Genetic background determines response to hemostasis and thrombosis
© Hoover-Plow et al; licensee BioMed Central Ltd. 2006
Received: 07 July 2006
Accepted: 05 October 2006
Published: 05 October 2006
Thrombosis is the fatal and disabling consequence of cardiovascular diseases, the leading cause of mortality and morbidity in Western countries. Two inbred mouse strains, C57BL/6J and A/J, have marked differences in susceptibility to obesity, atherosclerosis, and vessel remodeling. However, it is unclear how these diverse genetic backgrounds influence pathways known to regulate thrombosis and hemostasis. The objective of this study was to evaluate thrombosis and hemostasis in these two inbred strains and determine the phenotypic response of A/J chromosomes in the C57BL/6J background.
A/J and C57Bl/6J mice were evaluated for differences in thrombosis and hemostasis. A thrombus was induced in the carotid artery by application of the exposed carotid to ferric chloride and blood flow measured until the vessel occluded. Bleeding and rebleeding times, as surrogate markers for thrombosis and hemostasis, were determined after clipping the tail and placing in warm saline. Twenty-one chromosome substitution strains, A/J chromosomes in a C57BL/6J background, were screened for response to the tail bleeding assay.
Thrombus occlusion time was markedly decreased in the A/J mice compared to C57BL/6J mice. Tail bleeding time was similar in the two strains, but rebleeding time was markedly increased in the A/J mice compared to C57BL/6J mice. Coagulation times and tail morphology were similar, but tail collagen content was higher in A/J than C57BL/6J mice. Three chromosome substitution strains, B6-Chr5A/J, B6-Chr11A/J, and B6-Chr17A/J, were identified with increased rebleeding time, a phenotype similar to A/J mice. Mice heterosomic for chromosomes 5 or 17 had rebleeding times similar to C57BL/6J mice, but when these two chromosome substitution strains, B6-Chr5A/J and B6-Chr17A/J, were crossed, the A/J phenotype was restored in these doubly heterosomic progeny.
These results indicate that susceptibility to arterial thrombosis and haemostasis is remarkably different in C57BL/and A/J mice. Three A/J chromosome substitution strains were identified that expressed a phenotype similar to A/J for rebleeding, the C57Bl/6J background could modify the A/J phenotype, and the combination of two A/J QTL could restore the phenotype. The diverse genetic backgrounds and differences in response to vascular injury induced thrombosis and the tail bleeding assay, suggest the potential for identifying novel genetic determinants of thrombotic risk.
Family history  is the strongest risk factor for cardiovascular diseases (CVD). While a number of genetic mutations have been identified, these account for only a small percentage of the CVD in human populations. Thrombus formation on fissured atherosclerotic plaques is the precipitating event in the transition from a stable or subclinical atherosclerotic disease and leads to acute myocardial infarction, ischemic stroke or peripheral arterial occlusion. Arterial and venous thrombosis are complex responses and are influenced by multiple genetic and environmental factors [2–5]. Polymorphisms and mutations in coagulation factors, fibrinolytic factors, platelet surface receptors, methylenetetrahydrofalate reductase, endothelial nitric oxide synthase, and antioxidant enzymes have been implicated as genetic determinants of susceptibility to thrombosis [6, 7]. Great strides have been made in the diagnosis and treatment of thrombosis in the last decade. However, strategies to prevent thrombosis have lagged far behind due, in part, to the contribution of multiple, and as yet undefined, genetic factors that lead to thrombotic risk. Moreover, it remains unclear how genetic background influences the function of molecules and pathways known to regulate thrombus formation and lysis and, thereby, contributes to the risk of thrombotic disease.
Identification of mice susceptible or resistant to obesity, atherosclerosis, and vascular injury
S (apoE-/-) 
R (transplant) 
A panel of chromosome substitution strains (CSS) was generated  by a "marker-assisted" breeding program where the progeny of a B6 × A/J cross were successively backcrossed to the B6 mice. Genetic markers were used to identify homozygosity in the background (B6) and the individual A/J chromosome. These strains have one chromosome from A/J mice in a B6 background. This allows the identification of a trait in one or more CSS and implies that at least one QTL resides on this chromosome. Use of this panel requires fewer mice to determine the QTL than does a genome-wide scan. Another advantage is the ability for detection of QTL in the presence of other QTL. In addition, initial screens with the CSS simplify the fine-mapping of QTL. Several studies of this CSS panel, B6 Chr1-19, X, YA/J, have been reported for behavior , weight gain , sterols , and plasma amino acids levels [13, 14], and have identified many more QTL than studies of the same traits using a genome-wide scan.
The purpose of this study was to evaluate the two inbred strains of mice for prothrombotic risk, utilizing a ferric chloride vascular injury model, tail bleeding assay, and measures of coagulation and fibrinolysis. The results of this study indicate that the two inbred strains, B6 and A/J mice, have diverse prothrombotic phenotypes, unrelated to coagulation or platelet aggregation. In addition, in the CSS, expression of the A/J phenotypes, rebleeding time and arterial occlusion, was modified in the B6 background, and suggested that interactions occurred among the A/J QTL. Thus, it should be possible to identify independent genetic determinants of susceptibility to pathological haemostasis and thrombosis.
