₂-Antiplasmin Gene Deficiency in Mice Is Associated With Enhanced Fibrinolytic Potential Without Overt Bleeding

THE MAMMALIAN fibrinolytic system contains a proenzyme, plasminogen, that is converted to the active serine proteinase plasmin by tissue-type (t-PA) or urokinase-type (u-PA) plasminogen activator. Inhibition of the system may occur through neutralization of the plasminogen activators by plasminogen activator inhibitors (mainly PAI-1) or through neutralization of plasmin.1 α₂-antiplasmin (α₂-AP) is the main physiologic plasmin inhibitor in mammalian plasma, whereas excess plasmin may be inhibited by α₂-macroglobulin.2-4 α₂-AP is synthesized in the liver and is present in plasma at a concentration of about 1 μmol/L.2-4 Human and murine α₂-AP are serpins (serine proteinase inhibitors) with molecular weight (M_r) 65 to 70 kD,2-5 which inhibit plasmin in a very rapid reaction resulting in the formation of a stable inactive complex.6 The cDNA and deduced amino acid sequence,7,8 as well as the gene organization9,10 of both human and murine α₂-AP have been elucidated.

The mouse α₂-AP gene encodes a 491–amino acid protein, with the NH₂-terminus Val of the mature protein corresponding to residue 28,5,10 whereas mature human α₂-AP also consists of 464 residues with Met as NH₂-terminus.11 The reactive site peptide bond consists of Arg-Met in the inhibitor of both species.7,8 In human and murine plasma, α₂-AP occurs as a plasminogen-binding (60% to 70%) and as a non–plasminogen-binding (30% to 40%) form lacking a COOH-terminal fragment, which contains structures with affinity for the lysine-binding sites of plasminogen.5,12-14 The plasminogen-binding form cross-links to fibrin when it is clotted in the presence of Ca²⁺ and activated factor XIII.15 Binding of α₂-AP to plasminogen may prevent subsequent binding of plasminogen to fibrin, and thus result in an antifibrinolytic effect. Low M_r forms of α₂-AP have been detected at low concentration (0.05% of the plasma concentration) in the α-granules of blood platelets; their function remains unknown.16 

Besides in the removal of fibrin, the fibrinolytic system may also play a role in phenomena such as ovulation, embryogenesis, intima proliferation, atherosclerosis, tumorigenesis, and metastasis.17 α₂-AP may thus exert an inhibitory effect at different levels on fibrinolysis, as well as on several other plasmin-mediated biologic processes. Therefore, this inhibitor appears to be an interesting target to study its biologic role directly with the use of mice with specific inactivation of the α₂-AP gene. This strategy has successfully been applied to study the biologic function of most other components of the fibrinolytic system.17 We have previously characterized the murine α₂-AP gene and constructed a targeting vector for homologous recombination in embryonic stem (ES) cells.10 In this study, we report the generation of homozygous α₂-AP–deficient mice and evaluate the biologic effects of α₂-APgene disruption.

Mice were kept in microisolation cages on a 12-hour day-night cycle and fed a regular chow. They were anesthetized by intraperitoneal injection of 60 mg/kg Nembutal (Abbott Laboratories, North Chicago, IL), and blood was collected by vena cava puncture with a 24-gauge needle. Platelet-poor plasma was prepared by centrifugation of blood collected on citrate at 4,000g for 5 minutes.

Murine α₂-AP, plasminogen and plasmin, human plasmin, and two-chain urokinase-type plasminogen activator (tcu-PA) were prepared and characterized as described elsewhere.5 Polyclonal antibodies against purified murine α₂-AP and plasminogen were raised in rabbits. Before use, the antiserum against murine α₂-AP was adsorbed with 10% to 20% (vol/vol) murine α₂-AP–deficient plasma. α₂-AP and plasminogen antigen levels were quantitated by enzyme-linked immunosorbent assay (ELISA) using the purified murine proteins for calibration.5 α₂-AP activity levels in murine plasma were determined by addition of human plasmin (final concentration 5 nmol/L in 500-fold diluted plasma) and measurement of residual plasmin with the chromogenic substrate S-2403 (final concentration 0.3 mmol/L) (Chromogenix, Antwerp, Belgium) after incubation at 37°C for 10 seconds,5 using a calibration curve constructed with pooled plasma obtained from wild-type C57BL6/J mice. Plasma PAI-1 antigen levels were determined with a specific ELISA using monoclonal antibodies produced in gene-inactivated mice.18 Fibrinogen levels were determined with a clotting rate assay using human thrombin. Plasma levels of murine α₂-macroglobulin were determined by rocket electroimmunassay, as described,19 using a polyclonal rabbit antiserum kindly provided by Dr F. Van Leuven (Center for Human Genetics, University of Leuven, Belgium). Calibration curves were constructed using pooled plasma from wild-type male or female mice, for determination of α₂-macroglobulin levels in plasma samples from males or females, respectively. White blood cell, red blood cell, and platelet counts, hemoglobin and hematocrit levels, mean corpuscular value, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were determined on blood collected in trisodium citrate (final concentration, 10 mmol/L) using an automated analyzer.

