The inbred strains, B6 (#000664), Lep^ob (#000632), A/J (#000646) and gene-targeted plasminogen (Plg) activator inhibitor deficient mice (PAI-1-/-) (#002507) 15 mice in a B6 background were obtained from Jackson Laboratory (Bar Harbor, Maine) at 6 wks-of-age. The Plg-/- mice were generated as previously described 16 and maintained in the B6 background, generated by crossing mice from the original mixed (B6:129) background for eight generations with B6 mice. The Plg-/- mice do not reproduce well and Plg heterozygous pairs were used for breeding. Genotypes (Plg+/+, Plg+/-, Plg-/-) of the offspring were determined by a PCR assay at 3–4 weeks-of-age from an ear punch 17. Breeding pairs of the CSS mice were transferred from Dr. Nadeau's laboratory. All mice were housed and bred in the Biological Resource Unit at the Cleveland Clinic Foundation (CCF). Mice were housed in sterilized isolator cages, maintained on a 14 hr light/10 hr dark cycle, and were provided sterilized food and water ad libitum. Mice were fed a standard autoclavable laboratory diet consisting of: 23% protein, 4.5% fat, 6% fiber, (Purina, St. Louis, MO), and tested between 7 and 9 wks-of-age. All animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Research Advisory Committee at CCF.

To induce thrombosis formation in the carotid artery, a ferric chloride (FeCl₃) model of vessel injury 1819 was employed. Mice were anesthetized with ketamine/xylazine (80 mg/kg, 5 mg/kg), a midline cervical incision was made and the left common carotid artery isolated by blunt dissection. The flow probe (Transonic Systems, model 0.5PSB) was placed under the artery and when a stable baseline was reached, a 0.5 × 2 mm strip of filter paper saturated with 10% FeCl₃ solution was applied to the surface of the carotid artery for 3 min. Occlusion time was determined from the addition of the FeCl₃ solution to the occlusion of the artery (minimum blood flow). The flow probe was in place from the establishment of the baseline until several min after the stable occlusion had been reached or stopped at 30 min if it had not occluded. Blood flow was recorded every 10 sec (Transonic Systems, model TS420). There was no difference in body weight among the mice that were tested in the vascular injury model.

For the tail bleeding assay, mice were anesthetized with ketamine/xylazine (80 mg/kg, 5 mg/kg), the tail prewarmed for 5 min in 10 mL of saline at 37°C in a water bath. The tail was lifted from the saline and a 5 mm tail segment amputated and immediately returned to the saline. Bleeding time was measured as the time between the start of bleeding to cessation of bleeding. With the tail remaining in the same saline solution, the rebleeding time was measured from the time between bleeding cessation and the start of the second bleeding.

Mice were anesthetized with Isoflurane (Abbott), bled from the orbital sinus into uncoated capillary tubes, and a drop of blood immediately placed on a reagent strip (Synbiotics) for prothrombin time (PT) or activated partial thromboplastin time (aPTT) determinations and inserted into the precalibrated Coagulation Analyzer (SCA2000, Synbiotics). PAI-1 activity was determined according to the method of Chandler et al 20. PAI-1 antigen concentration was determined by ELISA 21 using sheep-anti mouse PAI-1 (American Diagnostica) as the capture antibody and mouse PAI-1 (American Diagnostica) as the standard (0–3.5 ng/mL). Plg was determined with a chromogenic assay 22 and the fibrinolytic activity determined by fibrin degradation 23. For the negative controls in these assays, plasma from PAI-1 or Plg deficient mice was used. Functionally active mouse α₂-antiplasmin was determined with a plasmin capture assay and detection with a mouse antibody (Molecular Innovations, Southfield, MI), and fibrinogen with an ELISA assay with antibodies that recognize mouse fibrinogen (Hyphen BioMed, France).