The inbred strains, B6 (#000664), Lepob (#000632), A/J (#000646) and gene-targeted plasminogen (Plg) activator inhibitor deficient mice (PAI-1-/-) (#002507)  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  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 . 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 [18, 19] 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.
Bleeding and rebleeding assay
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.
Coagulation, Plg, α2-antiplasmin, fibrinogen, fibrinolytic and PAI-1 assays
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 . PAI-1 antigen concentration was determined by ELISA  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  and the fibrinolytic activity determined by fibrin degradation . 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).
Histochemical and immunohistochemistry analysis
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, Lepob, 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 .
Marked differences in arterial thrombus formation in B6 and A/J mice
Marked differences in rebleeding time in B6 and A/J mice
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 Lepob 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.
No difference in blood coagulation between B6 and A/J mice
Coagulation and Plasminogen System Parameters in B6 and A/J inbred mouse strains
Prothrombin Time (sec)
17 ± 1
18 ± 1
aPTT Time (sec)
66 ± 3
61 ± 7
131 ± 5
176 ± 9**
Fibrinolytic Activity (nM)
2.6 ± 0.1
3.0 ± 0.1*
PAI-1 Antigen (ng/mL)
7.4 ± 0.7
7.5 ± 0.7
PAI-1 Activity (U/mL)
185 ± 10
229 ± 8**
170 ± 6
195 ± 5**
1.6 ± 0.04
1.7 ± 0.05*
Plg and PAI-1 higher in A/J than B6 mice
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.
Tail morphology in B6 and A/J
Tail bleeding assay identifies three chromosomes with QTL for rebleeding in CSS
Interactions of CSS for PAI-1 antigen and activity
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.
Interactions of CSS for arterial thrombus formation
In this study, thrombotic risk was systematically assessed in two inbred strains of mice that have marked differences in susceptibility to diet-induced obesity, diet-induced atherosclerosis, and ligation-induced neointimal hyperplasia and vessel remodeling. Arterial occlusion time, tail bleeding and rebleeding time were evaluated as potential predictors of thrombotic response. Assessments of the two inbred strains were compared to values from gene targeted mice of the Plg network with altered fibrinolytic responses, as well as in leptin deficient mice with a reduced platelet function. Marked differences were found in the thrombotic response among the two inbred strains, B6 and A/J, and the observed differences were not correlated with change in coagulation or platelet function. Screening the CSS identified three chromosomes that harbored genes which contributed to the A/J phenotype of increased rebleeding time. Mice homosomic for these chromosomes or doubly heterosomic for two of the chromosomes, 5 and 17, were required to express the A/J phenotype, elevated rebleeding time. PAI-1 antigen and activity were decreased in both CSS-5 and CSS-17 and the heterosomic mice, CSS-5F1 and CSS-17F1, but not in the CSS-11 strain. Values were restored in the doubly heterosomic for two of chromosomes, 5 and 17. Arterial occlusion time was similar to B6 in the CSS-5 and CSS-17 homozygous strains, but increased in doubly heterosomic for two of chromosomes, 5 and 17.
The FeCl3-induced model of vascular injury and thrombosis in mice is now widely used to evaluate genetic and pharmacological interventions . The two inbred strains had marked differences in the time for occlusion of the carotid artery after FeCl3 injury. After FeCl3 treatment, thrombus formation and occlusion was remarkably shortened in the A/J mice compared to the B6 mice. These results have not previously been reported. A recent study  of sheer stress in rats, found that the magnitude of changes in sheer stress with increased blood flow varied with the different strains. Further investigation, beyond the scope of this study would be necessary to determine the contribution in the differences in size and sheer stress of the carotids to arterial occlusion rates in the B6 and A/J mice. In preliminary studies, we have noted differences in the composition of the thrombus in B6 and A/J mice. The pattern of blood flow cessation for the two inbred strains was different than for the Lepob mice  with impaired platelet function, and coagulation time was similar in the two strains. In the mice with deficiencies of the Plg network , thrombus formation time was reduced in the Plg-/- mice, but increased in the PAI-/- mice, suggesting that alterations in plasmin activity that affect the rate of clot lysis, can modulate the events leading to occlusive thrombus formation.
The tail-bleeding assay has been used extensively in mice to assess the impact of deficiencies and over expression of platelet and coagulation proteins in mice. Sweeny et al  screened 25 strains of mice and found only the RIIS/J mice to have increased bleeding time due to reduced von Willebrand antigen. Broze et al  evaluated mice with gene deletions of the coagulation pathway and found that while bleeding time was not increased, rebleeding persisted despite electrocautery of the tail in FVIII and FIX deficient mice. This observation raised the possibility that rebleeding time may be a sensitive reporter of genetic influences on thrombus formation and stability. Mice that are deficient in factors required for normal platelet function, platelet glycoprotein Ib , and protease-activated receptor-4,  have increased tail bleeding time. The leptin deficiency with reduced platelet aggregation clearly shows a marked increase in bleeding time and the increased bleeding time in Plg-/- and uPA-/- mice suggests that Plg and uPA may exert an influence on platelet response to promote normal thrombus formation. Overall, the marked increase in rebleeding times observed in certain mouse strains suggests genetic factors other than coagulation may play a role in the stability of a thrombus.