Endotoxin (Escherichia coli lipopolysaccharide, serotype 0111: B4) was purchased from Sigma Chemical Co (St Louis, MO).

Liver or kidney extracts were prepared by homogenization and extraction with 0.1 mol/L Tris, 0.25% Triton X-100 (Merck, Darmstadt, Germany), pH 8.0, and protein concentration was determined using the BCA protein assay (Pierce, Rockford, IL).

SDS-PAGE without reduction was performed on 10% to 15% gradient gels using the PhastSystem (Pharmacia, Uppsala, Sweden). Immunoblotting, after transfer to nitrocellulose sheets, was performed using antisera against murine α₂-AP or plasminogen.

Bleeding times in mice were recorded after amputation of a standardized fragment of the tail (2 cm) or of a toe. Data are reported as mean ± SEM and statistical analysis was performed using the two-tailedt-test (nonparametric, Mann Whitney) for comparison between two groups. Genotype distributions and histopathologic data on fibrin deposition in kidney sections after endotoxin injection were compared by Chi-square analysis.

The targeting vector pPNT.α₂-AP, in which the neomycin resistance expression cassette replaces a 7-kb genomic fragment comprising exon 2 through part of exon 10 (including the stop codon), which represents the entire sequence encoding mature murine α₂-AP, was described previously.10 

Electroporation of 129R1 ES cells (obtained from A. Nagy, Samuel Lunenfeld Research Institute, Toronto, Canada) or of 129/SvJ RW4 ES cells (Genome Systems Inc, St Louis, MO) with the linearized targeting vector yielded, respectively, three (out of 127) or eight (out of 93) correctly targeted clones as confirmed by Southern blot analysis of purified genomic DNA with appropriate restriction enzymes and probes.10 

Targeted clones were used for aggregation with Swiss morulas (R1 ES clones) or C57BL6/J morulas (RW4 ES clones). Chimeric offspring, identified by the presence of agouti (R1 ES cells) or agouti and/or white (RW4 ES cells) coat pigmentation, were obtained (for both R1 and RW4 targeted clones), whereas germline transmission of ES-cell DNA was only obtained with RW4 ES clones (five germline-competent chimeras originating from three independently targeted clones). Heterozygous α₂-AP–deficient germline offspring, identified by Southern blot analysis of tail-tip genomic DNA, were intercrossed to generate α₂-AP^−/− progeny (yielding a genetic background of 50% 129/SvJ and 50% C57BL6/J).

DNA was isolated from mouse tail tips, digested with KpnI and analyzed by Southern blotting using a 3′ probe as described.10 

Polyadenylated RNA (polyA RNA) was extracted from kidney and liver using the Quick Prep mRNA purification kit (Pharmacia Biotech Benelux, Roosendaal, The Netherlands) and was submitted to first strand cDNA synthesis by oligo(dT) priming using the Ready-to-Go T-primed first strand kit (Pharmacia). The reaction products (RT-cDNA) were then used in PCR amplification with primers annealing in the coding part of exon 10 (sense primer: 5′-AATTGTTCCAGGGCCCAGACCTTCGT-3′, nucleotides (nt) 1109-1134 of murine α₂-AP cDNA, GenBank accession numberZ36774⁸; and antisense primer: 5′-GTCCTCCATGATGAAGAAGAGGAAGGG-3′, nt 1302-1276 of murine α₂-AP cDNA). The RT-PCR products were analyzed by separation on a 1% agarose gel.

α₂-AP^+/+ and α₂-AP^−/− mice (2 males and 2 females each at 6 and 20 weeks of age) were anesthetized and perfused through cardiac puncture with 0.9% NaCl followed by 4% formalin in 0.07 mol/L sodium phosphate buffer, pH 7.0.