The frozen tails or injured carotids in OCT were sectioned at 5 μm thickness and stained with Masson's Trichrome (HT 15, Sigma Diagnostics). For quantitative analysis of collagen area in the tail sections, four sections from 2–3 sites were measured using computer assisted image analysis (Image-Pro Plus, Cybernetics). For the area determination of the injured carotid lumen, 3–4 sections from different sites approximately 100 μm apart were analyzed and averaged for each mouse.

Data are presented as mean ± SEM. The statistical analysis for comparisons between A/J and B6 mice and between wild-type (WT) and Plg-/- littermates was compared with a two-tailed t-test. Differences among B6, Lep^ob, PAI-1-/- mice were determined by a one-way ANOVA and a Newman-Keuls post-test. A value of P < 0.05 was considered significant. The statistical analysis for comparisons between B6 mice and CSS were determined with two-tail t-tests and the level of the significant P-values (see figure legends) determined with a Bonferroni correction to account for multihypothesis testing 13.

The FeCl₃ cartoid artery injury model 24, a well-characterized arterial thrombosis model, was used to induce an occlusive thrombus in the B6 and A/J mouse strains under investigation. A marked difference in arterial thrombus occlusion time was found between the B6 and the A/J mice (Figure 1A). The occlusion time in the A/J mice was 2-fold less than for the B6 mice. A 5% dose of FeCl₃ was tested in the A/J and B6 mice and the occlusion time was lower in the A/J than the B6 mice, the same results as with the 10% dose. The occlusion time (min for both B6 (15 ± 3, n = 3) and A/J (8 ± 1, n = 3) at 5% were longer than at 10% for B6 (10 ± 1, n = 17 and A/J (5 ± 2, n = 14). The curves of blood flow after FeCl₃ application indicated a pattern with a gradual decrease before occlusion for the B6 mice (Figure 1B), but for the A/J mice the blood flow decreased abruptly to zero (Figure 1C), demonstrating a marked difference from the B6 strain. The mean occlusion times were significantly different between the two strains (Figure 1A). The size of the carotids in the two strains was noticeably different. The area (mm²) of the lumen (0.066 ± 0.007, n = 6) was significantly less in the A/J mice than for B6 mice (0.128 ± 0.007, n = 6). However, the ratio of the thrombus to lumen was similar for A/J (0.63 ± 0.03) compared to B6 mice (0.67 ± 0.09). Calculated sheer stress rates 25 were 14% less in the A/J mice when compared to B6 mice.

The bleeding time, an indicator of hemostatic activity, was determined after prewarming the tail, clipping a 5 mm segment, placing the tail in warm saline and measuring the time for the bleeding to stop. We found this procedure was consistent and reproducible in different mouse strains and was not gender dependent. Bleeding time was not different in A/J mice compared to B6 mice (Figure 2A). In order to determine if specific pathways were altered in the B6 and A/J strains, mice with known alterations in hemostasis, Lep^ob mice, or thrombosis, Plg-/- and PAI-/- mice, were also tested. The Lep^ob mice have impaired platelet aggregation 26 and were evaluated as a reference for platelet function. In the Lep^ob mice (Figure 2A) there was nearly a 10-fold increase in bleeding time compared to B6 mice. Bleeding time was also significantly (P = 0.0001) increased by 66% in the Plg-/- mice compared to WT mice littermates. Bleeding time was not different in PAI-1-/- mice compared to B6 mice. In contrast to the Lep^ob mice with a platelet defect and 10-fold increase in bleeding time, there was no difference in the bleeding time between B6 and A/J mice and suggested platelet aggregation is not altered in these two strains.

