured in an assay in which 50 μL of a 1:1 mixture of factor X–depleted rabbit plasma and barium-adsorbed rabbit plasma, 50 μL of a 1:40 dilution of test plasma in TBS/BSA, and 50 μL of rabbit brain thromboplastin were incubated together at 37°C for 3 minutes, and clotting was triggered by the addition of 50 μL of 35 mmol/L CaCl2. Prothrombin was measured as for factor X, except that rabbit plasma immunodepleted of prothrombin was used in the 1:1 substrate mixture.
Electroimmunoassay for Factor X Antigen
Electroimmunoassay was carried out on an 11.5×20.5-cm glass plate in 1% Seakem agarose ME in Tris-tricine buffer at pH 8.6 and a goat anti-rabbit factor X IgG concentration of 2%.
Hematocrit, WBC counts, and platelet counts were determined with a Coulter ST counter (Coulter Electronic Inc) in the clinical hematology laboratory of the UCSD Medical Center.
Eighteen female New Zealand rabbits of about 2 kg weight were used for this study in protocols approved by the Animal Subjects Committee of the University of California, San Diego. They were placed in a rabbit restrainer, and a Jelco 22-gauge IV catheter (Critikon) was inserted into a marginal ear vein and connected by way of a basic solution set to a sterile infusion bag containing 100 mL of 0.9% NaCl (Baxter Healthcare Corp). The catheter was kept open by a slow infusion of ≈10 mL/h. Immediately after beginning the infusion, 12 rabbits received over 4 to 5 minutes by way of the side arm of the infusion set from 70 to 170 IU/kg of anti-rabbit ATIII IgG (from 35 to 85 mg/kg) in a volume of 5 to 10 mL of HEPES buffer. Six control rabbits received from 27 to 50 mg/kg of nonimmune goat IgG in ≈3 to 5 mL of HEPES buffer. One hour later, 2.5 μg/kg of RVV-X in 10 mL of 0.9% sterile NaCl was added to the infusion fluid. The infusion rate was then increased to 20 to 25 mL/h so that the infusion of the RVV-X was completed in ≈4 hours.
Blood samples (1 to 2 mL) were obtained from the marginal ear vein of the opposite ear and processed as described earlier.8 An initial sample was drawn before the injection of IgG for measurement of clotting factors, hematocrit, WBC, and platelet counts. A second sample was drawn 1 hour later before starting the RVV-X infusion. Samples were drawn hourly thereafter until 1 hour after completion of the RVV-X infusion.
Rectal temperature was measured as a sensitive indicator of the effect of infused endotoxin at the beginning, middle, and end of the experiment. It did not rise significantly in any rabbit.
Data Processing and Statistical Methods
Because multiple blood samples were taken, the hematocrit fell during the course of an experiment as the plasma volume expanded to compensate for loss of whole blood. If one assumes that the fluid entering the plasma contained little protein, then it would dilute the plasma proteins in a manner analogous to dilution by the citrate anticoagulant. Therefore, values obtained in activity assays and immunoassays were corrected according to the following correction factor (Cf), derived as described earlier,12 in which Cf=(PHCti+11.1)/PHCto, where PHCti is the plasma hematocrit of the sample and PHCto is the plasma hematocrit of the initial blood sample.
Values obtained on the initial sample drawn before the injection of either control nonimmune or anti-ATIII IgG were assigned a value of 100%. Values obtained from subsequent samples were converted into percent of the initial value and are reported for each group of animals as mean±SEM.
Control group (n=6)
Rabbits were given nonimmune IgG. Mean plasma ATIII activity fell gradually from a mean value of 91±3% at the beginning of the infusion of RVV-X (hour 1) to a value of 82±5% at the end of the experiment (hour 6).
Moderate ATIII–depletion group (n=5)
Rabbits were given between 70 and 94 IU/kg of anti-ATIII IgG. Mean plasma ATIII activity level had fallen to 39±6% before beginning the infusion of RVV-X and had risen slightly to 48±5% by the end of the experiment. Corresponding values for plasma ATIII antigen were 52±11% and 62±4%.
Severe ATIII–depletion group
Rabbits were given between 110 and 170 IU/kg of anti-ATIII IgG. Mean plasma ATIII activity level had fallen to 10±2% before beginning the infusion of RVV-X and had risen to 21±4% by the end of the experiment. Corresponding values for plasma ATIII antigen were 20±2% and 28±5%.
The mean initial measured values for activity and antigen of the hemostatic factors assayed in this study are listed for each group in Table 1⇓. As mentioned earlier, all values from subsequent samples are expressed as percent of these initial mean values.