Organs were removed, postfixed in the same fixative for 20 hours, and embedded in paraffin. Representative 7-μm sections of all tissues were examined microscopically after staining with hematoxylin/eosin. The tissue sections included cross sections of brain, heart, thymus, lung, liver, spleen, kidney, small and large intestine, stomach, cecum, leg muscle, reproductive organs (vas deferens, testis and epididymis, or uterus and ovaries), lymph node, adrenal gland, and pancreas.

Immunostaining for fibrin(ogen) was performed by incubating the sections with goat antiserum against murine fibrinogen/fibrin (Nordic, Tilburg, The Netherlands: working dilution 1/500) in 0.01 mol/L Tris, pH 7.6, containing 0.9% NaCl and 0.1% Triton X-100 for 3 hours at room temperature. After rinsing, the sections were incubated consecutively for 60 minutes with biotinylated rabbit antigoat IgG (Dako, Prosan, Ghent, Belgium; dilution 1/400) and with peroxidase-labeled avidin-biotin complex (Dako). Antibody binding was visualized with diaminobenzidine, resulting in a brown staining of the immunoreactive sites. All sections were briefly counterstained with Harris’ hematoxylin (BDH Laboratory Supplies, Poole, England), dehydrated, and mounted with DePex (Prosan, Gentbrugge, Belgium). Specificity of the primary antibodies was tested by adsorption of the antisera with murine fibrinogen.

Lysis of ¹²⁵I-fibrin–labeled murine plasma clots, injected into age- and weight-matched α₂-AP^+/+ or α₂-AP^−/− mice through a jugular vein (and embolized into the pulmonary arteries) was determined essentially as described previously.20 Therefore, 25 μL¹²⁵I-fibrin–labeled plasma clots, containing ≈70,000 cpm human ¹²⁵I-labeled fibrinogen, were prepared from a plasma pool of α₂-AP^+/+ or α₂-AP^−/− mice, by addition of thrombin (final concentration, 1.5 NIH U/mL) and CaCl₂ (final concentration 70 mmol/L). Clot lysis was evaluated by measurement of the residual radioactivity in the heart and lungs ex vivo at 2 hours and 4 hours after injection, and was defined as the amount of radioactivity that had disappeared, expressed as a percent of the total amount of radioactivity injected.

Mice matched for sex, age (12 to 17 weeks), and weight (25 ± 1 g and 25 ± 2 g for α₂-AP^+/+ and α₂-AP^−/− mice; mean ± SEM, n = 8) were injected intraperitoneally with endotoxin (2 mg/kg, dissolved in sterile saline). Four or 8 hours after injection, the mice were sacrificed by injection of Nembutal and immediately perfused for 15 to 30 minutes with saline. For protein extraction, one kidney was removed and immediately frozen at −80°C. For immunohistochemical analysis, the other kidney was fixed in 1% paraformaldehyde overnight at 4°C, washed with phosphate-buffered saline, incubated in 70% ethanol overnight at 4°C, and embedded in paraffin.

After immunostaining, the extent of fibrin deposition was given a severity score of 0 to 3.20 Score 0 indicated the absence of fibrin deposits; score 1, the appearance of a few small fibrin deposits, stained very weakly; score 2, the presence of small clearly stained fibrin deposits; score 3, the presence of large and strongly stained fibrin deposits.

Inactivation of the murine α₂-AP gene was achieved by replacing, through homologous recombination in ES cells, a 7-kb genomic fragment comprising the entire coding sequence, with a neomycin resistance cassette.10 Morula aggregation of recombinant RW4 cell clones harboring a disrupted α₂-AP gene yielded germline-competent chimeras (male), as indicated by the presence of agouti pups among their offspring after mating with C57BL6/J females. Heterozygous α₂-AP–deficient (α₂-AP^+/−) mice among the agouti offspring were identified by Southern blot analysis of tail-tip DNA (not shown), and were intercrossed, yielding α₂-AP^+/+, α₂-AP^+/−, and α₂-AP^−/− F₂ littermates (Fig1A).

Deficiency in α₂-AP in the α₂-AP^−/− progeny was confirmed at the mRNA level by the absence of signal in RT-PCR analyses of kidney and liver polyA RNA using primers annealing in the coding part of exon 10 (Fig 1B).

Among 161 F₂ littermates from heterozygous crosses that were genotyped at 4 to 5 weeks of age, 23% were α₂-AP^+/+, 53% were α₂-AP^+/−, and 24% were α₂-AP^−/− (Table1). This distribution is similar for males and females, and is not significantly different (by Chi-square analysis) from the expected Mendelian 1:2:1 ratio, thus indicating equal viability.