Rebleeding time, an indicator of thrombus stability, was measured as the time between the cessation of bleeding and the start of the second bleeding (Figure 2B). Rebleeding times were significantly higher in A/J (2.6-fold, P < 0.001) mice than the B6 mice. In contrast, the rebleeding time was nearly 8.5-fold less in the Lep^ob mice than the B6 mice. To determine how alterations in the fibrinolytic system may affect rebleeding time, Plg-/- and PAI-1-/- mice were also tested (Figure 2B). Rebleeding time was not different in Plg-/- mice compared to WT littermates, but PAI-1-/- mice had a significantly (P < 0.05) reduced rebleeding time, which was 1.8-fold lower than for the B6 mice. Although a Plg deficiency did not result in a prolonged rebleeding time as anticipated, a PAI-1 deficiency resulted in a shortened time. The markedly increased rebleeding time in the A/J may suggest differences in the regulation of the Plg network.

To determine if differences in coagulation were contributing factors to the bleeding and rebleeding results, prothrombin time (PT) and activated partial thromboplastin time (aPTT) were measured. PT is a measurement of the extrinsic coagulation pathway (activation of Factor VII by tissue factor) while aPTT is a measurement of the intrinsic pathway (activation of Factors XII, XI, IX and VIII). The products of both pathways activate Factor X, which initiates the common pathway leading to thrombin generation and fibrin clot formation. PT and aPTT were measured in the B6 and A/J mice and values were not different between the two mouse strains (Table 2). These results suggest that the coagulation pathway is not different in the two inbred strains.

Plg concentration was 34% higher in the A/J mice than in B6 mice. In addition, fibrinolytic activity, PAI-1 activity, and α₂-anitplasmin were also increased in the A/J mice when compared to the B6 mice (Table 2). The functional consequences of these differences are not clear, since fibrinogen is also higher in the A/J mice. To determine whether there were protein structure changes of Plg from the A/J mice, plasma from the B6 and the A/J mice were subjected to electrophoresis under reduced, non-reduced, and non-denaturing conditions. No difference in migration for immunostained Plg under these conditions was observed (data not shown). The density of the Western blot from SDS-PAGE under reducing conditions of plasma of varying volumes from B6 and A/J mice was compared and the density of the bands was 56% higher in the A/J than the B6 mice (six individual mice were tested on at least two occasions) from the same amount of plasma (Fig. 1B). Thus, the concentration of Plg in plasma from the A/J mice was higher than plasma from the B6 mice as determined by two methods and no difference in migration after electrophoresis was observed, suggesting no marked changes in the structure of Plg in the two mouse strains.

The tails of the mice were examined for differences that might account for the variations in the bleeding and rebleeding values. Sections of the tail from the B6 and A/J mice were stained with Hematoxylin/Eosin or Masson's Trichrome and representative sections are shown in Figure 3. No obvious qualitative differences were observed in hair follicles, sebaceous glands, or center bone/cartilage in Hematoxylin/Eosin (Figure 3A) stained sections. Sections of the tails were stained with Masson's Trichrome (Figure 3B) that detects collagen, the major platelet adhesive substratum for initiation of thrombus formation. Collagen content (% tail area) was quantified (Figure 3C) from 3–4 sections from 2–3 areas/mouse. The amount of collagen was greater in the A/J than the B6 mice (P < 0.05). Collagen in vessels in adipose tissue in the A/J mice is also increased compared to the B6 mice (data not shown).

Using the tail bleeding/rebleeding assay as a surrogate marker for hemostasis and thrombosis, the CSS were screened. The bleeding times (Figure 4A) for the B6 and the A/J mice were not different, but 6six of the strains, B6-Chr5, 6, 8, 14, 15, and Y^A/J, had lower (P < 0.002) bleeding times (49–79 seconds) compared to the value for the B6 mice (121 sec). The rebleeding time (Figure 4B), determined as the time between bleeding cessation and initiation of the second bleeding, was markedly increased in the A/J mice compared to the B6 mice and two strains, B6-Chr11^A/J (CSS-11) and B6-Chr17^A/J (CSS-17), had significantly (P < 0.002) longer rebleeding times compared to the B6 mice that were similar to values from the A/J mice. Another strain, B6-Chr5^A/J (CSS-5), approached significance at P < 0.008. The rebleeding time was stopped at 600 sec, and the percentage of mice with this value determined for the three strains: CSS-5–60%, CSS-11–60% and CSS-A17–46%. The percentage of B6 mice with a rebleeding time of 600 sec was 7% and for the A/J mice the percentage was 85%.