Three rabbits given between 120 and 150 IU of anti-ATIII IgG/kg died at from 105 to 165 minutes after beginning the infusion of RVV-X, two with no recognized warning signs of distress and one after a brief convulsion. The ATIII activity of the plasma sample preceding death as percent of initial plasma activity in these three animals was 7%, 18%, and 10%. Had these rabbits survived the RVV-X infusion, they would have fallen into the severely depleted plasma ATIII group. Levels of other factors in the last plasma sample as percent of initial activity were as follows: for factor X activity, 57%, 52%, and 56%; for prothrombin, 62%, 73%, and 73%; for fibrinogen 19%, 52%, and 61%. These falls in plasma prothrombin and fibrinogen levels exceeded the mean decreases at comparable RVV-X infusion times of the rabbits in the severely depleted plasma ATIII group (Fig 2A and 2B⇓⇓). We suspect that these three animals died as a result of fibrin deposition within the pulmonary vasculature, but autopsies were not performed to confirm this.
Plasma factor X levels fell in all animals infused with RVV-X. Serial values for mean factor X activity and antigen for each group are plotted in Fig 1C and 1D⇑⇑. Mean falls in plasma factor X activity from the beginning of the infusion (hour 1) to the end of the experiment (hour 6) were: for the control group, 65%; for the moderate ATIII–depletion group, 63%; for the severe ATIII–depletion group, 59%. For factor X antigen, the corresponding mean falls were: for the control group, 55%; for the moderate ATIII–depletion group, 46%; for the severe ATIII–depletion group, 50%.
Serial values for mean plasma prothrombin activity for the three groups are plotted in Fig 2A⇑. In the control and moderate ATIII–depletion groups, mean plasma prothrombin activity fell only minimally over the 5 hours after beginning the infusion of RVV-X. Mean plasma prothrombin activity for the six animals of the control group were: at the beginning of the RVV-X infusion, 96±3%; at the end of the experiment (hour 6), 79±6%; mean decrease, 17%. Mean values for the five animals in the moderate ATIII–depletion group were: at the beginning of the RVV-X infusion, 91±5%; at the end of the experiment, 85±5%; mean decrease, 6%. In both groups, individual rabbits varied in the extent of the fall in their plasma prothrombin activity. In the control group, plasma prothrombin activity fell by 29% in one rabbit yet in another rabbit did not fall at all. In the moderate ATIII–depletion group, plasma prothrombin activity fell by 30% in one rabbit yet in two other rabbits did not fall at all.
Successive mean plasma prothrombin activity values for the four animals in the severe ATIII–depletion group appeared to diverge from the mean values for the other two groups as the infusion of RVV-X progressed (Fig 2A⇑). Mean values were: at the beginning of the RVV-X infusion, 91±2%; at the end of the experiment (hour 6), 67±9%; mean decrease, 24%. Decreases in individual animals were: 5%, 22%, 27%, and 42%. Because of the small number of surviving animals in the severe ATIII–depletion group, values obtained at hours 5 and 6 were combined for statistical analysis: control group, 80±3.5% (mean±SEM), n=12; severe ATIII–depletion group, 68±6.0%, n=8). These means differed significantly (P=.046, one-tailed t test).
Infusing RVV-X resulted in equivalent moderate falls in the mean fibrinogen level of the control and moderate ATIII–depletion groups (Fig 3B⇓). For the six control-group rabbits, mean values were: at the beginning of the RVV-X infusion, 96±4%; at the end of the experiment, 70±5.4%; mean decrease, 26%. The decreases in individual rabbits were: 2%, 18%, 21%, 23%, 42%, and 52%. For the five animals in the moderate ATIII–depletion group, mean values were: at the beginning of the RVV-X infusion, 100±7%; at the end of the experiment, 69±14%; mean decrease, 31%. The extent of the fall in fibrinogen level varied widely in this group of animals as well. In two animals there was essentially no fall, whereas in the other three animals there were decreases of 39%, 50%, and 67%.
In sharp contrast, infusing RVV-X resulted in a progressive marked fall in the mean plasma fibrinogen level of the four animals in the severe ATIII–depletion group. Mean values were: at the beginning of the RVV-X infusion, 87±2%; at the end of the experiment, 21±5%; mean decrease, 66%. The individual decreases for the four animals were: 60%, 77%, 72%, and 57%.
The fibrinogen assay we used is based on measuring the clotting time with thrombin of a test sample.11 Therefore, although unlikely, one could account for the above decreases if our RVV-X preparation contained a trace protease that retained its catalytic activity in vivo when plasma ATIII was severely depleted and could cleave fibrinogen with consequent impairment of its clotability.