α₂-AP deficiency did not affect the growth rate of the mice, as evidenced by weighing the mice at weekly intervals (not shown). Body weights at 5 weeks of age were (mean ± SEM; n = 4), 17 ± 2 g and 15 ± 1 g for α₂-AP^+/+ and α₂-AP^−/− mice, respectively, with corresponding values of 22 ± 2 g and 23 ± 1 g at 10 weeks. Mean body weights of α₂-AP^+/− mice (mean ± SEM; n = 13) were 16 ± 1 g and 22 ± 4 g at 5 and 10 weeks of age, respectively. No macroscopic abnormalities were observed. α₂-AP^−/− mice (F₂ and F₃ generations) produced similar sizes of litters^+/− as α₂-AP^+/+ mice with similar time intervals between the litters (Table2).

α₂-AP antigen levels and other hemostatic parameters are summarized in Table 3. α₂-AP antigen levels in plasma were gene-dose–dependent. The functional assay showed an unexpectedly high level of antiplasmin activity in α₂-AP^−/− plasma as compared with α₂-AP^+/− and α₂-AP^+/+ plasma: 22% ± 3% (mean ± SEM; n = 14) versus 47% ± 5% (n = 10) and 94% ± 5% (n = 13), respectively. The levels in male or female α₂-AP^−/− mice were not significantly different: 18% ± 4.4% versus 26% ± 4.0% (n = 7;P = .16). This rapid reacting plasmin inhibitory activity cannot be due to murine α₂-AP activity, because no complexes corresponding to plasmin–α₂-AP could be detected on addition of murine or human plasmin to α₂-AP^−/− plasma (see below).

Plasma levels of α₂-macroglobulin antigen were higher in α₂-AP^−/− mice than in α₂-AP^+/+ mice, for females (128% ± 7%, n = 10, v 101% ± 5%, n = 7, P = .02), and for males (134% ± 13%, n = 10, v 100% ± 9%, n = 6; P = .12). All other measured hemostasis parameters and cell counts were comparable for wild-type mice and for heterozygous and homozygous α₂-AP–deficient mice.

Bleeding times after amputation of a toe were variable (range, 0.5 to 7 minutes), but were on average (mean ± SEM) not significantly different between α₂-AP^+/+ (210 ± 64 seconds; n = 5), α₂-AP^+/− (180 ± 37 seconds; n = 12) and α₂-AP^−/−(210 ± 72 seconds; n = 5) mice. Also after tail amputation, bleeding stopped spontaneously in all three genotypes. No significant rebleeding was observed.

Microscopic analysis of cross-sections through different organs of 6- or 20-week-old α₂-AP^−/− mice, as described above, did not show any apparent abnormalities or differences with corresponding sections of α₂-AP^+/+ mice.

Western blotting of plasma using polyclonal rabbit anti-murine α₂-AP antibodies (Fig 2A), showed a positive band with an estimated M_rof 65 kD in plasma of α₂-AP^+/+ mice but not in plasma of α₂-AP^−/− mice. In urokinase-activated plasma (incubation with 50 nmol/L tcu-PA for 1 hour at 37°C) of α₂-AP^+/+ mice, but not of α₂-AP^−/− mice, an additional band was observed with M_r about 140 kD, which represents plasmin–α₂-AP complex, as confirmed by blotting with affinity-purified polyclonal rabbit anti-murine plasminogen antibodies (Fig 2C). After addition of purified plasmin, (1 μmol/L) and incubation for 10 seconds at 37°C, plasmin–α₂-AP complex was also detected in α₂-AP^+/+ but not in α₂-AP^−/− plasma (Fig 2B). The faint band at M_r about 140 kD observed in α₂-AP^−/− plasma (Fig 2B, lane 2 and Fig2C, lane 4) does not correspond to plasmin–α₂-AP complex, as it is not recognized by the antibodies against murine α₂-AP (not shown).

α₂-AP antigen levels in liver extracts (expressed in ng/mg protein) were 37 ± 4 (n = 5) in α₂-AP^+/+ mice, 24 ± 1 (n = 11) in α₂-AP^+/− mice, and below the 1 ng/mg detection level (n = 5) in α₂-AP^−/−mice.