Rebleeding time in the parent strains and CSS were compared to values from the progeny of mice that were crossed with B6 mice to produce heterosomic mice for the CSS-5, (B6xCSS-5)F1 and CSS-17 (B6xCSS-17)F1 strains (Figure 5). The rebleeding values for CSS-5F1 and CSS-17F1 mice were similar to the B6 rather than the A/J mice, suggesting the A/J trait is recessive. Bleeding and rebleeding in the progeny of the cross, B6-Chr5^A/J × B6-Chr17^A/J (CSS-5 × CSS-17) resulted in the recovery of the A/J phenotype in the progeny of these double heterosomic strains. Thus, despite the heterosomic chromosomes of 5 and 17 in the progeny of the CSS-5 × CSS-17, the phenotype was now similar to A/J. This suggested that traits on the A/J chromosomes were interacting to produce the phenotype.

Components of the Plg system reside on chromosomes CSS-5 (PAI-1), CSS-11 (α₂-antiplasmin), and CSS-17 (Plg). As reported in Table 2, the A/J mice had increased Plg antigen, fibrinolytic activity, α₂-antiplasmin, PAI-1 activity and fibrinogen when compared to B6 mice. In CSS-17 mice, the Plg antigen was also significantly (P = 0.015) increased (152 ± 6, n = 12) when compared to the B6 mice, but for CSS-5 and CSS-5 × 17, the Plg antigen was similar to values for the B6 mice. Fibrinolytic activity was not different in the CSS-5 or CSS-17 strains compared to the B6 mice. α₂-antiplasmin was significantly (P = < 0.01) reduced in CSS-5 mice (142 ± 4 μg/mL, n = 5), significantly (P < 0.05) higher in the CSS-11 (201 ± 6, n = 8), but not different in CSS-17 or CSS-5 × 17 mice compared to the B6 mice.

PAI-1 antigen and activity were determined in the CSS with increased rebleeding times, CSS-5, CSS-11, and CSS-17 (Figure 6), the heterosomic, CSS-5F1, CSS-17F1, and double hetersomic, CSS-5 × 17. CSS-5, CSS-17, CSS-5F1, and CSS-17F1 generally had reduced PAI-1 antigen when compared to B6 mice. The reduced PAI-1 antigen was a dominant trait due to the fact that the values were similar in the homosomic and heterosomic CSS-5 and CSS-17. The CSS-5 × CSS-17 double heterosomic strains had values similar to the B6 and A/J parent strains. PAI-1 activity was significantly reduced in the CSS-5 and CSS-17F1 mice, but again in the CSS-5 × CSS-17 strain the value for PAI-1 activity was restored to the value of the B6 and A/J parent strains. The PAI-1 antigen (5.4 ng/mL ± 0.5, n = 7) and activity (170 U/mL ± 20, n = 6) in the CSS-11 mice was not different than for B6 mice.

The tail bleeding assay was rapid and facilitated the screening of the 21 CSS strains, and subsequently the identified strains were tested for arterial occlusion after the FeCl₃ injury (Figure 7). The CSS-5 and CSS-17 had occlusion times similar to the B6 mice, but the doubly heterosomic CSS-5 × CSS-17 progeny had occlusion times higher than either the B6 mice or the heterosomic CSS-5F1 and CSS-17F1 or the A/J strains. In double heterosomic CSS-5 × CSS-17 mice, only one or three mice tested had occluded by 30 min; the other two mice did not occlude by 30 min. While only a small number of CSS mice have been tested for the arterial occlusion, the results suggest that interaction of the genes on chromosome 5 and 17 also determines arterial occlusion time.