In the tail bleeding assay, A/J mice did not have changes in bleeding time, but rebleeding time was higher compared to B6 mice. In our study, Plg-/- mice had increased bleeding time, PAI-1-/- had decreased rebleeding time, and uPA-/- mice had an increased bleeding time similar to the Plg-/- mice (J. Hoover-Plow, A. Shchurin, and E. Hart, unpublished results). Increased bleeding or rebleeding times have not been reported in any of the Plg network targeted mice. Matsuno et al  reported no difference in bleeding time for tPA-/-, uPA-/-, PAI-1-/- mice compared to WT mice, and the assay was similar, but the tail clip segment was considerably shorter and bleeding times reduced compared to the times reported in our study. Bleeding time has not been previously reported for Plg-/- mice. We have found similar values for Plg-/- mice in a 50%B6:50%129 background (J. Hoover-Plow, A. Shchurin, and E. Hart, unpublished results). The results of this study suggest that not only is bleeding time genetically determined by background, but also that tail rebleeding time is genetically determined.
While blood pressure could conceivably be higher in mice with elevated tail bleeding time, several studies suggest that this is not the case. Studies of A/J [33–36] mice have reported either decreased blood pressure or no difference compared to the B6 mice. Blood pressure was measured in the Plg-/- mice (D. Hellard, J. Hoover-Plow, and E. Plow, unpublished results), and these mice have reduced blood pressure when compared to WT littermates, and uPA-/-  and Lepob [38, 39] mice also have reduced blood pressure, but there is no correspondence between blood pressure and tail bleeding or rebleeding times. Morphometric analysis of the tail indicated a difference in the collagen content of the tail in the A/J mice. Differences in the structure and/or metabolism of collagen or extracellular matrix proteins may contribute to the increased rebleeding and reduced occlusion time in the A/J mice and warrant further investigation.
The genes for Plg, PAI-1, and α2-antiplasmin reside on the three chromosomes identified for QTL for rebleeding time and may or may not be coincidental to the observed phenotypes found in the A/J mice and CSS. It is not likely that merely the concentration of these components explains the increased rebleeding time and reduced arterial occlusion time in the A/J mice. When compared to the B6 mice, the A/J mice and the three strains with elevated rebleeding time had variable Plg, PAI-1, and α2-antiplasmin. Two allelic forms of the mouse Plg gene have been reported for B6 and A/J mice . Plg concentration was increased in A/J mice compared to B6 mice. Although we did not detect differences in Plg structure, this would not exclude possible functional differences. A congenic strain (specific for a homosomic region from A/J and the remaining Chr and background is from B6 mice) of A/J chromosome 17 that includes the Plg gene was tested in the bleeding assay. Values for bleeding and rebleeding in this congenic strain were similar to values from B6 mice, indicating that the QTL is not Plg. There are genes for other proteases and matrix proteins that reside on chromosome 17 in addition to unknown genes. DNA base pair sequence data for the PAI-1 gene or the α2-antiplasmin gene in B6 and A/J mice have reported no differences , but regulatory genes upstream of the coding region may be important. Our results suggest that novel or unknown genes may interact with the Plg system components to modify their function.
In summary, this study reports marked differences in two inbred strains of mice, B6 and A/J, in arterial thrombosis formation in a FeCl3 vascular injury model, rebleeding time in a tail bleeding assay, plasminogen function, and tissue and vessel collagen deposition. Three chromosomes from A/J mice were identified that had QTL for rebleeding time. The genes may interact with components in the Plg network and modulate arterial occlusion time.
The marked independent differences demonstrated in the B6 and A/J mice can be exploited to identify genetic determinants of thrombosis and haemostasis. Our results of the CSS screening suggest that novel or unknown genes can be used to identify the genes responsible for the traits related to arterial thrombosis, tail bleeding/rebleeding and vessel extracellular matrix. Identification of differences in the parent strains, such as those described in this study, and screening of the CSS is a first-step in the discovery of new genetic determinants of thrombotic risk.
chromosome substitution strains
(B6 × CSS-#)F1
plasminogen activator inhibitor-1
tissue plasminogen activator
urokinase plasminogen activator
standard error of the mean
quantitative trait locus
The authors thank Dr. A. Vasanji, Shiyang Wang, and Jenna Saraniti for assistance with image analyses, David Hellard for performing the blood pressure measurements, Dr. Y.Shchurina for the plasminogen analyses, Drs. Lindsey Burrage and Jonathan Smith for helpful discussions, and Robin Lewis for assistance with manuscript preparation. This study was supported by grants from NIH, HL17964, HL65204, HL078701 (JHP), T32 HL07914 (AS), and RR12305 (JHN).
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