A supplemental in vitro experiment was performed to evaluate this possibility. Rabbit plasma depleted of vitamin K–dependent clotting factors by barium adsorption was diluted 1/10 in TBS/BSA to reduce ATIII and other plasma proteinase inhibitors to 10% of intact plasma concentrations. Then, either TBS/BSA or RVV-X in TBS/BSA in a final concentration of 0.05 μg/mL was added to the plasma. The latter was the estimated in vivo RVV-X plasma concentration if all RVV-X infused over 4 hours persisted within the plasma. The adsorbed plasma was then incubated at 37°C in the presence or absence of CaCl2, 15 mmol/L final concentration. Subsamples removed at increasing times were clotted with thrombin according to the method used for the fibrinogen assay. The incubation with RVV-X was without effect on the clotting times. For example, in one experiment in which Ca2+ was present, the clotting times of subsamples incubated with control buffer or RVV-X were as follows: at 5 minutes' incubation time, 22.8 and 22.6 seconds; at 120 minutes' incubation time, 26.0 and 26.0 seconds.
ATIII/Xa complexes accumulated in the plasma of all rabbits infused with RVV-X. Their concentration was expressed (Fig 3⇑) relative to a 100% value assigned to the concentration of complexes formed when a pooled reference plasma was clotted in vitro with RVV-X and Ca2+ in the presence of heparin to assure their maximum formation. In the control group (mean plasma ATIII level of 91% at the beginning of the RVV-X infusion), the mean consumption over 5 hours of 0.65 U/mL of factor X activity (Fig 1C⇑) resulted in a progressive accumulation in the plasma of about 34% of the concentration of ATIII/Xa complexes that could be maximally formed on activating the factor X of the reference plasma in vitro. For both the moderate ATIII–depletion group and the severe ATIII–depletion group, a mean consumption over 5 hours of 0.63 U/mL of factor X in the former and 0.59 U/mL of factor X in the latter resulted in the accumulation of close to 25% of the ATIII/Xa complexes formed maximally in vitro. Complexes accumulated more slowly than in the control group (Fig 3⇑).
Serial mean values for mean plasma TFPI antigen and activity are plotted in Fig 4A and 4B⇓⇓. Antigen levels rose after the injection of anti-rabbit ATIII IgG but not after injection of nonimmune goat IgG. Thus, mean antigen levels at the beginning of the RVV-X infusion were: for the control group, 96±6%; for the moderate ATIII–depletion group, 125±9%; for the severe ATIII–depletion group, 139±11%. Antigen levels fell to a varying degree during the infusion of RVV-X in all groups and by the end of the experiment were: for the control group, 79±10%; for the moderate ATIII–depletion group, 74±7%; for the severe ATIII group, 52±18%.
TFPI antigen and activity assays were discordant in that animals given anti-ATIII IgG had only very questionable if any mean rise in plasma TFPI activity. Moreover, mean plasma TFPI activity levels did not fall during the infusion of RVV-X in either the control group or the moderate ATIII–depletion group. Whereas TFPI activity of the four animals in the severe ATIII–depletion group was plotted as means that did fall during the infusion of RVV-X, the late values for plasma TFPI activity of the individual rabbits varied markedly. At 6 hours, the values were: 9%, 32%, 71%, and 134% of the initial value before the injection of anti-ATIII IgG.
Platelet and WBC Counts
The mean platelet and WBC counts for the three groups of animals are summarized in Table 2⇓. The mean platelet counts for the control animals did not change during the course of the experiment. However, the mean platelet count, obtained at either 1 hour or 2 hours, fell in both the low-dose and high-dose anti-ATIII IgG groups, presumably as a consequence of the formation of ATIII/anti-ATIII immune complexes. In the high-dose group, the mean platelet count, obtained at hours 5 or 6, had fallen further, possibly reflecting an additional effect of thrombin on platelets in these animals.
In experiments performed over 30 years ago, rabbits given a large intravenous injection of warfarin to block synthesis of vitamin K–dependent clotting factors were infused 4 hours later with either rabbit TF or saline. A 30-minute infusion of TF resulted in a mean excess fall over controls in plasma factor X of 20% and in plasma prothrombin of only 10%. Nevertheless, the TF infusion resulted in extensive intravascular coagulation, with consumption of about 50% of plasma fibrinogen.5 These findings contrast strikingly with Aronson's observation 20 years later4 that a bolus injection into rabbits of a concentration of RVV-X essentially depleting the plasma of factor X over 30 minutes caused virtually no consumption of plasma fibrinogen. After confirming Aronson's finding,3 we carried out the experiments reported here to evaluate the effect of infusing RVV-X into control rabbits, rabbits moderately immunodepleted of ATIII (40% activity), and rabbits severely immunodepleted of ATIII (10% to 20% activity).