Spontaneous lysis within 2 to 4 hours of a¹²⁵I-fibrin–labeled pulmonary plasma clot was always higher in α₂-AP^−/− than in α₂-AP^+/+ mice (Fig3). In α₂-AP^+/+ mice, lysis of a clot produced from α₂-AP^+/+ or from α₂-AP^−/− plasma was comparable, indicating that the α₂-AP content of the clot does not significantly inhibit lysis in plasma with normal inhibitor concentration. Also in α₂-AP^−/−mice, lysis of both clot types was not significantly different.

Histopathologic examination and immunostaining of kidney sections after endotoxin injection in α₂-AP^+/+ mice showed the occasional presence of fibrin deposits in the glomeruli of the outer cortex (Fig 4, IB) and, more frequently, in the capillaries of the medulla (Fig 4, IIB). In α₂-AP^+/+ mice without endotoxin injection and in α₂-AP^−/− mice after endotoxin injection, significantly less fibrin deposits were detected in the glomeruli (Fig 4, IA and IC) and in the medulla (Fig 4, IIA and IIC). Semi-quantitative analysis of fibrin deposition in the glomeruli indicated that, 8 hours after endotoxin injection, all 15 sections of α₂-AP^−/− mice (4 animals) were free of fibrin (score 0), whereas only 9 of 16 sections of α₂-AP^+/+ were devoid of fibrin (Table 4). In the capillaries of the medulla, fibrin deposits were detected in 4 of 15 sections of α₂-AP^−/− mice, whereas 14 of 16 sections of α₂-AP^+/+ mice showed fibrin deposition (Table 4). Chi-square analysis using a two by four contingency table indicated a significant reduction of fibrin deposits in α₂-AP^−/− mice as compared with α₂-AP^+/+ mice (P = .11 at 4 hours and P < .05 at 8 hours for the glomeruli; p ≤ .005 at 4 hours and at 8 hours for the medulla).

PAI-1 levels (mean ± SEM) in extracts of kidney sections taken at the end of the experiments were not significantly different for α₂-AP^+/+ or α₂-AP^−/− mice (2.8 ± 0.44 or 2.3 ± 0.32 ng/mg protein at 4 hours, and 0.96 ± 0.34 or 0.90 ± 0.07 ng/mg protein at 8 hours, as compared with <0.1 ng/mg protein in both genotypes without endotoxin injection). Plasma PAI-1 levels after endotoxin injection in α₂-AP^+/+or α₂-AP^−/− mice were also comparable (140 ± 26 or 110 ± 14 ng/mL at 4 hours, and 190 ± 93 and 77 ± 14 ng/mL at 8 hours), and were increased more than 50-fold over baseline levels.

α₂-AP levels in kidney extracts of α₂-AP^+/+ mice were not increased after endotoxin injection (7.5 ± 0.12 and 5.6 ± 3.3 ng/mg protein at 4 hours and 8 hours after injection, as compared with 7.0 ± 0.12 ng/mg protein without endotoxin injection). Plasma α₂-AP levels in α₂-AP^+/+ mice were also comparable before (81 ± 5 μg/mL) and 4 hours (73 ± 4 μg/mL) or 8 hours (56 ± 3 μg/mL) after endotoxin injection.

Congenital homozygous α₂-AP deficiency was reported in patients who presented with hemorrhagic diathesis, whereas several cases of heterozygosity in different families have been described with no or only mild bleeding symptoms.21-29 In all heterozygotes described so far, both α₂-AP antigen and activity levels ranged between 40% and 60% of normal, suggesting that the deficiency is due to decreased synthesis of a normal α₂-AP molecule. A single case of dysfunctional α₂-AP (α₂-AP Enschede) associated with severe bleeding tendency has been reported in two siblings in a Dutch family.30 The ability of this α₂-AP to bind reversibly to plasminogen was not affected, but it was converted from an inhibitor of plasmin into a substrate, as a result of the insertion of an extra alanine residue in the reactive center loop.31The bleeding tendency in patients with α₂-AP deficiency is most likely due to premature lysis of hemostatic plugs, because the half-life of plasmin molecules generated at the fibrin surface may be considerably prolonged in the absence of α₂-AP. Acquired α₂-AP deficiency associated with enhanced fibrinolysis has been reported in patients with liver disease,32,33disseminated intravascular coagulation,32 and acute promyelocytic leukemia.34 Furthermore, α₂-AP levels may be significantly reduced in patients undergoing thrombolytic therapy, especially with nonfibrin-specific agents, as a result of extensive systemic generation of plasmin.35 After exhaustion of plasma α₂-AP, excess plasmin may degrade several plasma proteins, including fibrinogen, and eventually lead to bleeding complications. Pathophysiologic observations in humans thus support the relevant role of α₂-AP in regulating and controlling plasmin activity. In addition, studies in mice with deficiency of the main components of the fibrinolytic system indicate an important role of plasmin in fibrin surveillance and in maintenance of an intact hemostatic balance.17 To further substantiate these findings, we have generated mice with homozygous deficiency of α₂-AP, the main plasmin inhibitor in mammalian plasma.