A concentration of RVV-X was infused over 4 hours into these three groups of rabbits that caused a progressive mean fall over 5 hours of 59% to 65% of plasma factor X activity and 46% to 55% of plasma factor X antigen. The RVV-X–catalyzed fluid-phase activation of plasma factor X was independent of plasma ATIII concentration. This was anticipated from an earlier in vitro observation of Gitel and colleagues13 in a purified system that ATIII, either in the presence or absence of heparin, did not affect the rate of RVV-X–catalyzed activation of factor X. The modestly higher mean values obtained for consumption of factor X measured as activity than as antigen could have stemmed at least partially from the accumulation in the plasma of ATIII/Xa complexes capable of reacting with our goat anti-rabbit factor X IgG (Fig 3⇑).
Individual animals in both the control and moderate ATIII–depleted groups varied somewhat in the responses of their plasma prothrombin and fibrinogen levels to a continuing 5-hour fluid-phase generation of factor Xa. However, mean falls in plasma prothrombin and fibrinogen of the animals with a moderately reduced plasma ATIII activity of about 40% of initial activity did not exceed the minimal mean falls observed in the control group of animals (Fig 2⇑).
In rabbits in which ATIII was immunodepleted to 10% to 20% of initial mean activity—the four rabbits in the severe-immunodepletion group and the three rabbits that died during the RVV-X infusion—the falls in prothrombin in some individual animals also did not exceed those observed in some individual animals in the control and moderate ATIII–depletion groups. Nevertheless, the serial mean plasma prothrombin values of the four surviving animals in the severe-immunodepletion group progressively diverged from the mean values of the other two rabbit groups (Fig 2A⇑). However, the striking finding was that this evidence for a modestly increased mean generation of thrombin was associated with a substantially increased mean consumption of plasma fibrinogen (Fig 2B⇑).
When ATIII reacts with a cognate proteinase such as factor Xa, the resultant complex is cleared from the circulation by its binding to a receptor present on hepatocytes designated as serpin receptor 1.14 15 If one assumes a rabbit plasma factor X concentration of 140 nmol/L and a plasma volume of 50 mL/kg, then a progressive mean consumption of about 60% of circulating factor X over 5 hours reflected activation of about 14 pmol·kg−1·min−1. This rate of fluid-phase generation of factor Xa (an underestimate, since some new factor X was also made during the 5-hour interval) was associated with a progressive rise in plasma ATIII/Xa complexes (Fig 3⇑). In many rabbits, the level of complexes continued to increase after the RVV-X infusion was stopped, which undoubtedly reflected a continuing activation of factor X by circulating RVV-X (Fig 1C⇑).
In the control group with a normal plasma ATIII concentration, the rise in plasma ATIII/Xa complexes appeared to be leveling off at a concentration about one third of that measured when rabbit plasma was clotted in vitro under conditions maximizing formation of ATIII/Xa complexes.10 Thus, although injected ATIII/proteinase complexes have been reported to be cleared rapidly from the circulation in mice,15 a substantial plasma ATIII/Xa concentration had to be achieved in these rabbits before the presumed rate of release into plasma from microvascular endothelium of ATIII/Xa complexes (see below) and the rate of clearance from plasma of the complexes by serpin receptor 1 approached equivalence.
The plateau level of the progress curve of accumulation of plasma ATIII/Xa complexes was reduced in the rabbits immunodepleted to a mean of 40% of initial ATIII activity (Fig 3⇑). Nevertheless, this indirect evidence for a reduced generation and subsequent release from microvascular endothelium of ATIII/Xa complexes was not associated with a factor Xa–catalyzed increase in the mean rate of fall of plasma prothrombin (Fig 2A⇑). This could conceivably reflect an ability of α1-proteinase inhibitor and α2-macroglobulin to compensate for a moderately reduced inhibition of factor Xa by ATIII.16
When plasma ATIII concentration was immunodepleted to 10% to 20% of normal, the rate of accumulation of circulating plasma ATIII/Xa complexes was substantially diminished (Fig 3⇑). This indirect evidence of a further impairment in the ability of ATIII to neutralize factor Xa that was being generated continuously in the fluid phase was associated with a modestly increased mean rate of fall of plasma prothrombin (Fig 2A⇑).
Despite the activation of about 60% of the circulating factor X over 5 hours, mean plasma TFPI activity levels did not fall in either the control group or the moderately depleted ATIII group (Fig 4B⇑). However, in three of the four rabbits of the severely ATIII–depleted group, plasma TFPI activity did fall over the later hours of the experiment to values of 9%, 32%, and 71% of initial plasma activity. It appeared that plasma ATIII levels had to be markedly reduced before enough TFPI/Xa complexes were formed and subsequently cleared to reduce substantially the activity of plasma TFPI as measured in a capacity assay.