α₂-AP^−/− mice develop and reproduce normally. Macroscopic examination and microscopic analysis of cross-sections of different organs did not show significant hemorrhage in 6- to 20-week-old α₂-AP^−/− mice. Furthermore, after amputation of tail or toe tips, bleeding stopped spontaneously in α₂-AP^−/−, as well as in α₂-AP^+/− and α₂-AP^+/+ mice. The absence of an overt bleeding phenotype in α₂-AP^−/− mice appears somewhat surprising in view of the observations in humans described above. Several factors may contribute to this apparent difference between humans and mice. First, although the main components of the fibrinolytic system are similar in both humans and mice, important quantitative differences were observed, as a result of which the fibrinolytic system in mice appeared to be very resistant to activation.5,36 Second, the spectrum of proteinase inhibitors in murine plasma, other than α₂-AP, may be more efficient toward plasmin than in human plasma, or additional inhibitory mechanisms may contribute. Inhibition of plasmin has indeed been observed by several other plasma-proteinase inhibitors, including α₂-macroglobulin and α₁-antitrypsin. The residual rapid-reacting plasmin-inhibitory activity in α₂-AP–deficient plasma may thus be explained by alternative inhibitory pathways. We have shown that it is not due to interaction with α₂-AP. Furthermore, the levels of antiplasmin activity are comparable in male and female α₂-AP^−/− mice and thus do not correlate with potential sex-related differences in α₂-macroglobulin levels. The apparent antiplasmin activity observed in the functional assay, which persisted in the presence of higher S-2403 concentrations or with the use of murine plasmin, may reflect an interaction with plasma proteins rendering the plasmin unavailable for the chromogenic substrate.

The absence of a bleeding phenotype in α₂-AP^−/− mice, as in many heterozygous patients, probably reflects the fact that the coagulation system adequately prevents bleeding under circumstances where the fibrinolytic system is not dramatically challenged. Extensive activation of the system, in the absence of α₂-AP, will, however, result in efficient fibrin degradation, and may thus cause lysis of hemostatic plugs and hemorrhagic complications.

In vivo clot lysis experiments confirm that the endogenous thrombolytic potential is significantly enhanced in α₂-AP^−/− mice, indicating a physiologic role of α₂-AP in fibrin surveillance. Furthermore, the experiments with cross-linked α₂-AP^+/+ and α₂-AP^−/− plasma clots in α₂-AP^+/+ and α₂-AP^−/− mice suggest that the efficiency of spontaneous thrombolysis is determined primarily by the α₂-AP concentration in circulating blood, and not by the amount that is cross-linked to fibrin. This suggests that cross-linking of α₂-AP to fibrin, which renders a fibrin clot less susceptible to degradation by plasmin, does not dramatically affect the lysability of the clot by the murine endogenous fibrinolytic system.

Injection of endotoxin in mice was previously shown to result in enhanced PAI-1 levels and in significant fibrin deposition in the kidneys, within 4 to 8 hours after injection.37 Using this model, fibrin deposition was found to be significantly reduced in α₂-AP^−/− mice as compared with α₂-AP^+/+ mice. This is most likely not due to a different degree of fibrin formation in both genotypes; furthermore, PAI-1 levels in kidney and plasma were enhanced to a similar degree after endotoxin injection. The observed difference, therefore, would appear to be due to a higher endogenous fibrinolytic capacity in α₂-AP^−/− mice, again confirming the role of α₂-AP in fibrin clearance.

In conclusion, α₂-AP^−/− mice survive, develop, and reproduce normally, but show an enhanced endogenous fibrinolytic capacity without overt bleeding phenotype.

Skillful technical assistance by K. Bijnens, E. Gils, L. Kieckens, T. Vancoetsem, B. Van Hoef, I. Vanlinthout, A. Van Nuffelen, M. Verstreken, and G. Wallays is gratefully acknowledged.