For an unknown reason, values for plasma TFPI activity and TFPI antigen were discordant in that antigen levels but not activity levels clearly rose after the injection of goat anti-rabbit ATIII IgG. In addition, a modest fall in plasma TFPI antigen to about 75% of its initial mean concentration was observed in both the control and moderate ATIII–depletion rabbit groups. Viewed as a whole, the data here and from our earlier observations in rabbits immunodepleted of TFPI3 provide convincing evidence that TFPI plays little if any role in preventing factor Xa generated in fluid phase by a TF-independent mechanism from triggering intravascular coagulation.
It was observed many years ago in a rabbit stasis model that a thrombus would form in a contralateral jugular vein segment that was isolated within seconds after injecting 0.12 nmol of factor Xa into a marginal ear vein.13 In a 2-kg rabbit with an assumed 100-mL plasma volume, the plasma concentration of factor Xa within the isolated vein segment would be considerably lower than 1.2 nmol/L because of substantial clearance of factor Xa during its passage through the microvasculature of the lung. Although the concentration of factor Xa within the isolated contralateral vein segment would be equivalent to activating not more than a trace of the 140-nmol/L concentration of plasma factor X, inhibitory reactions within the isolated vein segment were unable to prevent thrombosis.
In contrast, fluid-phase activation of most of the circulating factor X, by either bolus injection3 4 or infusion of RVV-X as described here, failed to induce substantial consumption of either plasma prothrombin or fibrinogen. This presumably reflected a highly effective flow-dependent, continuing neutralization within the microvasculature of both factor Xa and any thrombin that it might have generated. The loss of protection against intravascular coagulation observed in the present experiments when ATIII was immunodepleted to 10% to 20% of its initial plasma level fits with a hypothesis that the protection stemmed from a physical approximation of factor Xa and ATIII on heparin-like glycosaminoglycans, presumably heparan sulfate, expressed on microvasculature endothelium. This would enhance the ability of the ATIII both to limit factor Xa–catalyzed activation of prothrombin on microvascular endothelium and, in keeping with the earlier evidence of Lollar and Owen,17 to quench the catalytic activity of whatever thrombin was formed.
This analysis of our present findings highlights for us the importance of the participation of heparin-like glycosaminoglycans in the physiological function of ATIII as a natural anticoagulant. It helps us to understand better why in our earlier experiments, small amounts of thrombin generated by infusing rabbits with TF, ie, generated by reactions initiated by VIIa/TF complexes on a mixed phospholipid particle lacking heparin-like glycosaminoglycans, could initiate extensive intravascular coagulation despite a normal plasma ATIII concentration.5 6
We believe that the above analysis can also be extrapolated to human hemostasis to clarify why heparin is required to suppress the catastrophic coagulopathy of Trousseau's syndrome.18 Substantial evidence summarized earlier19 leads us to conclude that a continuing exposure of the circulating blood to TF expressed on tumor cells or vesicles shed from them triggers the continuing low-grade intravascular coagulation18 19 20 and multiple thrombotic events of Trousseau's syndrome. As in the rabbit infused with TF, normal plasma levels of ATIII fail to suppress the intravascular coagulation of Trousseau's syndrome. However, if heparin in a therapeutic dosage is given, then intravascular coagulation and thromboses cease.18 19 ATIII/heparin complexes can then suppress by two mechanisms the initiation of coagulation on a surface expressing TF but devoid of heparin-like glycosaminoglycans. The first mechanism is the neutralization and removal of factor VIIa molecules bound to TF by ATIII/heparin.21 The second is the inhibition of factor Xa–catalyzed back activation to VIIa of factor VII molecules bound to TF.22
It is unknown whether it is also important in Trousseau's syndrome, as it was in the present experiments, for ATIII/glycosaminoglycans complexes to possess the capability of neutralizing any thrombin that is generated. Data addressing this question are required before one could accept the use of low-molecular-weight heparin in place of unfractionated heparin in the management of Trousseau's syndrome or other human coagulopathies of similar pathogenesis.
Selected Abbreviations and Acronyms
|RVV-X||=||factor X–activating fraction of Russell's viper venom|
|TFPI||=||TF pathway inhibitor|
|WBC||=||white blood cell|
This study was supported by grant HL27234 from the National Heart, Lung, and Blood Institute. We thank Dr Ariella Zivelin for helpful suggestions during the course of these experiments.
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